This family has been recognized as alpha/beta hydrolase by Kamil et al. Previously associated with Lipoprotein_lipase family but is also close to phospholipase. vLIP may not serve as a traditional lipase enzyme, but the serine nucleophile position is essential in vivo for the viral functions of vLIP. Could be example of repurposing alpha/beta hydrolase fold toward a nonenzymatic role, possibly in lipid bonding
The genome of Marek's disease virus (MDV) has been predicted to encode a secreted glycoprotein, vLIP, which bears significant homology to the alpha/beta hydrolase fold of pancreatic lipases. Here it is demonstrated that MDV vLIP mRNA is produced via splicing and that vLIP is a late gene, due to its sensitivity to inhibition of DNA replication. While vLIP was found to conserve several residues essential to hydrolase activity, an unfavorable asparagine substitution is present at the lipase catalytic triad acid position. Consistent with structural predictions, purified recombinant vLIP did not show detectable activity on traditional phospholipid or triacylglyceride substrates. Two different vLIP mutant viruses, one bearing a 173-amino-acid deletion in the lipase homologous domain, the other having an alanine point mutant at the serine nucleophile position, caused a significantly lower incidence of Marek's disease in chickens and resulted in enhanced survival relative to two independently produced vLIP revertants or parental virus. These data provide the first evidence that vLIP enhances the replication and pathogenic potential of MDV. Furthermore, while vLIP may not serve as a traditional lipase enzyme, the data indicate that the serine nucleophile position is nonetheless essential in vivo for the viral functions of vLIP. Therefore, it is suggested that this particular example of lipase homology may represent the repurposing of an alpha/beta hydrolase fold toward a nonenzymatic role, possibly in lipid bonding.
Lipase's thermostability and organic solvent tolerance are two crucial properties that enable it to function as a biocatalyst. This study examined the characteristics of two recombinant thermostable lipases (Lk2, Lk3) based on transesterification activity. Conversion of C12-C18 methyl ester with paranitrophenol was investigated in various organic solvent. Both lipases exhibited activity on difference carbon chain length (C12 - C18, C18:1, C18:2) of substrates. The activity of Lk2 was higher in each of substrate compared to that the Lk3. Experimental findings showed that the best substrates for Lk2 and Lk3 are C18:1 and C18:2 respectively, in agreement with the computational analysis. The activity of both enzymes prefers on nonpolar solvent. On nonpolar solvent the enzymes are able to keep its native folding shown by the value of radius gyration, solvent-enzyme interaction and orientation of triad catalytic residues. Lk3 appeared to be more thermostable, with maximum activity at 55 degreesC. The presence of Fe3+ increased the activity of Lk2 and Lk3. However, the activity of both enzymes were dramatically decreased by the present of Ca2+ despite of the enzymes belong to family I.1 lipase known as calcium dependent enzyme. Molecular analysis on His loop of Lk2 and Lk3 on the present of Ca2+ showed that there were shifting on the orientation of catalytic triad residues. All the data suggest that Lk2 and Lk3 are novel lipase on the family I.1 and both lipase available as a biocatalyst candidate.
Title: Structure, mechanism, and enantioselectivity shifting of lipase LipK107 with a simple way Zhang L, Gao B, Yuan Z, He X, Yuan YA, Zhang JZ, Wei D Ref: Biochimica & Biophysica Acta, 1844:1183, 2014 : PubMed
Because of the complex mechanisms of enzymatic reactions, no precise and simple method of understanding and controlling the chiral selectivity of enzymes has been developed. However, structure-based rational design is a powerful approach to engineering enzymes with desired catalytic activities. In this work, a simple, structure-based, large-scale in silico design and virtual screening strategy was developed and successfully applied to enzyme engineering. We first performed protein crystallization and X-ray diffraction to determine the structure of lipase LipK107, which is a novel family I.1 lipase displaying activity for both R and S isomers in chiral resolution reactions. The catalytic mechanism of family I.1, which includes LipK107, was ascertained first through comparisons of the sequences and structures of lipases from other families. The binding states of LipK107, including the energy and the conformation of complexes with the R and S enantiomers, have been evaluated by careful biocomputation to figure out the reason for the higher S selectivity. Based on this study, a simple strategy for manipulating the chiral selectivity by modulating a crucial distance in the enzyme-substrate complex and judging virtual mutations in silico is recommended. Then, a novel electrostatic interaction analysis protocol was used to design LipK107 mutants to validate our strategy. Both positive and negative mutations determined using this theoretical protocol have been implemented in wet experiments and were proved to produce the desired enantioselectivity, showing a 176% increase or 50% decrease in enantioselectivity as desired. Because of its accuracy and versatility, the strategy is promising for practical applications.
BACKGROUND:
Biodiesels are methyl esters of fatty acids that are usually produced by base catalyzed transesterification of triacylglyerol with methanol. Some lipase enzymes are effective catalysts for biodiesel synthesis and have many potential advantages over traditional base or acid catalyzed trasesterification. Natural lipases are often rapidly inactivated by the high methanol concentrations used for biodiesel synthesis, however, limiting their practical use. The lipase from Proteus mirabilis is a particularly promising catalyst for biodiesel synthesis as it produces high yields of methyl esters even in the presence of large amounts of water and expresses very well in Escherichia coli. However, since the Proteus mirabilis lipase is only moderately stable and methanol tolerant, these properties need to be improved before the enzyme can be used industrially.
RESULTS:
We employed directed evolution, resulting in a Proteus mirabilis lipase variant with 13 mutations, which we call Dieselzyme 4. Dieselzyme 4 has greatly improved thermal stability, with a 30-fold increase in the half-inactivation time at 50[degree sign]C relative to the wild-type enzyme. The evolved enzyme also has dramatically increased methanol tolerance, showing a 50-fold longer half-inactivation time in 50% aqueous methanol. The immobilized Dieselzyme 4 enzyme retains the ability to synthesize biodiesel and has improved longevity over wild-type or the industrially used Brukholderia cepacia lipase during many cycles of biodiesel synthesis. A crystal structure of Dieselzyme 4 reveals additional hydrogen bonds and salt bridges in Dieselzyme 4 compared to the wild-type enzyme, suggesting that polar interactions may become particularly stabilizing in the reduced dielectric environment of the oil and methanol mixture used for biodiesel synthesis.
CONCLUSIONS:
Directed evolution was used to produce a stable lipase, Dieselzyme 4, which could be immobilized and re-used for biodiesel synthesis. Dieselzyme 4 outperforms the industrially used lipase from Burkholderia cepacia and provides a platform for still further evolution of desirable biodiesel production properties.
Title: Crystal Structure of Proteus mirabilis Lipase, a Novel Lipase from the Proteus/Psychrophilic Subfamily of Lipase Family I.1 Korman TP, Bowie JU Ref: PLoS ONE, 7:e52890, 2012 : PubMed
Bacterial lipases from family I.1 and I.2 catalyze the hydrolysis of triacylglycerol between 25-45 degrees C and are used extensively as biocatalysts. The lipase from Proteus mirabilis belongs to the Proteus/psychrophilic subfamily of lipase family I.1 and is a promising catalyst for biodiesel production because it can tolerate high amounts of water in the reaction. Here we present the crystal structure of the Proteus mirabilis lipase, a member of the Proteus/psychrophilic subfamily of I.1lipases. The structure of the Proteus mirabilis lipase was solved in the absence and presence of a bound phosphonate inhibitor. Unexpectedly, both the apo and inhibitor bound forms of P. mirabilis lipase were found to be in a closed conformation. The structure reveals a unique oxyanion hole and a wide active site that is solvent accessible even in the closed conformation. A distinct mechanism for Ca(2+) coordination may explain how these lipases can fold without specific chaperones.
Title: Lipases for biotechnology Jaeger KE, Eggert T Ref: Curr Opin Biotechnol, 13:390, 2002 : PubMed
Lipases constitute the most important group of biocatalysts for biotechnological applications. The high-level production of microbial lipases requires not only the efficient overexpression of the corresponding genes but also a detailed understanding of the molecular mechanisms governing their folding and secretion. The optimisation of industrially relevant lipase properties can be achieved by directed evolution. Furthermore, novel biotechnological applications have been successfully established using lipases for the synthesis of biopolymers and biodiesel, the production of enantiopure pharmaceuticals, agrochemicals, and flavour compounds.
Title: Crystal structure of pseudomonas aeruginosa lipase in the open conformation. The prototype for family I.1 of bacterial lipases Nardini M, Lang DA, Liebeton K, Jaeger KE, Dijkstra BW Ref: Journal of Biological Chemistry, 275:31219, 2000 : PubMed
The x-ray structure of the lipase from Pseudomonas aeruginosa PAO1 has been determined at 2.54 A resolution. It is the first structure of a member of homology family I.1 of bacterial lipases. The structure shows a variant of the alpha/beta hydrolase fold, with Ser(82), Asp(229), and His(251) as the catalytic triad residues. Compared with the "canonical" alpha/beta hydrolase fold, the first two beta-strands and one alpha-helix (alphaE) are not present. The absence of helix alphaE allows the formation of a stabilizing intramolecular disulfide bridge. The loop containing His(251) is stabilized by an octahedrally coordinated calcium ion. On top of the active site a lid subdomain is in an open conformation, making the catalytic cleft accessible from the solvent region. A triacylglycerol analogue is covalently bound to Ser(82) in the active site, demonstrating the position of the oxyanion hole and of the three pockets that accommodate the sn-1, sn-2, and sn-3 fatty acid chains. The inhibited enzyme can be thought to mimic the structure of the tetrahedral intermediate that occurs during the acylation step of the reaction. Analysis of the binding mode of the inhibitor suggests that the size of the acyl pocket and the size and interactions of the sn-2 binding pocket are the predominant determinants of the regio- and enantio-preference of the enzyme.
Title: Bacterial lipolytic enzymes: classification and properties Arpigny JL, Jaeger KE Ref: Biochemical Journal, 343:177, 1999 : PubMed
Knowledge of bacterial lipolytic enzymes is increasing at a rapid and exciting rate. To obtain an overview of this industrially very important class of enzymes and their characteristics, we have collected and classified the information available from protein and nucleotide databases. Here we propose an updated and extensive classification of bacterial esterases and lipases based mainly on a comparison of their amino acid sequences and some fundamental biological properties. These new insights result in the identification of eight different families with the largest being further divided into six subfamilies. Moreover, the classification enables us to predict (1) important structural features such as residues forming the catalytic site or the presence of disulphide bonds, (2) types of secretion mechanism and requirement for lipase-specific foldases, and (3) the potential relationship to other enzyme families. This work will therefore contribute to a faster identification and to an easier characterization of novel bacterial lipolytic enzymes.
Lipases constitute the most important group of biocatalysts for biotechnological applications. The high-level production of microbial lipases requires not only the efficient overexpression of the corresponding genes but also a detailed understanding of the molecular mechanisms governing their folding and secretion. The optimisation of industrially relevant lipase properties can be achieved by directed evolution. Furthermore, novel biotechnological applications have been successfully established using lipases for the synthesis of biopolymers and biodiesel, the production of enantiopure pharmaceuticals, agrochemicals, and flavour compounds.
Title: Bacterial lipolytic enzymes: classification and properties Arpigny JL, Jaeger KE Ref: Biochemical Journal, 343:177, 1999 : PubMed
Knowledge of bacterial lipolytic enzymes is increasing at a rapid and exciting rate. To obtain an overview of this industrially very important class of enzymes and their characteristics, we have collected and classified the information available from protein and nucleotide databases. Here we propose an updated and extensive classification of bacterial esterases and lipases based mainly on a comparison of their amino acid sequences and some fundamental biological properties. These new insights result in the identification of eight different families with the largest being further divided into six subfamilies. Moreover, the classification enables us to predict (1) important structural features such as residues forming the catalytic site or the presence of disulphide bonds, (2) types of secretion mechanism and requirement for lipase-specific foldases, and (3) the potential relationship to other enzyme families. This work will therefore contribute to a faster identification and to an easier characterization of novel bacterial lipolytic enzymes.
This family corresponds to family I.3 of the classification of Arpigny and Jaeger (1999) The N-catalytic domain (residues 1370) contains the active site residues, Ser207, Asp255, and His313 4, 5. The C-domain contains several repeats of the RTX motif and a putative secretion signal near the C-terminus. The Cdomain contains two beta-roll motifs, laterally stacked together forming the so called beta-roll sandwich. The first beta-roll motif consists of residues 373417, containing five RTX repeats and binds three Ca2+ ions. The second beta-roll motif consists of residues 493-568, containing eight RTX repeats and binds five Ca2+ ions. HemolysinCabind (PF00353). This family contains Polyurethanases (PURase)
Title: X-ray crystallographic and MD simulation studies on the mechanism of interfacial activation of a family I.3 lipase with two lids Angkawidjaja C, Matsumura H, Koga Y, Takano K, Kanaya S Ref: Journal of Molecular Biology, 400:82, 2010 : PubMed
The interfacial activation mechanism of family I.3 lipase from Pseudomonas sp. MIS38 (PML), which has two alpha-helical lids (lid1 and lid2), was investigated using a combination of X-ray crystallography and molecular dynamics (MD) simulation. The crystal structure of PML in an open conformation was determined at 2.1 A resolution in the presence of Ca(2+) and Triton X-100. Comparison of this structure with that in the closed conformation indicates that both lids greatly change their positions and lid1 is anchored by the calcium ion (Ca1) in the open conformation. This structure was not seriously changed even when the protein was dialyzed extensively against the Ca(2+)-free buffer containing Triton X-100 before crystallization, indicating that the open conformation is fairly stable unless a micellar substance is removed. The crystal structure of the PML derivative, in which the active site serine residue (Ser207) is diethylphosphorylated by soaking the crystal of PML in the open conformation in a solution containing diethyl p-nitrophenyl phosphate, was also determined. This structure greatly resembles that in the open conformation, indicating that PML structure in the open conformation represents that in the active form. MD simulation of PML in the open conformation in the absence of micelles showed that lid2 closes first, while lid1 maintains its open conformation. Likewise, MD simulation of PML in the closed conformation in the absence of Ca(2+) and in the presence of octane or trilaurin micelles showed that lid1 opens, while lid2 remains closed. These results suggest that Ca1 functions as a hook for stabilization of a fully opened conformation of lid1 and for initiation of subsequent opening of lid2.
A family I.3 lipase from Pseudomonas sp. MIS38 (PML) contains three Ca(2+)-binding sites (Ca1-Ca3) in the N-catalytic domain. Of them, the Ca1 site is formed only in an open conformation. To analyze the role of these Ca(2+)-binding sites, three mutant proteins D157A-PML, D275A-PML and D337A-PML, which are designed to remove the Ca1, Ca2 and Ca3 sites, respectively, were constructed. Of them, the crystal structures of D157A-PML and D337A-PML in a closed conformation were determined. Both structures are nearly identical to that of the wild-type protein, except that the Ca3 site is missing in the D337A-PML structure. D157A-PML was as stable as the wild-type protein. Nevertheless, it exhibited little lipase and very weak esterase activities. D275A-PML was less stable than the wild-type protein by approximately 5 degrees C in T(1/2). It exhibited weak but significant lipase and esterase activities when compared with the wild-type protein. D337A-PML was also less stable than the wild-type protein by approximately 5 degrees C in T(1/2) but was fully active. These results suggest that the Ca1 site is required to make the active site fully open by anchoring lid 1. The Ca2 and Ca3 sites contribute to the stabilization of PML. The Ca2 site is also required to make PML fully active.
The crystal structure of a family I.3 lipase from Pseudomonas sp. MIS38 in a closed conformation was determined at 1.5A resolution. This structure highly resembles that of Serratia marcescens LipA in an open conformation, except for the structures of two lids. Lid1 is anchored by a Ca2+ ion (Ca1) in an open conformation, but lacks this Ca1 site and greatly changes its structure and position in a closed conformation. Lid2 forms a helical hairpin in an open conformation, but does not form it and covers the active site in a closed conformation. Based on these results, we discuss on the lid-opening mechanism.
Title: A calcium-gated lid and a large beta-roll sandwich are revealed by the crystal structure of extracellular lipase from Serratia marcescens Meier R, Drepper T, Svensson V, Jaeger KE, Baumann U Ref: Journal of Biological Chemistry, 282:31477, 2007 : PubMed
Lipase LipA from Serratia marcescens is a 613-amino acid enzyme belonging to family I.3 of lipolytic enzymes that has an important biotechnological application in the production of a chiral precursor for the coronary vasodilator diltiazem. Like other family I.3 lipases, LipA is secreted by Gram-negative bacteria via a type I secretion system and possesses 13 copies of a calcium binding tandem repeat motif, GGXGXDXUX (U, hydrophobic amino acids), in the C-terminal part of the polypeptide chain. The 1.8-A crystal structure of LipA reveals a close relation to eukaryotic lipases, whereas family I.1 and I.2 enzymes appear to be more distantly related. Interestingly, the structure shows for the N-terminal lipase domain a variation on the canonical alpha/beta hydrolase fold in an open conformation, where the putative lid helix is anchored by a Ca(2+) ion essential for activity. Another novel feature observed in this lipase structure is the presence of a helical hairpin additional to the putative lid helix that exposes a hydrophobic surface to the aqueous medium and might function as an additional lid. The tandem repeats form two separated parallel beta-roll domains that pack tightly against each other. Variations of the consensus sequence of the tandem repeats within the second beta-roll result in an asymmetric Ca(2+) binding on only one side of the roll. The analysis of the properties of the beta-roll domains suggests an intramolecular chaperone function.
Title: Lipases for biotechnology Jaeger KE, Eggert T Ref: Curr Opin Biotechnol, 13:390, 2002 : PubMed
Lipases constitute the most important group of biocatalysts for biotechnological applications. The high-level production of microbial lipases requires not only the efficient overexpression of the corresponding genes but also a detailed understanding of the molecular mechanisms governing their folding and secretion. The optimisation of industrially relevant lipase properties can be achieved by directed evolution. Furthermore, novel biotechnological applications have been successfully established using lipases for the synthesis of biopolymers and biodiesel, the production of enantiopure pharmaceuticals, agrochemicals, and flavour compounds.
Title: Bacterial lipolytic enzymes: classification and properties Arpigny JL, Jaeger KE Ref: Biochemical Journal, 343:177, 1999 : PubMed
Knowledge of bacterial lipolytic enzymes is increasing at a rapid and exciting rate. To obtain an overview of this industrially very important class of enzymes and their characteristics, we have collected and classified the information available from protein and nucleotide databases. Here we propose an updated and extensive classification of bacterial esterases and lipases based mainly on a comparison of their amino acid sequences and some fundamental biological properties. These new insights result in the identification of eight different families with the largest being further divided into six subfamilies. Moreover, the classification enables us to predict (1) important structural features such as residues forming the catalytic site or the presence of disulphide bonds, (2) types of secretion mechanism and requirement for lipase-specific foldases, and (3) the potential relationship to other enzyme families. This work will therefore contribute to a faster identification and to an easier characterization of novel bacterial lipolytic enzymes.
This family correspond to family I.5 of the classification of Arpigny and Jaeger (1999) and abH15 of the LED database. It includes lipases from gram positive bacteria, organic-solvent tolerant, showing thermoalkalophilic properties and high molecular weight resulting from extra domain where a zinc ion is coordinatively bound to the enzyme. The family also contains poly (butylene adipate-co-terephthalate)-hydrolyzing lipase from Pelosinus fermentans
Certain alpha/beta hydrolases have the ability to hydrolyze synthetic polyesters. While their partial hydrolysis has a potential for surface functionalization, complete hydrolysis allows recycling of valuable building blocks. Although knowledge about biodegradation of these materials is important regarding their fate in the environment, it is currently limited to aerobic organisms. A lipase from the anaerobic groundwater organism Pelosinus fermentans DSM 17108 (PfL1) was cloned and expressed in Escherichia coli BL21-Gold(DE3) and purified from the cell extract. Biochemical characterization with small substrates showed thermoalkalophilic properties (T opt = 50 degrees C, pHopt = 7.5) and higher activity towards para-nitrophenyl octanoate (12.7 U mg-1) compared to longer and shorter chain lengths (C14 0.7 U mg-1 and C2 4.3 U mg-1, respectively). Crystallization and determination of the 3-D structure displayed the presence of a lid structure and a zinc ion surrounded by an extra domain. These properties classify the enzyme into the I.5 lipase family. PfL1 is able to hydrolyze poly(1,4-butylene adipate-co-terephthalate) (PBAT) polymeric substrates. The hydrolysis of PBAT showed the release of small building blocks as detected by liquid chromatography-mass spectrometry (LC-MS). Protein dynamics seem to be involved with lid opening for the hydrolysis of PBAT by PfL1.
Title: Improvement of thermal stability via outer-loop ion pair interaction of mutated T1 lipase from Geobacillus zalihae strain T1 Ruslan R, Rahman RNZRA, Leow ATC, Ali MSM, Basri M, Salleh AB Ref: Int J Mol Sci, 13:943, 2012 : PubMed
Mutant D311E and K344R were constructed using site-directed mutagenesis to introduce an additional ion pair at the inter-loop and the intra-loop, respectively, to determine the effect of ion pairs on the stability of T1 lipase isolated from Geobacillus zalihae. A series of purification steps was applied, and the pure lipases of T1, D311E and K344R were obtained. The wild-type and mutant lipases were analyzed using circular dichroism. The T(m) for T1 lipase, D311E lipase and K344R lipase were approximately 68.52 degrees C, 70.59 degrees C and 68.54 degrees C, respectively. Mutation at D311 increases the stability of T1 lipase and exhibited higher T(m) as compared to the wild-type and K344R. Based on the above, D311E lipase was chosen for further study. D311E lipase was successfully crystallized using the sitting drop vapor diffusion method. The crystal was diffracted at 2.1 A using an in-house X-ray beam and belonged to the monoclinic space group C2 with the unit cell parameters a = 117.32 A, b = 81.16 A and c = 100.14 A. Structural analysis showed the existence of an additional ion pair around E311 in the structure of D311E. The additional ion pair in D311E may regulate the stability of this mutant lipase at high temperatures as predicted in silico and spectroscopically.
Title: High level expression and characterization of a novel thermostable, organic solvent tolerant, 1,3-regioselective lipase from Geobacillus sp. strain ARM Ebrahimpour A, Rahman RNZRA, Basri M, Salleh AB Ref: Bioresour Technol, 102:6972, 2011 : PubMed
The mature ARM lipase gene was cloned into the pTrcHis expression vector and over-expressed in Escherichia coli TOP10 host. The optimum lipase expression was obtained after 18 h post induction incubation with 1.0mM IPTG, where the lipase activity was approximately 1623-fold higher than wild type. A rapid, high efficient, one-step purification of the His-tagged recombinant lipase was achieved using immobilized metal affinity chromatography with 63.2% recovery and purification factor of 14.6. The purified lipase was characterized as a high active (7092 U mg(-1)), serine-hydrolase, thermostable, organic solvent tolerant, 1,3-specific lipase with a molecular weight of about 44 kDa. The enzyme was a monomer with disulfide bond(s) in its structure, but was not a metalloenzyme. ARM lipase was active in a broad range of temperature and pH with optimum lipolytic activity at pH 8.0 and 65 degrees C. The enzyme retained 50% residual activity at pH 6.0-7.0, 50 degrees C for more than 150 min.
Title: Crystallization and preliminary X-ray crystallographic analysis of a thermostable organic solvent-tolerant lipase from Bacillus sp. strain 42 Khusaini MS, Rahman RNZRA, Ali MSM, Leow ATC, Basri M, Salleh AB Ref: Acta Crystallographica Sect F Struct Biol Cryst Commun, 67:401, 2011 : PubMed
An organic solvent-tolerant lipase from Bacillus sp. strain 42 was crystallized using the capillary-tube method. The purpose of studying this enzyme was in order to better understand its folding and to characterize its properties in organic solvents. By initially solving its structure in the native state, further studies on protein-solvent interactions could be performed. X-ray data were collected at 2.0 A resolution using an in-house diffractometer. The estimated crystal dimensions were 0.09x0.19x0.08 mm. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a=117.41, b=80.85, c=99.44 A, beta=96.40 degrees .
Title: Crystallization and preliminary X-ray crystallographic analysis of highly thermostable L2 lipase from the newly isolated Bacillus sp. L2 Shariff FM, Rahman RNZRA, Ali MSM, Chor AL, Basri M, Salleh AB Ref: Acta Crystallographica Sect F Struct Biol Cryst Commun, 66:715, 2010 : PubMed
Purified thermostable recombinant L2 lipase from Bacillus sp. L2 was crystallized by the counter-diffusion method using 20% PEG 6000, 50 mM MES pH 6.5 and 50 mM NaCl as precipitant. X-ray diffraction data were collected to 2.7 A resolution using an in-house Bruker X8 PROTEUM single-crystal diffractometer system. The crystal belonged to the primitive orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 87.44, b = 94.90, c = 126.46 A. The asymmetric unit contained one single molecule of protein, with a Matthews coefficient (V(M)) of 2.85 A(3) Da(-1) and a solvent content of 57%.
The bacterial thermoalkalophilic lipases that hydrolyze saturated fatty acids at 60-75 degrees C and pH 8-10 are grouped as the lipase family I.5. We report here the crystal structure of the lipase from Geobacillus thermocatenulatus, the first structure of a member of the lipase family I.5 showing an open configuration. Unexpectedly, enzyme activation involves large structural rearrangements of around 70 amino acids and the concerted movement of two lids, the alpha6- and alpha7-helices, unmasking the active site. Central in the restructuring process of the lids are both the transfer of bulky hydrophobic residues out of the N-terminal end of the alpha6-helix and the incorporation of short side chain residues to the alpha6 C-terminal end. All these structural changes are stabilized by the Zn(2+)-binding domain, which is characteristic of this family of lipases. Two detergent molecules are placed in the active site, mimicking chains of the triglyceride substrate, demonstrating the position of the oxyanion hole and the three pockets that accommodate the sn-1, sn-2, and sn-3 fatty acids chains. The combination of structural and biochemical studies indicate that the lid opening is not mediated by temperature but triggered by interaction with lipid substrate.
Lipases constitute the most important group of biocatalysts for biotechnological applications. The high-level production of microbial lipases requires not only the efficient overexpression of the corresponding genes but also a detailed understanding of the molecular mechanisms governing their folding and secretion. The optimisation of industrially relevant lipase properties can be achieved by directed evolution. Furthermore, novel biotechnological applications have been successfully established using lipases for the synthesis of biopolymers and biodiesel, the production of enantiopure pharmaceuticals, agrochemicals, and flavour compounds.
Title: Novel zinc-binding center and a temperature switch in the Bacillus stearothermophilus L1 lipase Jeong ST, Kim HK, Kim SJ, Chi SW, Pan JG, Oh TK, Ryu SE Ref: Journal of Biological Chemistry, 277:17041, 2002 : PubMed
The bacterial thermoalkalophilic lipases optimally hydrolyze saturated fatty acids at elevated temperatures. They also have significant sequence homology with staphylococcal lipases, and both the thermoalkalophilic and staphylococcal lipases are grouped as the lipase family I.5. We report here the first crystal structure of the lipase family I.5, the structure of a thermoalkalophilic lipase from Bacillus stearothermophilus L1 (L1 lipase) determined at 2.0-A resolution. The structure is in a closed conformation, and the active site is buried under a long lid helix. Unexpectedly, the structure exhibits a zinc-binding site in an extra domain that accounts for the larger molecular size of the family I.5 enzymes in comparison to other microbial lipases. The zinc-coordinated extra domain makes tight interactions with the loop extended from the C terminus of the lid helix, suggesting that the activation of the family I.5 lipases may be regulated by the strength of the interactions. The unusually long lid helix makes strong hydrophobic interactions with its neighbors. The structural information together with previous biochemical observations indicate that the temperature-mediated lid opening is triggered by the thermal dissociation of the hydrophobic interactions.
We describe the first lipase structure from a thermophilic organism. It shares less than 20% amino acid sequence identity with other lipases for which there are crystal structures, and shows significant insertions compared with the typical alpha/beta hydrolase canonical fold. The structure contains a zinc-binding site which is unique among all lipases with known structures, and which may play a role in enhancing thermal stability. Zinc binding is mediated by two histidine and two aspartic acid residues. These residues are present in comparable positions in the sequences of certain lipases for which there is as yet no crystal structural information, such as those from Staphylococcal species and Arabidopsis thaliana. The structure of Bacillus stearothermophilus P1 lipase provides a template for other thermostable lipases, and offers insight into mechanisms used to enhance thermal stability which may be of commercial value in engineering lipases for industrial uses.
Title: Bacterial lipolytic enzymes: classification and properties Arpigny JL, Jaeger KE Ref: Biochemical Journal, 343:177, 1999 : PubMed
Knowledge of bacterial lipolytic enzymes is increasing at a rapid and exciting rate. To obtain an overview of this industrially very important class of enzymes and their characteristics, we have collected and classified the information available from protein and nucleotide databases. Here we propose an updated and extensive classification of bacterial esterases and lipases based mainly on a comparison of their amino acid sequences and some fundamental biological properties. These new insights result in the identification of eight different families with the largest being further divided into six subfamilies. Moreover, the classification enables us to predict (1) important structural features such as residues forming the catalytic site or the presence of disulphide bonds, (2) types of secretion mechanism and requirement for lipase-specific foldases, and (3) the potential relationship to other enzyme families. This work will therefore contribute to a faster identification and to an easier characterization of novel bacterial lipolytic enzymes.
This family correspond to family I.6 of the classification of Arpigny and Jaeger (1999). These lipases differ from other bacterial lipases. They present high phospholipase A1 activity. The substrate-binding cavity contains two large hydrophobic acyl chain-binding pockets and a shallow and more polar third pocket that is capable of binding either a (short) fatty acid or a phospholipid head-group.
Staphylococcus hyicus lipase differs from other bacterial lipases in its high phospholipase A1 activity. Here, we present the crystal structure of the S. hyicus lipase at 2.86 A resolution. The lipase is in an open conformation, with the active site partly covered by a neighbouring molecule. Ser124, Asp314 and His355 form the catalytic triad. The substrate-binding cavity contains two large hydrophobic acyl chain-binding pockets and a shallow and more polar third pocket that is capable of binding either a (short) fatty acid or a phospholipid head-group. A model of a phospholipid bound in the active site shows that Lys295 is at hydrogen bonding distance from the substrate's phosphate group. Residues Ser356, Glu292 and Thr294 hold the lysine in position by hydrogen bonding and electrostatic interactions. These observations explain the biochemical data showing the importance of Lys295 and Ser356 for phospholipid binding and phospholipase A1 activity.
Title: Lipases for biotechnology Jaeger KE, Eggert T Ref: Curr Opin Biotechnol, 13:390, 2002 : PubMed
Lipases constitute the most important group of biocatalysts for biotechnological applications. The high-level production of microbial lipases requires not only the efficient overexpression of the corresponding genes but also a detailed understanding of the molecular mechanisms governing their folding and secretion. The optimisation of industrially relevant lipase properties can be achieved by directed evolution. Furthermore, novel biotechnological applications have been successfully established using lipases for the synthesis of biopolymers and biodiesel, the production of enantiopure pharmaceuticals, agrochemicals, and flavour compounds.
Title: Bacterial lipolytic enzymes: classification and properties Arpigny JL, Jaeger KE Ref: Biochemical Journal, 343:177, 1999 : PubMed
Knowledge of bacterial lipolytic enzymes is increasing at a rapid and exciting rate. To obtain an overview of this industrially very important class of enzymes and their characteristics, we have collected and classified the information available from protein and nucleotide databases. Here we propose an updated and extensive classification of bacterial esterases and lipases based mainly on a comparison of their amino acid sequences and some fundamental biological properties. These new insights result in the identification of eight different families with the largest being further divided into six subfamilies. Moreover, the classification enables us to predict (1) important structural features such as residues forming the catalytic site or the presence of disulphide bonds, (2) types of secretion mechanism and requirement for lipase-specific foldases, and (3) the potential relationship to other enzyme families. This work will therefore contribute to a faster identification and to an easier characterization of novel bacterial lipolytic enzymes.
This family correspond to family I.1, I.2, I.3, I.5, I.6, (families I.4 and 1.7 are more related to Lipase_2) of the classification of Arpigny and Jaeger (1999).(also included in IPR000734) Also close to Lipase_2 (2lip). Pseudomonas cepacia lipase is in the same family of scop as 1I6W bacillus subtilis lipase).
Title: A cell wall-degrading esterase of Xanthomonas oryzae requires a unique substrate recognition module for pathogenesis on rice Aparna G, Chatterjee A, Sonti RV, Sankaranarayanan R Ref: Plant Cell, 21:1860, 2009 : PubMed
Xanthomonas oryzae pv oryzae (Xoo) causes bacterial blight, a serious disease of rice (Oryza sativa). LipA is a secretory virulence factor of Xoo, implicated in degradation of rice cell walls and the concomitant elicitation of innate immune responses, such as callose deposition and programmed cell death. Here, we present the high-resolution structural characterization of LipA that reveals an all-helical ligand binding module as a distinct functional attachment to the canonical hydrolase catalytic domain. We demonstrate that the enzyme binds to a glycoside ligand through a rigid pocket comprising distinct carbohydrate-specific and acyl chain recognition sites where the catalytic triad is situated 15 A from the anchored carbohydrate. Point mutations disrupting the carbohydrate anchor site or blocking the pocket, even at a considerable distance from the enzyme active site, can abrogate in planta LipA function, exemplified by loss of both virulence and the ability to elicit host defense responses. A high conservation of the module across genus Xanthomonas emphasizes the significance of this unique plant cell wall-degrading function for this important group of plant pathogenic bacteria. A comparison with the related structural families illustrates how a typical lipase is recruited to act on plant cell walls to promote virulence, thus providing a remarkable example of the emergence of novel functions around existing scaffolds for increased proficiency of pathogenesis during pathogen-plant coevolution.
Title: Structure of the alkalohyperthermophilic Archaeoglobus fulgidus lipase contains a unique C-terminal domain essential for long-chain substrate binding Chen CK, Lee GC, Ko TP, Guo RT, Huang LM, Liu HJ, Ho YF, Shaw JF, Wang AH Ref: Journal of Molecular Biology, 390:672, 2009 : PubMed
Several crystal structures of AFL, a novel lipase from the archaeon Archaeoglobus fulgidus, complexed with various ligands, have been determined at about 1.8 A resolution. This enzyme has optimal activity in the temperature range of 70-90 degrees C and pH 10-11. AFL consists of an N-terminal alpha/beta-hydrolase fold domain, a small lid domain, and a C-terminal beta-barrel domain. The N-terminal catalytic domain consists of a 6-stranded beta-sheet flanked by seven alpha-helices, four on one side and three on the other side. The C-terminal lipid binding domain consists of a beta-sheet of 14 strands and a substrate covering motif on top of the highly hydrophobic substrate binding site. The catalytic triad residues (Ser136, Asp163, and His210) and the residues forming the oxyanion hole (Leu31 and Met137) are in positions similar to those of other lipases. Long-chain lipid is located across the two domains in the AFL-substrate complex. Structural comparison of the catalytic domain of AFL with a homologous lipase from Bacillus subtilis reveals an opposite substrate binding orientation in the two enzymes. AFL has a higher preference toward long-chain substrates whose binding site is provided by a hydrophobic tunnel in the C-terminal domain. The unusually large interacting surface area between the two domains may contribute to thermostability of the enzyme. Two amino acids, Asp61 and Lys101, are identified as hinge residues regulating movement of the lid domain. The hydrogen-bonding pattern associated with these two residues is pH dependent, which may account for the optimal enzyme activity at high pH. Further engineering of this novel lipase with high temperature and alkaline stability will find its use in industrial applications.
Title: Selectivity of inhibitors of endocannabinoid biosynthesis evaluated by activity-based protein profiling Hoover HS, Blankman JL, Niessen S, Cravatt BF Ref: Bioorganic & Medicinal Chemistry Lett, 18:5838, 2008 : PubMed
The endocannabinoid 2-arachidonoylglycerol (2-AG) has been implicated as a key retrograde mediator in the nervous system based on pharmacological studies using inhibitors of the 2-AG biosynthetic enzymes diacyglycerol lipase alpha and beta (DAGL-alpha/beta). Here, we show by competitive activity-based protein profiling that the DAGL-alpha/beta inhibitors, tetrahydrolipstatin (THL) and RHC80267, block several brain serine hydrolases with potencies equal to or greater than their inhibitory activity against DAGL enzymes. Interestingly, a minimal overlap in target profiles was observed for THL and RHC80267, suggesting that pharmacological effects observed with both agents may be viewed as good initial evidence for DAGL-dependent events.
A family I.3 lipase from Pseudomonas sp. MIS38 (PML) contains three Ca(2+)-binding sites (Ca1-Ca3) in the N-catalytic domain. Of them, the Ca1 site is formed only in an open conformation. To analyze the role of these Ca(2+)-binding sites, three mutant proteins D157A-PML, D275A-PML and D337A-PML, which are designed to remove the Ca1, Ca2 and Ca3 sites, respectively, were constructed. Of them, the crystal structures of D157A-PML and D337A-PML in a closed conformation were determined. Both structures are nearly identical to that of the wild-type protein, except that the Ca3 site is missing in the D337A-PML structure. D157A-PML was as stable as the wild-type protein. Nevertheless, it exhibited little lipase and very weak esterase activities. D275A-PML was less stable than the wild-type protein by approximately 5 degrees C in T(1/2). It exhibited weak but significant lipase and esterase activities when compared with the wild-type protein. D337A-PML was also less stable than the wild-type protein by approximately 5 degrees C in T(1/2) but was fully active. These results suggest that the Ca1 site is required to make the active site fully open by anchoring lid 1. The Ca2 and Ca3 sites contribute to the stabilization of PML. The Ca2 site is also required to make PML fully active.
The crystal structure of a family I.3 lipase from Pseudomonas sp. MIS38 in a closed conformation was determined at 1.5A resolution. This structure highly resembles that of Serratia marcescens LipA in an open conformation, except for the structures of two lids. Lid1 is anchored by a Ca2+ ion (Ca1) in an open conformation, but lacks this Ca1 site and greatly changes its structure and position in a closed conformation. Lid2 forms a helical hairpin in an open conformation, but does not form it and covers the active site in a closed conformation. Based on these results, we discuss on the lid-opening mechanism.
Title: Lipases for biotechnology Jaeger KE, Eggert T Ref: Curr Opin Biotechnol, 13:390, 2002 : PubMed
Lipases constitute the most important group of biocatalysts for biotechnological applications. The high-level production of microbial lipases requires not only the efficient overexpression of the corresponding genes but also a detailed understanding of the molecular mechanisms governing their folding and secretion. The optimisation of industrially relevant lipase properties can be achieved by directed evolution. Furthermore, novel biotechnological applications have been successfully established using lipases for the synthesis of biopolymers and biodiesel, the production of enantiopure pharmaceuticals, agrochemicals, and flavour compounds.
In a series of four racemic phenoxyalkyl-alkyl carbinols, 1-phenoxy-2-hydroxybutane (1) is enantioselectively acetylated by Burkholderia cepacia (formerly Pseudomonas cepacia) lipase with an E value > or = 200, whereas for the other three racemates E was found to be < or = 4. To explain the high preference of B. cepacia lipase for (R)-(+)-1, a precursor of its transition state analogue with a tetrahedral P-atom, (R(P),S(P))-O-(2R)-(1-phenoxybut-2-yl)methylphosphonic acid chloride was prepared and crystallized in complex with B. cepacia lipase. The X-ray structure of the complex was determined, allowing to compare the conformation of the inhibitor with results of molecular modelling.
Title: Bacterial lipolytic enzymes: classification and properties Arpigny JL, Jaeger KE Ref: Biochemical Journal, 343:177, 1999 : PubMed
Knowledge of bacterial lipolytic enzymes is increasing at a rapid and exciting rate. To obtain an overview of this industrially very important class of enzymes and their characteristics, we have collected and classified the information available from protein and nucleotide databases. Here we propose an updated and extensive classification of bacterial esterases and lipases based mainly on a comparison of their amino acid sequences and some fundamental biological properties. These new insights result in the identification of eight different families with the largest being further divided into six subfamilies. Moreover, the classification enables us to predict (1) important structural features such as residues forming the catalytic site or the presence of disulphide bonds, (2) types of secretion mechanism and requirement for lipase-specific foldases, and (3) the potential relationship to other enzyme families. This work will therefore contribute to a faster identification and to an easier characterization of novel bacterial lipolytic enzymes.
The lead of this family is Candida antarctica (Trichosporon oryzae) (yeast) Lipase B. It was previously embedded in Lipase_3. The family corresponds to the _abH37 - Candida antarctica lipase like_ family of the LED database. Lipase B from Candida antarctica (CALB) has broad substrate specificity and high enantioselectivity. It can function in aqueous and organic environments and is used for a wide range of applications such as transesterification, and polymerization reactions, asymmetric synthesis PANTHER db family PTHR37574
Candida antarctica lipase B (CAL-B) exhibits remarkable enantioselectivity for various chiral sec-alcohols, and the enantioselectivity is structurally well-understood. Two substituents at the chiral center of a sec-alcohol separately bind two pockets, namely, large and medium binding pockets. It has been believed that the medium pocket is too small to accommodate a large substituent (larger than an ethyl group), and thus, bulky sec-alcohols bearing two large substituents have been regarded as a poor substrate for CAL-B. However, we found that CAL-B can catalyze the transesterification of N-Boc-protected rac-2-amino-1-phenylethanol (1a) enantioselectively with a moderate reaction rate. X-ray crystallography and computer modeling revealed that the rotation of the Leu278 side chain creates a space to accept the N-Boc-aminomethylene group of 1a. Moreover, a sec-alcohol substrate with less than one hydrogen atom at the gamma-position from the hydroxyl group is required to achieve a moderate reaction rate. On the basis of this observation, we diversified bulky N-Boc-protected rac-2-amino-1-arylethanols for the transesterifications with high enantioselectivities (E > 200).
Title: Biocatalytic ammonolysis of (5S)-4,5-dihydro-1H-pyrrole-1,5-dicarboxylic acid, 1-(1,1-dimethylethyl)-5-ethyl ester: preparation of an intermediate to the dipeptidyl peptidase IV inhibitor Saxagliptin Gill I, Patel R Ref: Bioorganic & Medicinal Chemistry Lett, 16:705, 2006 : PubMed
An efficient biocatalytic method has been developed for the conversion of (5S)-4,5-dihydro-1H-pyrrole-1,5-dicarboxylic acid, 1-(1,1-dimethylethyl)-5-ethyl ester (1) into the corresponding amide (5S)-5-aminocarbonyl-4,5-dihydro-1H-pyrrole-1-carboxylic acid, 1-(1,1-dimethylethyl)ester (2), which is a critical intermediate in the synthesis of the dipeptidyl peptidase IV (DPP4) inhibitor Saxagliptin (3). Candida antartica lipase B mediates ammonolysis of the ester with ammonium carbamate as ammonia donor to yield up to 71% of the amide. The inclusion of Ascarite and calcium chloride as adsorbents for carbon dioxide and ethanol byproducts, respectively, increases the yield to 98%, thereby offering an efficient and practical alternative to chemical routes which yield 57-64%.
Title: Understanding structure-stability relationships of Candida antartica lipase B in ionic liquids De Diego T, Lozano P, Gmouh S, Vaultier M, Iborra JL Ref: Biomacromolecules, 6:1457, 2005 : PubMed
Two different water-immiscible ionic liquids (ILs), 1-ethyl-3-methylimidizolium bis(trifluoromethylsulfonyl)imide and butyltrimethylammonium bis(trifluoromethylsulfonyl)imide, were used for butyl butyrate synthesis from vinyl butyrate catalyzed by Candida antarctica lipase B (CALB) at 2% (v/v) water content and 50 degrees C. Both the synthetic activity and stability of the enzyme in these ILs were enhanced as compared to those in hexane. Circular dichroism and intrinsic fluorescence spectroscopic techniques have been used over a period of 4 days to determine structural changes in the enzyme associated with differences in its stability for each assayed medium. CALB showed a loss in residual activity higher than 75% after 4 days of incubation in both water and hexane media at 50 degrees C, being related to great changes in both alpha-helix and beta-strand secondary structures. The stabilization of CALB, which was observed in the two ILs studied, was associated with both the maintenance of the 50% of initial alpha-helix content and the enhancement of beta-strands. Furthermore, intrinsic fluorescence studies clearly showed how a classical enzyme unfolding was occurring with time in both water and hexane media. However, the structural changes associated with the incubation of the enzyme in both ILs might be attributed to a compact and active enzyme conformation, resulting in an enhancement of the stability in these nonaqueous environments.
Candida antarctica lipase B (CAL-B) catalyzes the regioselective acylation of natural thymidine with oxime esters and also the regioselective acylation of an analogue, 3',5'-diamino-3',5'-dideoxythymidine with nonactivated esters. In both cases, acylation favors the less hindered 5'-position over the 3'-position by upto 80-fold. Computer modeling of phosphonate transition-state analogues for the acylation of thymidine suggests that CAL-B favors acylation of the 5'-position because this orientation allows the thymine ring to bind in a hydrophobic pocket and forms stronger key hydrogen bonds than acylation of the 3'-position. On the other hand, computer modeling of phosphonamidate analogues of the transition states for acylation of either the 3'- or 5'-amino groups in 3',5'-diamino-3',5'-dideoxythymidine shows similar orientations and hydrogen bonds and, thus, does not explain the high regioselectivity. However, computer modeling of inverse structures, in which the acyl chain binds in the nucleophile pocket and vice versa, does rationalize the observed regioselectivity. The inverse structures fit the 5'-, but not the 3'-intermediate thymine ring, into the hydrophobic pocket, and form a weak new hydrogen bond between the O-2 carbonyl atom of the thymine and the nucleophile amine only for the 5'-intermediate. A water molecule might transfer a proton from the ammonium group to the active-site histidine. As a test of this inverse orientation, we compared the acylation of thymidine and 3',5'-diamino-3',5'-dideoxythymidine with butyryl acyl donors and with isosteric methoxyacetyl acyl donors. Both acyl donors reacted at equal rates with thymidine, but the methoxyacetyl acyl donor reacted four times faster than the butyryl acyl donor with 3',5'-diamino-3',5'-dideoxythymidine. This faster rate is consistent with an inverse orientation for 3',5'-diamino-3',5'-dideoxythymidine, in which the ether oxygen atom of the methoxyacetyl group can form a similar hydrogen bond to the nucleophilic amine. This combination of modeling and experiments suggests that such lipase-catalyzed reactions of apparently close substrate analogues like alcohols and amines might follow different pathways.
The active site of Candida antarctica lipase B (CALB) hosts the catalytic triad (Ser-His-Asp), an oxyanion hole and a stereospecificity pocket. During catalysis, the fast-reacting enantiomer of secondary alcohols places its medium-sized substituent in the stereospecificity pocket and its large substituent towards the active-site entrance. The largest group to fit comfortably in the stereospecificity pocket is ethyl, and this restricts the number of secondary alcohols that are good substrates for CALB. In order to overcome this limitation, the size of the stereospecificity pocket was redesigned by changing Trp104. The substrate specificity of the Trp104Ala mutant compared to that of the wild-type lipase increased 270 times towards heptan-4-ol and 5500 times towards nonan-5-ol; this resulted in the high specificity constants 1100 and 830 s(-1) M(-1), respectively. The substrate selectivity changed over 400,000 times for nonan-5-ol over propan-2-ol with both Trp104Ala and the Trp104Gln mutations.
Title: Improving the catalytic activity of Candida antarctica lipase B by circular permutation Qian Z, Lutz S Ref: Journal of the American Chemical Society, 127:13466, 2005 : PubMed
Lipases (EC 3.1.1.3) play an important role in asymmetric biocatalysis. Tailoring these enzymes to novel, unnatural substrates is one of the primary challenges of protein engineering. We have used circular permutation, the intramolecular relocation of a protein's N- and C-termini, to explore the effects of altered active site accessibility and protein backbone flexibility on the catalytic performance of lipase B from Candida antarctica (CALB). Our combinatorial approach identified 63 unique functional protein permutants of CALB, and kinetic analysis of selected candidates indicated that a majority of enzyme variants either retained or surpassed wild-type CALB activity on a series of standard substrates. Beyond the potential benefits of these tailor-made lipases as new catalysts for unnatural substrates, our study validates circular permutation as a promising general method for lipase engineering.
Title: Synthesis of flavor and fragrance esters using Candida antarctica lipase Larios A, Garcia HS, Oliart RM, Valerio-Alfaro G Ref: Applied Microbiology & Biotechnology, 65:373, 2004 : PubMed
Candida antarctica lipase fraction B (CAL-B) showed substrate specificity in the synthesis of esters in hexane involving reactions of short-chain acids having linear (acetic and butyric acids) and branched chain (isovaleric acid) structures, an unsaturated (tiglic acid) fatty acid, and phenylacetic acid with n-butanol and geraniol. The variation in the conversion to the esters was ca. 10%. Similar results were observed in a study of the alcohol specificity of the enzyme for esterification of acetic and butyric acids with four alcohols: n-butyl, isopentyl, 2-phenylethyl, and geraniol. Enantioselectivity of CAL-B in hexane with a range of chiral alpha-substituted or beta-substituted carboxylic acids and n-butyl alcohol was analyzed. The results show that CAL-B can be employed as a robust biocatalyst in esterification reactions due to the high conversions obtained in the synthesis of short-chain flavor esters in an organic solvent, although this enzyme exhibited modest enantioselectivity with chiral short-chain carboxylic acids.
Glucuronic acid n-alkyl esters, a novel class of promising biosurfactants and their corresponding glucose esters with the same side-chain length, were synthesized by direct esterification in a non-aqueous phase (tert-butanol) using an immobilized lipase.
Title: Kinetic resolution of rac-2-pentanol catalyzed by Candida antarctica lipase B in the ionic liquid, 1-butyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]amide Noel M, Lozano P, Vaultier M, Iborra JL Ref: Biotechnol Lett, 26:301, 2004 : PubMed
The ionic liquid, l-butyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]amide ([Bmim] [NTf2]), was used as a reaction medium for the kinetic resolution of rac-2-pentanol catalyzed by free Candida antarctica lipase B, using vinyl propionate at 2% (v/v) water content. The synthetic activity of lipase in [Bmim] [NTf2] was up 2.5-times greater than in hexane, and showed high enantioselectivity (ee > 99.99%). The optimal temperature and pH were 60 degrees C and 7, respectively. A decrease in water activity (aw) produced a decay in synthetic activity, and an exponential increase in selectivity.
Title: Regioselective acylation of flavonoids catalyzed by immobilized Candida antarctica lipase under reduced pressure Passicos E, Santarelli X, Coulon D Ref: Biotechnol Lett, 26:1073, 2004 : PubMed
A single-step acylation of rutin and naringin, catalyzed by immobilized Candida antarctica lipase B in 2-methyl-2-butanol, occurred preferentially on the primary hydroxyl group. Using palmitic methyl ester as acyl donor, the acylation rate of naringin was 10-fold higher than that of rutin. Under optimal conditions, i.e. a molar ratio acyl donor/naringin of 7:1 and 200 mbar, 92% naringin was acylated.
Title: Stability improvement of immobilized Candida antarctica lipase B in an organic medium under microwave radiation Rejasse B, Lamare S, Legoy MD, Besson T Ref: Org Biomol Chem, 2:1086, 2004 : PubMed
The influence of microwave heating on the stability of immobilized Candida antarctica lipase B was studied at 100 degrees in an organic medium. The microwave radiation was carried out before enzymatic reaction (storage conditions) or during the enzymatic catalysis (use conditions). In both cases, enzymatic stability was higher under microwave heating than under conventional thermal heating, in strictly identical operating conditions. Furthermore, the gain of enzymatic stability under microwave heating appears to be higher in a more polar solvent, which interacts strongly with the microwave field. Our results suggest that microwave radiation has an effect, not related to temperature, on the process of enzymatic inactivation.
Immobilized lipase from Candida antarctica was employed to convert triglycerides to biodiesel using alcohol. Immobilized lipase is frequently deactivated by lower alcohols with deactivation being caused by the immiscibility between triglycerides and methanol or ethanol. When the lower alcohol is adsorbed to the immobilized enzyme, the entry of triglycerides is blocked, which causes the reaction to stop. An alcohol with three or more carbon atoms, preferably 2-butanol or tert-butanol, can regenerate the deactivated immobilized enzyme. The present work established that the activity of immobilized lipase could be significantly increased when such alcohols were used for an immersion pretreatment of the enzyme. The activity of the commercially available immobilized enzyme, Novozyme 435, increased about tenfold in comparison to the enzyme not subjected to any pretreatment. Following complete deactivation of the enzyme by methanol, washing with 2-butanol and tert-butanol successfully regenerated the enzyme and restored it to about 56% and 75% of its original activity level, respectively.
A mixture of oil/ethanol (1:3, w/w) was shaken at 30 degrees C with 4% immobilized Candida antarctica lipase by weight of the reaction mixture. The reaction regiospecifically converted FA at the 1- and 3-positions to FA ethyl esters, and the lipase acted on C14-C24 FA to a similar degree. The content of 2-MAG reached a maximum after 4 h; the content was 28-29 mol% based on the total amount of FA in the reaction mixture at 59-69% ethanolysis. Only 2-MAG were present in the reaction mixture during the first 4 h, and 1(3)-MAG were detected after 7 h. After removal of ethanol from the 4-h reaction mixture by evaporation, 2-MAG were fractionated by silica gel column chromatography. The contents of FA in the 2-MAG obtained by ethanolysis of several oils coincided well with FA compositions at the 2-position, which was analyzed by Grignard degradation. It was shown that ethanolysis of oil with C. antarctica lipase can be applied to analysis of FA composition at the 2-position in TAG.
Title: Improving tolerance of Candida antarctica lipase B towards irreversible thermal inactivation through directed evolution Zhang N, Suen WC, Windsor W, Xiao L, Madison V, Zaks A Ref: Protein Engineering, 16:599, 2003 : PubMed
To expand the functionality of lipase B from Candida antarctica (CALB) we have used directed evolution to create CALB mutants with improved resistance towards irreversible thermal inactivation. Two mutants, 23G5 and 195F1, were generated with over a 20-fold increase in half-life at 70 degrees C compared with the wild-type CALB (WT-CALB). The increase in half-life was attributed to a lower propensity of the mutants to aggregate in the unfolded state and to an improved refolding. The first generation mutant, 23G5, obtained by error-prone PCR, had two amino acid mutations, V210I and A281E. The second generation mutant, 195F1, derived from 23G5 by error-prone PCR, had one additional mutation, V221D. Amino acid substitutions at positions 221 and 281 were determined to be critical for lipase stability, while the residue at position 210 had only a marginal effect. The catalytic efficiency of the mutants with p-nitrophenyl butyrate and 6,8-difluoro-4-methylumbelliferyl octanoate was also found to be superior to that of WT-CALB.
Ethyl docosahexaenoate (EtDHA) is regarded as a potentially useful pharmaceutical substance on account of its beneficial physiological activities. We attempted the ethyl esterification of docosahexaenoic acid (DHA) in an organic solvent-free system using Candida antarctica lipase, which acts strongly on DHA and ethanol. Esterification of 88% was attained by shaking a mixture of DHA/ethanol (1:1, mol/mol) and 2 wt% immobilized C. antarctica lipase at 30 degrees C for 24 h. However, even in the presence of an excess amount of ethanol, the extent of esterification could not be raised above 90%. To attain a higher level of esterification, a two-step reaction was found to be effective. The first step was performed in a mixture of DHA/ethanol (1:1, mol/mol), and the reaction mixture was then dehydrated. In the second step, the resulting mixture was shaken at 30 degrees C for 24 h with 5 molar equivalents of ethanol against the remaining DHA using 2 wt% immobilized lipase. By means of this two-step procedure, 96% esterification was attained. Repetition of the first and second reactions showed that the immobilized lipase was reusable for at least 50 cycles. In addition, DHA remaining in the second-step reaction mixture was removed by a conventional alkali refining process, giving purified EtDHA with a high yield.
The effects of the pretreatment of immobilized Candida antarctica lipase enzyme (Novozym 435) on methanolysis for biodiesel fuel production were investigated. Methanolysis progressed much faster when Novozym 435 was preincubated in methyl oleate for 0.5 h and subsequently in soybean oil for 12 h. The initial reaction rate of methanolysis catalyzed by both the non-treated and preincubated enzyme decreased significantly with increasing water content. The initial reaction rate increased with increasing methanol content, showed a maximum, and thereafter decreased when the methanol content was increased further. The variation of the initial reaction rate with the methanol content was therefore analyzed using a Michaelis-Menten-type equation with substrate inhibition. Based on this equation, a procedure for the stepwise addition of methanol to the reaction mixture so as to maintain the desired methanol content was determined. When preincubated Novozym 435 was used, the ME content reached over 97% within 3.5 h by stepwise addition of 0.33 molar equivalent of methanol at 0.25-0.4 h intervals.
Ethanolysis of fish oil under mild conditions has been strongly desired for preparing the starting materials for the purification of ethyl docosahexaenoate. Thus, we attempted ethanolysis of tuna oil using immobilized Candida antarctica lipase. The immobilized lipase was inactivated in the presence of 2 3 molar equivalent of ethanol against the total fatty acids in tuna oil. To avoid such inactivation, the first step of ethanolysis was conducted at 40 degrees C in a mixture of tuna oil and 1 3 molar equivalent of ethanol using 4% immobilized lipase. After a 10-h reaction, ethanol was consumed and 33% of tuna oil was converted to its corresponding ethyl esters (E-FAs). The reactant is named Gly/E-FA33. The lipase was not inactivated in the presence of 2 3 molar equivalent of ethanol against the total fatty acids in Gly/E-FA33. These findings and the consideration of several factors affecting ethanolysis of tuna oil led to the development of the two- and three-step ethanolyses. The two-step reaction was performed as follows: the first step was carried out at 40 degrees C for 12 h in a mixture of tuna oil and 1 3 molar equivalent of ethanol with 4% immobilized lipase; the second step was performed for 36 h (total reaction period, 48 h) after adding 2 3 molar equivalent of ethanol. On the other hand, the three-step reaction was conducted as follows: the first step was conducted under the same conditions as those in the two-step ethanolysis; in the second and third steps, 1 3 molar equivalent of ethanol was added after 12 and 24 h, respectively; and in the third step, the mixture was shaken for 24 h (total, 48 h). Both types of ethanolyses achieved the conversion of 95% or more of tuna oil to its corresponding E-FAs. To investigate the lipase stability, the two- and three-step ethanolyses were repeated by transferring the enzyme to a fresh substrate mixture of the first step after finishing one cycle of reaction. The two- and three-step reactions maintained over 95% of the conversion for 70 d and over 100 d, respectively.
Many lipases are potent catalysts of stereoselective reactions and are therefore of interest for use in chemical synthesis. The crystal structures of lipases show a large variation in the shapes of their active site environments that may explain the large variation in substrate specificity of these enzymes. We have determined the three-dimensional structure of Candida antarctica lipase B (CALB) cocrystallized with the detergent Tween 80. In another crystal form, the structure of the enzyme in complex with a covalently bound phosphonate inhibitor has been determined. In both structures, the active site is exposed to the external solvent. The potential lid-forming helix alpha 5 in CALB is well-ordered in the Tween 80 structure and disordered in the inhibitor complex. The tetrahedral intermediates of two chiral substrates have been modeled on the basis of available structural and biochemical information. The results of this study provide a structural explanation for the high stereoselectivity of CALB toward many secondary alcohols.
Title: Crystallization and preliminary X-ray studies of lipase B from Candida antarctica Uppenberg J, Patkar S, Bergfors T, Jones TA Ref: Journal of Molecular Biology, 235:790, 1994 : PubMed
Lipase B from Candida antarctica has been crystallized in five different crystal forms. The space groups and cell dimensions have been determined by X-ray diffraction methods. Four of the crystal forms have been judged to be of good quality for further X-ray studies. The best crystals diffract to 1.7 Angstrom.
Title: The sequence, crystal structure determination and refinement of two crystal forms of lipase B from Candida antarctica Uppenberg J, Hansen MT, Patkar S, Jones TA Ref: Structure, 2:293, 1994 : PubMed
BACKGROUND: Lipases constitute a family of enzymes that hydrolyze triglycerides. They occur in many organisms and display a wide variety of substrate specificities. In recent years, much progress has been made towards explaining the mechanism of these enzymes and their ability to hydrolyze their substrates at an oil-water interface. RESULTS: We have determined the DNA and amino acid sequences for lipase B from the yeast Candida antarctica. The primary sequence has no significant homology to any other known lipase and deviates from the consensus sequence around the active site serine that is found in other lipases. We have determined the crystal structure of this enzyme using multiple isomorphous replacement methods for two crystal forms. Models for the orthorhombic and monoclinic crystal forms of the enzyme have been refined to 1.55 A and 2.1 A resolution, respectively. Lipase B is an alpha/beta type protein that has many features in common with previously determined lipase structures and other related enzymes. In the monoclinic crystal form, lipid-like molecules, most likely beta-octyl glucoside, can be seen close to the active site. The behaviour of these lipid molecules in the crystal structure has been studied at different pH values. CONCLUSION: The structure of Candida antarctica lipase B shows that the enzyme has a Ser-His-Asp catalytic triad in its active site. The structure appears to be in an 'open' conformation with a rather restricted entrance to the active site. We believe that this accounts for the substrate specificity and high degree of stereospecificity of this lipase.
Chlorophyllase (Chlase) enzyme involved in chlorophylle (Chl) degradation and catalyses the hydrolysis of ester bond to yield chlorophyllide and phytol. This family correspond to bacterial enzymes close to chlorophyllases of plant Chlorophyllase_Plant. The activity of these enzymes are related to cutinase and Poly-ethylene-therephthalate (PET) hydrolases
This family consists of several plant specific Chlorophyllase proteins (EC: 3.1.1.14). Chlorophyllase (Chlase) is the first enzyme involved in chlorophylle (Chl) degradation and catalyses the hydrolysis of ester bond to yield chlorophyllide and phytol The family includes both plant bacteria and Amphioxus members. However Chlorophyllase is not localized to plastids, and double knockout mutant plants still are able to degrade chlorophyll during leaf senescence. So pheophytinase is a new pathway. Chlorophyllase could be more important in fruit rippening. Some bacterial enzyme are close to plant chlorophyllases but are now separated in another family Chlorophyllase. (Few sponge or marine invertebrates protein included). A structure-function analysis of chlorophyllase reveals a mechanism for activity regulation dependent on disulfide bonds(Jo et al.)
Title: A structure-function analysis of chlorophyllase reveals a mechanism for activity regulation dependent on disulfide bonds Jo M, Knapp M, Boggs DG, Brimberry M, Donnan PH, Bridwell-Rabb J Ref: Journal of Biological Chemistry, :102958, 2023 : PubMed
Chlorophyll (Chl) pigments are used by photosynthetic organisms to facilitate light capture and mediate the conversion of sunlight into chemical energy. Due to the indispensable nature of this pigment, and its propensity to form reactive oxygen species, organisms heavily invest in its biosynthesis, recycling, and degradation. One key enzyme implicated in these processes is chlorophyllase, an alpha/beta hydrolase that hydrolyzes the phytol tail of Chl pigments to produce chlorophyllide (Chlide) molecules. This enzyme was discovered a century ago, but despite its importance to diverse photosynthetic organisms, there are still many missing biochemical details regarding how chlorophyllase functions. Here, we present the 4.46- resolution crystal structure of chlorophyllase from Triticum aestivum. This structure reveals the dimeric architecture of chlorophyllase, the arrangement of catalytic residues, an unexpected divalent metal ion binding site, and a substrate binding site that can accommodate a diverse range of pigments. Further, this structure exhibits the existence of both intermolecular and intramolecular disulfide bonds. We investigated the importance of these architectural features using enzyme kinetics, mass spectrometry, and thermal shift assays. Through this work, we demonstrated that the oxidation state of the Cys residues is imperative to the activity and stability of chlorophyllase, illuminating a biochemical trigger for responding to environmental stress. Additional bioinformatics analysis of the chlorophyllase enzyme family reveals widespread conservation of key catalytic residues and the identified "redox switch" among other plant chlorophyllase homologs, thus revealing key details regarding the structure-function relationships in chlorophyllase.
Title: Chlorophyll degradation during senescence Hortensteiner S Ref: Annu Rev Plant Biol, 57:55, 2006 : PubMed
The catabolic pathway of chlorophyll (Chl) during senescence and fruit ripening leads to the accumulation of colorless breakdown products (NCCs). This review updates an earlier review on Chl breakdown published here in 1999 ( 69 ). It summarizes recent advances in the biochemical reactions of the pathway and describes the characterization of new NCCs and their formation inside the vacuole. Furthermore, I focus on the recent molecular identification of three chl catabolic enzymes, chlorophyllase, pheophorbide a oxygenase (PAO), and red Chl catabolite reductase (RCCR). The analysis of Chl catabolic mutants demonstrates the importance of Chl breakdown for plant development and survival. Mutants defective in PAO or RCCR develop a lesion mimic phenotype, due to the accumulation of breakdown intermediates. Thus, Chl breakdown is a prerequisite to detoxify the potentially phototoxic pigment within the vacuoles in order to permit the remobilization of nitrogen from Chl-binding proteins to proceed during senescence.
Title: Mechanistic analysis of wheat chlorophyllase Arkus KA, Cahoon EB, Jez JM Ref: Archives of Biochemistry & Biophysics, 438:146, 2005 : PubMed
Chlorophyllase catalyzes the initial step in the degradation of chlorophyll and plays a key role in leaf senescence and fruit ripening. Here, we report the cloning of chlorophyllase from Triticum aestivum (wheat) and provide a detailed mechanistic analysis of the enzyme. Purification of recombinant chlorophyllase from an Escherichia coli expression system indicates that the enzyme functions as a dimeric protein. Wheat chlorophyllase hydrolyzed the phytol moiety from chlorophyll (k(cat) = 566 min(-1); K(m) = 63 microM) and was active over a broad temperature range (10-75 degrees C). In addition, the enzyme displays carboxylesterase activity toward p-nitrophenyl (PNP)-butyrate, PNP-decanoate, and PNP-palmitate. The pH-dependence of the reaction showed the involvement of an active site residue with a pK(a) of approximately 6.5 for both k(cat) and k(cat)/K(m) with chlorophyll, PNP-butyrate, and PNP-decanoate. Using these substrates, solvent kinetic isotope effects ranging from 1.5 to 1.9 and from 1.4 to 1.9 on k(cat) and k(cat)/K(m), respectively, were observed. Proton inventory experiments suggest the transfer of a single proton in the rate-limiting step. Our analysis of wheat chlorophyllase indicates that the enzyme uses a charge-relay mechanism similar to other carboxylesterases for catalysis. Understanding the activity and mechanism of chlorophyllase provides insight on the biological and chemical control of senescence in plants and lays the groundwork for biotechnological improvement of this enzyme.
Chlorophyllases (Chlases), cloned so far, contain a lipase motif with the active serine residue of the catalytic triad of triglyceride lipases. Inhibitors specific for the catalytic serine residue in serine hydrolases, which include lipases effectively inhibited the activity of the recombinant Chenopodium album Chlase (CaCLH). From this evidence we assumed that the catalytic mechanism of hydrolysis by Chlase might be similar to those of serine hydrolases that have a catalytic triad composed of serine, histidine and aspartic acid in their active site. Thus, we introduced mutations into the putative catalytic residue (Ser162) and conserved amino acid residues (histidine, aspartic acid and cysteine) to generate recombinant CaCLH mutants. The three amino acid residues (Ser162, Asp191 and His262) essential for Chlase activity were identified. These results indicate that Chlase is a serine hydrolase and, by analogy with a plausible catalytic mechanism of serine hydrolases, we proposed a mechanism for hydrolysis catalyzed by Chlase.
Title: Cloning of chlorophyllase, the key enzyme in chlorophyll degradation: finding of a lipase motif and the induction by methyl jasmonate Tsuchiya T, Ohta H, Okawa K, Iwamatsu A, Shimada H, Masuda T, Takamiya K Ref: Proc Natl Acad Sci U S A, 96:15362, 1999 : PubMed
Chlorophyllase (Chlase) is the first enzyme involved in chlorophyll (Chl) degradation and catalyzes the hydrolysis of ester bond to yield chlorophyllide and phytol. In the present study, we isolated the Chlase cDNA. We synthesized degenerate oligo DNA probes based on the internal amino acid sequences of purified Chlase from Chenopodium album, screened the C. album cDNA library, and cloned a cDNA (CaCLH, C. album chlorophyll-chlorophyllido hydrolase). The deduced amino acid sequence (347 aa residues) had a lipase motif overlapping with an ATP/GTP-binding motif (P-loop). CaCLH possibly was localized in the extraplastidic part of the cell, because a putative signal sequence for endoplasmic reticulum is at the N terminus. The amino acid sequence shared 37% identity with a function-unknown gene whose mRNA is inducible by coronatine and methyl jasmonate (MeJA) in Arabidopsis thaliana (AtCLH1). We expressed the gene products of AtCLH1 and of CaCLH in Escherichia coli, and they similarly exhibited Chlase activity. Moreover, we isolated another full-length cDNA based on an Arabidopsis genomic fragment and expressed it in E. coli, demonstrating the presence of the second Arabidopsis CLH gene (AtCLH2). No typical feature of signal sequence was identified in AtCLH1, whereas AtCLH2 had a typical signal sequence for chloroplast. AtCLH1 mRNA was induced rapidly by a treatment of MeJA, which is known to promote senescence and Chl degradation in plants, and a high mRNA level was maintained up to 9 h. AtCLH2, however, did not respond to MeJA.
Pancreatic, hepatic and gastric/lingual lipase are closely related to each other and to lipoprotein lipase (EC: 3.1.1.34), which hydrolyses triglycerides of chylomicrons and very low density lipoproteins (VLDL). Familial human hepatic lipase deficiency is a rare recessive disorder Two variants (S267F and T383M) are rare mutations found to date only in HL deficient subjects and their relatives. Of the six HL variants described to date, only S267F and T383M are associated with hyperlipidemia. human-LIPC. The disease is characterised by premature atherosclerosis and abnormal circulating lipoproteins
Hepatic lipase (gene: LIPC; enzyme: HL; E.C.3.1.1.3) is one of three members of the triglyceride lipase family that contributes to vascular lipoprotein degradation and serves a dual role in triglyceride hydrolysis and in facilitating receptor-mediated lipoprotein uptake into the liver. Amino acid sequences, protein structures, and gene locations for vertebrate LIPC (or Lipc for mouse and rat) genes and proteins were sourced from previous reports and vertebrate genome databases. Lipc was distinct from other neutral lipase genes (Lipg encoding endothelial lipase and Lpl encoding lipoprotein lipase [LPL]) and was located on mouse chromosome 9 with nine coding exons on the negative strand. Exon 9 of human LIPC and mouse and rat Lipc genes contained "stop codons" in different positions, causing changes in C-termini length. Vertebrate HL protein subunits shared 58%-97% sequence identities, including active, signal peptide, disulfide bond, and N-glycosylation sites, as well as proprotein convertase ("hinge") and heparin binding regions. Predicted secondary and tertiary structures revealed similarities with the three-dimensional structure reported for horse and human pancreatic lipases. Potential sites for regulating LIPC gene expression included CpG islands near the 5''-untranslated regions of the mouse and rat LIPC genes. Phylogenetic analyses examined the relationships and potential evolutionary origins of the vertebrate LIPC gene family with other neutral triglyceride lipase gene families (LIPG and LPL). We conclude that the triglyceride lipase ancestral gene for vertebrate neutral lipase genes (LIPC, LIPG, and LPL) predated the appearance of fish during vertebrate evolution.
Title: Human hepatic lipase mutations and polymorphisms Hegele RA, Tu L, Connelly PW Ref: Hum Mutat, 1:320, 1992 : PubMed
Human hepatic lipase (HL) is a 477 residue glycoprotein that hydrolyzes triglycerides from plasma lipoproteins. Familial HL deficiency is a rare recessive disorder that is characterized by premature atherosclerosis and abnormal circulating lipoproteins. While studying the HL gene from the world's index family with HL deficiency, we identified four coding sequence variants of HL, one in each of exons 4, 5, 6, and 8. In this report we present the genetic basis for two new HL gene variants, one in each of exons 3 and 5. All six HL DNA variants are single base pair changes. Two variants (at codons 133 and 202) are diallelic DNA polymorphisms that are silent at the amino acid level. One variant (V73M) is an allele that defines an uncommon HL isoprotein. One variant (N193S) has two alleles of approximately equal frequency in the population that specify two common HL isoproteins. Two variants (S267F and T383M) are rare mutations found to date only in HL deficient subjects and their relatives. Of the six HL variants described to date, only S267F and T383M are associated with hyperlipidemia.
Phospholipase A(1) (PLA(1)) is an enzyme that hydrolyzes phospholipids and produces 2-acyl-lysophospholipids and fatty acids and is conserved in a wide range of organisms. Included in this family are Vespid venom allergen phospholipase A1. Vespid phospholipase A1 (vPLA1) is one of the primary venom components with local inflammatory effects. In addition to causing allergic reactions, vPLA1 can hydrolyze the sn-1 fatty acids in phospholipids and convert them into their corresponding lyso compounds. vPLA1 may disrupt the phospholipid packing of biological membranes, causing severe hemolysis and leading to cardiac dysfunction and death in animals
Title: Crystal structure of vespid phospholipase A1 reveals insights into the mechanism for cause of membrane dysfunction Hou MH, Chuang CY, Ko TP, Hu NJ, Chou CC, Shih YP, Ho CL, Wang AH Ref: Insect Biochemistry & Molecular Biology, 68:79, 2016 : PubMed
Vespid phospholipase A1 (vPLA1) from the black-bellied hornet (Vespa basalis) catalyzes the hydrolysis of emulsified phospholipids and shows potent hemolytic activity that is responsible for its lethal effect. To investigate the mechanism of vPLA1 towards its function such as hemolysis and emulsification, we isolated vPLA1 from V. basalis venom and determined its crystal structure at 2.5 A resolution. vPLA1 belongs to the alpha/beta hydrolase fold family. It contains a tightly packed beta-sheet surrounded by ten alpha-helices and a Gly-X-Ser-X-Gly motif, characteristic of a serine hydrolyase active site. A bound phospholipid was modeled into the active site adjacent to the catalytic Ser-His-Asp triad indicating that Gln95 is located at hydrogen-bonding distance from the substrate's phosphate group. Moreover, a hydrophobic surface comprised by the side chains of Phe53, Phe62, Met91, Tyr99, Leu197, Ala167 and Pro169 may serve as the acyl chain-binding site. vPLA1 shows global similarity to the N-terminal domain of human pancreatic lipase (HPL), but with some local differences. The lid domain and the beta9 loop responsible for substrate selectivity in vPLA1 are shorter than in HPL. Thus, solvent-exposed hydrophilic residues can easily accommodate the polar head groups of phospholipids, thereby accounting for the high activity level of vPLA1. Our result provides a potential explanation for the ability of vPLA1 to hydrolyze phospholipids of cell membrane.
This family groups insect lipases close to mammalian pancreatic, hepatic and gastric/lingual lipase which are closely related to each other and to lipoprotein lipase (EC: 3.1.1.34), which hydrolyses triglycerides of chylomicrons and very low density lipoproteins (VLDL). These are neutral lipases distinct from Acidic lipases and higher dipteran yolk proteins
Lipases have key roles in insect lipid acquisition, storage and mobilisation and are also fundamental to many physiological processes underpinning insect reproduction, development, defence from pathogens and oxidative stress, and pheromone signalling. We have screened the recently sequenced genomes of five species from four orders of holometabolous insects, the dipterans Drosophila melanogaster and Anopheles gambiae, the hymenopteran Apis mellifera, the moth Bombyx mori and the beetle Tribolium castaneum, for the six major lipase families that are also found in other organisms. The two most numerous families in the insects, the neutral and acid lipases, are also the main families in mammals, albeit not in Caenorhabditis elegans, plants or microbes. Total numbers of the lipases vary two-fold across the five insect species, from numbers similar to those in mammals up to numbers comparable to those seen in C. elegans. Whilst there is a high degree of orthology with mammalian lipases in the other four families, the great majority of the insect neutral and acid lipases have arisen since the insect orders themselves diverged. Intriguingly, about 10% of the insect neutral and acid lipases have lost motifs critical for catalytic function. Examination of the length of lid and loop regions of the neutral lipase sequences suggest that most of the insect lipases lack triacylglycerol (TAG) hydrolysis activity, although the acid lipases all have intact cap domains required for TAG hydrolysis. We have also reviewed the sequence databases and scientific literature for insights into the expression profiles and functions of the insect neutral and acid lipases and the orthologues of the mammalian adipose triglyceride lipase which has a pivotal role in lipid mobilisation. These data suggest that some of the acid and neutral lipase diversity may be due to a requirement for rapid accumulation of dietary lipids. The different roles required of lipases at the four discrete life stages of holometabolous insects may also contribute to the diversity of lipases required by insects. In addition, insects use lipases to perform roles for which there are no correlates in mammals, including as yolk and male accessory gland proteins.
Title: Multiple tandem gene duplications in a neutral lipase gene cluster in Drosophila Horne I, Haritos VS Ref: Gene, 411:27, 2008 : PubMed
We have examined a highly dynamic section of the Drosophila melanogaster genome which contains neutral lipase family genes that have undergone multiple tandem duplication events. We have identified the orthologous clusters, encoding between five and eight apparently functional lipases, in other Drosophila genomes: yakuba, ananassae, pseudoobscura, virilis, mojavensis, persimilis, grimshawi and willistoni. We examined their gene structure, duplication and pseudogene formation, and the presence of transposable elements. Based on phylogenetic comparisons, the lipase genes contained in each of the clusters fall into four distinct clades. Clades I and II have distinct evolutionary constraints to clades III and IV. Multiple gene duplications have occurred in different lineages of clades I and II while clades III and IV contain a single lipase gene from each species. Compared with lipases from other clades, clade IV genes contain an additional 3' domain of tandemly repeated sequence of varying length and composition, and a substitution in the residue adjacent to the key catalytic serine in the encoded proteins. A comparison of non-synonymous to synonymous nucleotide substitution (dN/dS) rates within each clade showed the highest rate of divergence was between paralogous lipase gene pairs suggesting selection pressure on duplicated genes. Analysis of the encoded lipase protein sequences within each species using PAML identified positively selected sites; structure homology modeling based on human pancreatic lipase indicated many of these residues formed part of the active site of the enzyme. As some of the cluster lipase genes are known to be expressed in the insect midgut and respond to changes in dietary components, we propose that the lipase cluster has undergone dynamic evolutionary changes to maximize absorption of lipid nutrients from the diet.
Triglyceride lipases are lipases that hydrolyse ester linkages of triglycerides. These lipases are widely distributed in animals, plants and prokaryotes. This family was also called class 3 lipases as they are only distantly related to other lipase families. In some fungi DDHD domain Pfam PF02862 180 residues long containing four conserved residues that may form a metal binding site is associated with the Lipase_3. Bacterial enzymes (LipG Lee et al. 2006) belong to family XI of the classification of Arpigny and Jaeger 1999. The (phospho)lipase of F.solani has the highest microbial activity on galactolipids Jallouli et al. Feruloyl esterases are enzymes produced by micro-organisms to deconstruct plant cell walls by hydrolyzing phenolic groups involved in the cross-linking of arabinoxylan to other polymeric structures. The non-modular type-A feruloyl esterase from Aspergillus niger AnFaeA is similar to fungal lipases and different from other feruloyl esterases. Feruloyl esterases are distributed in different sub-classes type-A B C,D and E and fall respectively in the following families. Type-A in Lipase_3, Type-B in Esterase_phb (PHB depolymerase), Type-C in Tannase, Type-D in FaeC, Type-E in A85-Feruloyl-Esterase, Type-F in BD-FAE
Background: The endogenous cannabinoid system modulates inflammatory signaling in a variety of pathological states, including traumatic brain injury (TBI). The selective expression of diacylglycerol lipase-beta (DAGL-beta), the 2-arachidonylglycerol biosynthetic enzyme, on resident immune cells of the brain (microglia) and the role of this pathway in neuroinflammation, suggest that this enzyme may contribute to TBI-induced neuroinflammation. Accordingly, we tested whether DAGL-beta(-/-) mice would show a protective phenotype from the deleterious consequences of TBI on cognitive and neurological motor functions. Materials and Methods: DAGL-beta(-/-) and -beta(+/+) mice were subjected to the lateral fluid percussion model of TBI and assessed for learning and memory in the Morris water maze (MWM) Fixed Platform (reference memory) and Reversal (cognitive flexibility) tasks, as well as in a cued MWM task to infer potential sensorimotor/motivational deficits. In addition, subjects were assessed for motor behavior (Rotarod and the Neurological Severity Score assays) and in the light/dark box and the elevated plus maze to infer whether these manipulations affected anxiety-like behavior. Finally, we also examined whether brain injury disrupts the ceramide/sphingolipid lipid signaling system and if DAGL-beta deletion offers protection. Results: TBI disrupted all measures of neurological motor function and reduced body weight, but did not affect body temperature or performance in common assays used to infer anxiety. TBI also impaired performance in MWM Fixed Platform and Reversal tasks, but did not affect cued MWM performance. Although no differences were found between DAGL-beta(-/-) and -beta(+/+) mice in any of these measures, male DAGL-beta(-/-) mice displayed an unexpected survival-protective phenotype, which persisted at increased injury severities. In contrast, TBI did not elicit mortality in female mice regardless of genotype. TBI also produced significant changes in sphingolipid profiles (a family of lipids, members of which have been linked to both apoptotic and antiapoptotic pathways), in which DAGL-beta deletion modestly altered levels of select species. Conclusions: These findings indicate that although DAGL-beta does not play a necessary role in TBI-induced cognitive and neurological function, it appears to contribute to the increased vulnerability of male mice to TBI-induced mortality, whereas female mice show high survival rates irrespective of DAGL-beta expression.
Determining optimal conditions for the production of well diffracting crystals is a key step in every biocrystallography project. Here, a microfluidic device is described that enables the production of crystals by counter-diffusion and their direct on-chip analysis by serial crystallography at room temperature. Nine 'non-model' and diverse biomacromolecules, including seven soluble proteins, a membrane protein and an RNA duplex, were crystallized and treated on-chip with a variety of standard techniques including micro-seeding, crystal soaking with ligands and crystal detection by fluorescence. Furthermore, the crystal structures of four proteins and an RNA were determined based on serial data collected on four synchrotron beamlines, demonstrating the general applicability of this multipurpose chip concept.
Diacylglycerol lipases (DAGLalpha and DAGLbeta) convert diacylglycerol to the endocannabinoid 2-arachidonoylglycerol. Our understanding of DAGL function has been hindered by a lack of chemical probes that can perturb these enzymes in vivo. Here, we report a set of centrally active DAGL inhibitors and a structurally related control probe and their use, in combination with chemical proteomics and lipidomics, to determine the impact of acute DAGL blockade on brain lipid networks in mice. Within 2 h, DAGL inhibition produced a striking reorganization of bioactive lipids, including elevations in DAGs and reductions in endocannabinoids and eicosanoids. We also found that DAGLalpha is a short half-life protein, and the inactivation of DAGLs disrupts cannabinoid receptor-dependent synaptic plasticity and impairs neuroinflammatory responses, including lipopolysaccharide-induced anapyrexia. These findings illuminate the highly interconnected and dynamic nature of lipid signaling pathways in the brain and the central role that DAGL enzymes play in regulating this network.
The purified (phospho)lipase of Fusarium solani (FSL), was known to be active on both triglycerides and phospholipids. This study aimed at assessing the potential of this enzyme in hydrolyzing galactolipids. FSL was found to hydrolyze at high rates of synthetic medium chains monogalactosyldiacylglycerol (4658+/-146U/mg on DiC8-MGDG) and digalactosyldiacylglycerol (3785+/-83U/mg on DiC8-DGDG) and natural long chain monogalactosyldiacylglycerol extracted from leek leaves (991+/-85U/mg). It is the microbial enzyme with the highest activity on galactolipids identified so far with a level of activity comparable to that of pancreatic lipase-related protein 2. FSL maximum activity on galactolipids was measured at pH8. The analysis of the hydrolysis product of natural MGDG from leek showed that FSL hydrolyzes preferentially the ester bond at the sn-1 position of galactolipids. To investigate the structure-activity relationships of FSL, a 3D model of this enzyme was built. In silico docking of medium chains MGDG and DGDG and phospholipid in the active site of FSL reveals structural solutions which are in concordance with in vitro tests.
Title: Crystal structure of a secreted lipase from Gibberella zeae reveals a novel double-lock mechanism Lou Z, Li M, Sun Y, Liu Y, Liu Z, Wu W, Rao Z Ref: Protein Cell, 1:760, 2010 : PubMed
Fusarium graminearum (sexual stage: Gibberella zeae) is the causative agent of Fusarium Head Blight (FHB), which is one of the most destructive plant disease of cereals, accounting for high grain yield losses, especially for wheat and maize. Like other fungal pathogens, several extracellular enzymes secreted by G. zeae are known to be involved in host infection. Among these secreted lipases, G. zeae lipase (GZEL), which is encoded by the FGL1 gene, was demonstrated to be crucial to G. zeae pathogenicity. However, the precise mechanism of GZEL remains unclear due to a lack of detailed structural information. In this study, we report the crystal structure of GZEL at the atomic level. The structure of GZEL displays distinct structural differences compared to reported homologues and indicates a unique "double lock" enzymatic mechanism. To gain insight into substrate/inhibitor recognition, we proposed a model of GZEL in complex with substrate and the lipase inhibitor ebelactone B (based on the reported structures of GZEL homologues), which defines possible substrate binding sites within the catalytic cleft and suggests an "anti sn-l" binding mode. These results pave the way to elucidating the mechanism of GZEL and thus provide clues for the design of anti-FHB inhibitors.
Title: Structural redesign of lipase B from Candida antarctica by circular permutation and incremental truncation Qian Z, Horton JR, Cheng X, Lutz S Ref: Journal of Molecular Biology, 393:191, 2009 : PubMed
Circular permutation of Candida antarctica lipase B yields several enzyme variants with substantially increased catalytic activity. To better understand the structural and functional consequences of protein termini reorganization, we have applied protein engineering and x-ray crystallography to cp283, one of the most active hydrolase variants. Our initial investigation has focused on the role of an extended surface loop, created by linking the native N- and C-termini, on protein integrity. Incremental truncation of the loop partially compensates for observed losses in secondary structure and the permutants' temperature of unfolding. Unexpectedly, the improvements are accompanied by quaternary-structure changes from monomer to dimer. The crystal structures of one truncated variant (cp283 Delta 7) in the apo-form determined at 1.49 A resolution and with a bound phosphonate inhibitor at 1.69 A resolution confirmed the formation of a homodimer by swapping of the enzyme's 35-residue N-terminal region. Separately, the new protein termini at amino acid positions 282/283 convert the narrow access tunnel to the catalytic triad into a broad crevice for accelerated substrate entry and product exit while preserving the native active-site topology for optimal catalytic turnover.
The M37 lipase from Photobacterium lipolyticum shows an extremely low activation energy and strong activity at low temperatures, with optimum activity seen at 298 K and more than 75% of the optimum activity retained down to 278 K. Though the M37 lipase is most closely related to the filamentous fungal lipase, Rhizomucor miehei lipase (RML) at the primary structure level, their activity characteristics are completely different. In an effort to identify structural components of cold adaptation in lipases, we determined the crystal structure of the M37 lipase at 2.2 A resolution and compared it to that of nonadapted RML. Structural analysis revealed that M37 lipase adopted a folding pattern similar to that observed for other lipase structures. However, comparison with RML revealed that the region beneath the lid of the M37 lipase included a significant and unique cavity that would be occupied by a lid helix upon substrate binding. In addition, the oxyanion hole was much wider in M37 lipase than RML. We propose that these distinct structural characteristics of M37 lipase may facilitate the lateral movement of the helical lid and subsequent substrate hydrolysis, which might explain its low activation energy and high activity at low temperatures.
Title: Isolation and characterization of a novel lipase from a metagenomic library of tidal flat sediments: evidence for a new family of bacterial lipases Lee MH, Lee CH, Oh TK, Song JK, Yoon JH Ref: Applied Environmental Microbiology, 72:7406, 2006 : PubMed
We cloned lipG, which encoded a lipolytic enzyme, from a Korean tidal flat metagenomic library. LipG was related to six putative lipases previously identified only in bacterial genome sequences. These enzymes comprise a new family. We partially characterized LipG, providing the first experimental data for a member of this family.
Title: Feruloyl esterase: a key enzyme in biomass degradation Wong DWS Ref: Appl Biochem Biotechnol, 133:87, 2006 : PubMed
Feruloyl esterase forms a part of the enzyme complex that acts collectively and synergistically to completely hydrolyze xylan to its monomers. The enzyme has found potential uses in a wide variety of applications of interest to the agrifood and pharmaceutical industries. This review describes the enzymology of feruloyl esterases involved in xylan degradation. The occurrence of feruloyl esterases in various microorganisms and their physiochemical properties are presented. The nature of the enzyme substrates and products, the role of synergistic interactions with xylanases and other accessory enzymes, as well as the sequence-structure relating to the reaction mechanism are emphasized.
Title: The crystal structure of feruloyl esterase A from Aspergillus niger suggests evolutive functional convergence in feruloyl esterase family Hermoso JA, Sanz-Aparicio J, Molina R, Juge N, Gonzalez R, Faulds CB Ref: Journal of Molecular Biology, 338:495, 2004 : PubMed
As a component of the array of enzymes produced by micro-organisms to deconstruct plant cell walls, feruloyl esterases hydrolyze phenolic groups involved in the cross-linking of arabinoxylan to other polymeric structures. This is important for opening the cell wall structure, making material more accessible to glycosyl hydrolases. Here, we describe the first crystal structure of the non-modular type-A feruloyl esterase from Aspergillus niger (AnFaeA) solved at 2.5A resolution. AnFaeA displays an alpha/beta hydrolase fold similar to that found in fungal lipases and different from that reported for other feruloyl esterases. Crystallographic and site-directed mutagenesis studies allow us to identify the catalytic triad (Ser133-His247-Asp194) that forms the catalytic machinery of this enzyme. The active-site cavity is confined by a lid (residues 68-80), on the analogy of lipases, and by a loop (residues 226-244) that confers plasticity to the substrate-binding site. The lid presents a high ratio of polar residues, which in addition to a unique N-glycosylation site stabilises the lid in an open conformation, conferring the esterase character to this enzyme. A putative model for bound 5,5'-diferulic acid-linked arabinoxylan has been built, pointing to the more relevant residues involved in substrate recognition. Comparison with structurally related lipases reveals that subtle amino acid and conformational changes within a highly conserved protein fold may produce protein variants endowed with new enzymatic properties, while comparison with functionally related proteins points to a functional convergence after evolutionary divergence within the feruloyl esterases family.
Title: Structure of a feruloyl esterase from Aspergillus niger McAuley KE, Svendsen A, Patkar SA, Wilson KS Ref: Acta Crystallographica D Biol Crystallogr, 60:878, 2004 : PubMed
The crystallographic structure of feruloyl esterase from Aspergillus niger has been determined to a resolution of 1.5 A by molecular replacement. The protein has an alpha/beta-hydrolase structure with a Ser-His-Asp catalytic triad; the overall fold of the protein is very similar to that of the fungal lipases. The structure of the enzyme-product complex was determined to a resolution of 1.08 A and reveals dual conformations for the serine and histidine residues at the active site.
We report the cloning and characterization of a gene encoding a ferulic acid esterase, faeA, from Aspergillus niger and Aspergillus tubingensis. The A. niger and A. tubingensis genes have a high degree of sequence identity and contain one conserved intron. The gene product, FAEA, was overexpressed in wild-type A. tubingensis and a protease-deficient A. niger mutant. Overexpression of both genes in wild-type A. tubingensis and an A. niger protease-deficient mutant showed that the A. tubingensis gene product is more sensitive to degradation than the equivalent gene product from A. niger. FAEA from A. niger was identical to A. niger FAE-III (C. B. Faulds and G. Williamson, Microbiology 140:779-787, 1994), as assessed by molecular mass, pH and temperature optima, pI, N-terminal sequence, and activity on methyl ferulate. The faeA gene was induced by growth on wheat arabinoxylan and sugar beet pectin, and its gene product (FAEA) released ferulic acid from wheat arabinoxylan. The rate of release was enhanced by the presence of a xylanase. FAEA also hydrolyzed smaller amounts of ferulic acid from sugar beet pectin, but the rate was hardly affected by addition of an endo-pectin lyase.
Lipases from filamentous fungi have been studied extensively over many years. They exhibit properties attractive for industrial applications, e.g. in laundry detergents, tanning and paper industries and stereospecific organic synthesis. Enzymes from the fungi Rhizomucor miehei and Geotrichum candidum have been among the first neutral lipases to be characterized structurally by X-ray diffraction methods. In this paper we report a preliminary account of crystallographic studies of three other fungal lipases homologous to that from R. miehei and obtained from Humicola lanuginosa, Penicillium camembertii and Rhizopus delemar. These newly characterized structures have important implications for our understanding of structure-function relationships in lipases in general and the molecular basis of interfacial activation.
The stability of globular proteins arises largely from the burial of non-polar amino acids in their interior. These residues are efficiently packed to eliminate energetically unfavorable cavities. Contrary to these observations, high resolution X-ray crystallographic analyses of four homologous lipases from filamentous fungi reveal an alpha/beta fold which contains a buried conserved constellation of charged and polar side chains with associated cavities containing ordered water molecules. It is possible that this structural arrangement plays an important role in interfacial catalysis.
Title: Conformational lability of lipases observed in the absence of an oil-water interface: crystallographic studies of enzymes from the fungi Humicola lanuginosa and Rhizopus delemar Derewenda U, Swenson L, Wei Y, Green R, Kobos PM, Joerger R, Haas MJ, Derewenda ZS Ref: J Lipid Res, 35:524, 1994 : PubMed
Considerable controversy exists regarding the exact nature of the molecular mechanism of interfacial activation, a process by which most lipases achieve maximum catalytic activity upon adsorption to an oil water interface. X-ray crystallographic studies show that lipases contain buried active centers and that displacements of entire secondary structure elements, or "lids," take place when the enzymes assume active conformations [Derewenda, U., A. M. Brzozowski, D. M. Lawson, and Z. S. Derewenda. 1992. Biochemistry: 31: 1532-1541; van Tilbeurgh, H., M-P. Egloff, C. Martinez, N. Rugani, R. Verger, and C. Cambillau. 1993. Nature: 362: 814-820; Grochulski, P., L. Yunge, J. D. Schrag, F. Bouthillier, P. Smith, D. Harrison, B. Rubin, and M. Cygler. 1993. J. Biol. Chem. 268: 12843-12847]. A simple two-state model inferred from these results implies that the "closed" conformation is stable in an aqueous medium, rendering the active centers inaccessible to water soluble substrates. We now report that in crystals of the Humicola lanuginosa lipase the "lid" is significantly disordered irrespective of the ionic strength of the medium, while in a related enzyme from Rhizopus delemar, crystallized in the presence of a detergent, the two molecules that form the asymmetric unit show different "lid" conformations. These new results call into question the simplicity of the "enzyme theory" of interfacial activation.
Title: The sequence, crystal structure determination and refinement of two crystal forms of lipase B from Candida antarctica Uppenberg J, Hansen MT, Patkar S, Jones TA Ref: Structure, 2:293, 1994 : PubMed
BACKGROUND: Lipases constitute a family of enzymes that hydrolyze triglycerides. They occur in many organisms and display a wide variety of substrate specificities. In recent years, much progress has been made towards explaining the mechanism of these enzymes and their ability to hydrolyze their substrates at an oil-water interface. RESULTS: We have determined the DNA and amino acid sequences for lipase B from the yeast Candida antarctica. The primary sequence has no significant homology to any other known lipase and deviates from the consensus sequence around the active site serine that is found in other lipases. We have determined the crystal structure of this enzyme using multiple isomorphous replacement methods for two crystal forms. Models for the orthorhombic and monoclinic crystal forms of the enzyme have been refined to 1.55 A and 2.1 A resolution, respectively. Lipase B is an alpha/beta type protein that has many features in common with previously determined lipase structures and other related enzymes. In the monoclinic crystal form, lipid-like molecules, most likely beta-octyl glucoside, can be seen close to the active site. The behaviour of these lipid molecules in the crystal structure has been studied at different pH values. CONCLUSION: The structure of Candida antarctica lipase B shows that the enzyme has a Ser-His-Asp catalytic triad in its active site. The structure appears to be in an 'open' conformation with a rather restricted entrance to the active site. We believe that this accounts for the substrate specificity and high degree of stereospecificity of this lipase.
True lipases attach triacylglycerols and act at an oil-water interface; they constitute a ubiquitous group of enzymes catalysing a wide variety of reactions, many with industrial potential. But so far the three-dimensional structure has not been reported for any lipase. Here we report the X-ray structure of the Mucor miehei triglyceride lipase and describe the atomic model obtained at 3.1 A resolution and refined to 1.9 A resolution. It reveals a Ser..His..Asp trypsin-like catalytic triad with an active serine buried under a short helical fragment of a long surface loop.
Lipoprotein lipase (LPL) is a key enzyme of lipid metabolism that hydrolyses triglycerides, providing free fatty acids for cells and affecting the maturation of circulating lipoproteins. The enzyme is thought to play a role in the development of obesity and atherosclerosis. Defects in LPL are a cause of familial chylomicronemia syndrome (or type I hyperlipoproteinemia) and also of a form of deficiency characterised by hypertriglyceridemia. Familial chylomicronemia is a recessive disorder usually manifesting in childhood. On a normal diet, patients often present with abdominal pain, hepatosplenomegaly, lipemia retinalis, eruptive xanthomata, and massive hypertriglyceridemia, sometimes complicated with acute pancreatitis. Endothelial lipase (encoded by the LIPG gene) regulates the circulating level of high density lipoprotein cholesterol (HDL-C). It can also form a molecular bridge between endothelial cells and lipoproteins or circulating macrophages through interaction with heparan sulfate proteoglycans. This nonenzymatic action can increase cellular lipoprotein uptake and monocyte adhesion and contribute to atherosclerosis. LPL is a secreted glycoprotein that contains five disulfide bonds and requires an endoplasmic reticulum (ER) protein, lipase maturation factor 1 (LMF1), to successfully fold and traffic out of the ER to the Golgi. LPL is sorted into vesicles in an inactive state: helical LPL oligomer. LPL secretion is mediated by Syndecan-1 (SDC1), a heparan sulfate proteoglycan (HSPG). Stored LPL can be secreted into the interstitial space, where it interacts with HSPGs that bind to the multiple heparin binding sites on each LPL molecule . LPL is next bound by glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) and transported into the capillary, where it acts on chylomicrons and very-low-density lipoproteins (VLDLs) to hydrolyze packaged triglycerides and release FFAs. The angiopoietin-like (ANGPTL) family of proteins inhibit LPL in different tissues. Leth-Espensen et al. publish that the intrinsic instability of the hydrolase domain of lipoprotein lipase facilitates its inactivation by ANGPTL4-catalyzed unfolding.
The complex between lipoprotein lipase (LPL) and its endothelial receptor (GPIHBP1) is responsible for the lipolytic processing of triglyceride-rich lipoproteins (TRLs) along the capillary lumen, a physiologic process that releases lipid nutrients for vital organs such as heart and skeletal muscle. LPL activity is regulated in a tissue-specific manner by endogenous inhibitors (angiopoietin-like [ANGPTL] proteins 3, 4, and 8), but the molecular mechanisms are incompletely understood. ANGPTL4 catalyzes the inactivation of LPL monomers by triggering the irreversible unfolding of LPL's alpha/beta-hydrolase domain. Here, we show that this unfolding is initiated by the binding of ANGPTL4 to sequences near LPL's catalytic site, including beta2, beta3-alpha3, and the lid. Using pulse-labeling hydrogendeuterium exchange mass spectrometry, we found that ANGPTL4 binding initiates conformational changes that are nucleated on beta3-alpha3 and progress to beta5 and beta4-alpha4, ultimately leading to the irreversible unfolding of regions that form LPL's catalytic pocket. LPL unfolding is context dependent and varies with the thermal stability of LPL's alpha/beta-hydrolase domain (T (m) of 34.8 degreesC). GPIHBP1 binding dramatically increases LPL stability (T (m) of 57.6 degreesC), while ANGPTL4 lowers the onset of LPL unfolding by -20 degreesC, both for LPL and LPLGPIHBP1 complexes. These observations explain why the binding of GPIHBP1 to LPL retards the kinetics of ANGPTL4-mediated LPL inactivation at 37 degreesC but does not fully suppress inactivation. The allosteric mechanism by which ANGPTL4 catalyzes the irreversible unfolding and inactivation of LPL is an unprecedented pathway for regulating intravascular lipid metabolism.
Title: Lipoprotein Lipase and Its Regulators: An Unfolding Story Wu SA, Kersten S, Qi L Ref: Trends Endocrinol Metab, 32:48, 2021 : PubMed
Lipoprotein lipase (LPL) is one of the most important factors in systemic lipid partitioning and metabolism. It mediates intravascular hydrolysis of triglycerides packed in lipoproteins such as chylomicrons and very-low-density lipoprotein (VLDL). Since its initial discovery in the 1940s, its biology and pathophysiological significance have been well characterized. Nonetheless, several studies in the past decade, with recent delineation of LPL crystal structure and the discovery of several new regulators such as angiopoietin-like proteins (ANGPTLs), glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1), lipase maturation factor 1 (LMF1) and Sel-1 suppressor of Lin-12-like 1 (SEL1L), have completely transformed our understanding of LPL biology.
Lipases are enzymes necessary for the proper distribution and utilization of lipids in the human body. Lipoprotein lipase (LPL) is active in capillaries, where it plays a crucial role in preventing dyslipidemia by hydrolyzing triglycerides from packaged lipoproteins. Thirty years ago, the existence of a condensed and inactive LPL oligomer was proposed. Although recent work has shed light on the structure of the LPL monomer, the inactive oligomer remained opaque. Here we present a cryo-EM reconstruction of a helical LPL oligomer at 3.8-A resolution. Helix formation is concentration-dependent, and helices are composed of inactive dihedral LPL dimers. Heparin binding stabilizes LPL helices, and the presence of substrate triggers helix disassembly. Superresolution fluorescent microscopy of endogenous LPL revealed that LPL adopts a filament-like distribution in vesicles. Mutation of one of the helical LPL interaction interfaces causes loss of the filament-like distribution. Taken together, this suggests that LPL is condensed into its inactive helical form for storage in intracellular vesicles.
Lipoprotein lipase (LPL) plays a central role in triglyceride (TG) metabolism. By catalyzing the hydrolysis of TGs present in TG-rich lipoproteins (TRLs), LPL facilitates TG utilization and regulates circulating TG and TRL concentrations. Until very recently, structural information for LPL was limited to homology models, presumably due to the propensity of LPL to unfold and aggregate. By coexpressing LPL with a soluble variant of its accessory protein glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) and with its chaperone protein lipase maturation factor 1 (LMF1), we obtained a stable and homogenous LPL/GPIHBP1 complex that was suitable for structure determination. We report here X-ray crystal structures of human LPL in complex with human GPIHBP1 at 2.5-3.0 A resolution, including a structure with a novel inhibitor bound to LPL. Binding of the inhibitor resulted in ordering of the LPL lid and lipid-binding regions and thus enabled determination of the first crystal structure of LPL that includes these important regions of the protein. It was assumed for many years that LPL was only active as a homodimer. The structures and additional biochemical data reported here are consistent with a new report that LPL, in complex with GPIHBP1, can be active as a monomeric 1:1 complex. The crystal structures illuminate the structural basis for LPL-mediated TRL lipolysis as well as LPL stabilization and transport by GPIHBP1.
Lipoprotein lipase (LPL) is responsible for the intravascular processing of triglyceride-rich lipoproteins. The LPL within capillaries is bound to GPIHBP1, an endothelial cell protein with a three-fingered LU domain and an N-terminal intrinsically disordered acidic domain. Loss-of-function mutations in LPL or GPIHBP1 cause severe hypertriglyceridemia (chylomicronemia), but structures for LPL and GPIHBP1 have remained elusive. Inspired by our recent discovery that GPIHBP1's acidic domain preserves LPL structure and activity, we crystallized an LPL-GPIHBP1 complex and solved its structure. GPIHBP1's LU domain binds to LPL's C-terminal domain, largely by hydrophobic interactions. Analysis of electrostatic surfaces revealed that LPL contains a large basic patch spanning its N- and C-terminal domains. GPIHBP1's acidic domain was not defined in the electron density map but was positioned to interact with LPL's large basic patch, providing a likely explanation for how GPIHBP1 stabilizes LPL. The LPL-GPIHBP1 structure provides insights into mutations causing chylomicronemia.
Lipoprotein lipase (LPL), identified in the 1950s, has been studied intensively by biochemists, physiologists, and clinical investigators. These efforts uncovered a central role for LPL in plasma triglyceride metabolism and identified LPL mutations as a cause of hypertriglyceridemia. By the 1990s, with an outline for plasma triglyceride metabolism established, interest in triglyceride metabolism waned. In recent years, however, interest in plasma triglyceride metabolism has awakened, in part because of the discovery of new molecules governing triglyceride metabolism. One such protein-and the focus of this review-is GPIHBP1, a protein of capillary endothelial cells. GPIHBP1 is LPL's essential partner: it binds LPL and transports it to the capillary lumen; it is essential for lipoprotein margination along capillaries, allowing lipolysis to proceed; and it preserves LPL's structure and activity. Recently, GPIHBP1 was the key to solving the structure of LPL. These developments have transformed the models for intravascular triglyceride metabolism.
The intravascular processing of triglyceride-rich lipoproteins depends on lipoprotein lipase (LPL) and GPIHBP1, a membrane protein of endothelial cells that binds LPL within the subendothelial spaces and shuttles it to the capillary lumen. In the absence of GPIHBP1, LPL remains mislocalized within the subendothelial spaces, causing severe hypertriglyceridemia (chylomicronemia). The N-terminal domain of GPIHBP1, an intrinsically disordered region (IDR) rich in acidic residues, is important for stabilizing LPL's catalytic domain against spontaneous and ANGPTL4-catalyzed unfolding. Here, we define several important properties of GPIHBP1's IDR. First, a conserved tyrosine in the middle of the IDR is posttranslationally modified by O-sulfation; this modification increases both the affinity of GPIHBP1-LPL interactions and the ability of GPIHBP1 to protect LPL against ANGPTL4-catalyzed unfolding. Second, the acidic IDR of GPIHBP1 increases the probability of a GPIHBP1-LPL encounter via electrostatic steering, increasing the association rate constant (kon) for LPL binding by >250-fold. Third, we show that LPL accumulates near capillary endothelial cells even in the absence of GPIHBP1. In wild-type mice, we expect that the accumulation of LPL in close proximity to capillaries would increase interactions with GPIHBP1. Fourth, we found that GPIHBP1's IDR is not a key factor in the pathogenicity of chylomicronemia in patients with the GPIHBP1 autoimmune syndrome. Finally, based on biophysical studies, we propose that the negatively charged IDR of GPIHBP1 traverses a vast space, facilitating capture of LPL by capillary endothelial cells and simultaneously contributing to GPIHBP1's ability to preserve LPL structure and activity.
Lipoprotein lipase (LPL) is secreted into the interstitial spaces by adipocytes and myocytes but then must be transported to the capillary lumen by GPIHBP1, a glycosylphosphatidylinositol-anchored protein of capillary endothelial cells. The mechanism by which GPIHBP1 and LPL move across endothelial cells remains unclear. We asked whether the transport of GPIHBP1 and LPL across endothelial cells was uni- or bidirectional. We also asked whether GPIHBP1 and LPL are transported across cells in vesicles and whether this transport process requires caveolin-1. The movement of GPIHBP1 and LPL across cultured endothelial cells was bidirectional. Also, GPIHBP1 moved bidirectionally across capillary endothelial cells in live mice. The transport of LPL across endothelial cells was inhibited by dynasore and genistein, consistent with a vesicular transport process. Also, transmission electron microscopy (EM) and dual-axis EM tomography revealed GPIHBP1 and LPL in invaginations of the plasma membrane and in vesicles. The movement of GPIHBP1 across capillary endothelial cells was efficient in the absence of caveolin-1, and there was no defect in the internalization of LPL by caveolin-1-deficient endothelial cells in culture. Our studies show that GPIHBP1 and LPL move bidirectionally across endothelial cells in vesicles and that transport is efficient even when caveolin-1 is absent.
GPIHBP1, a glycosylphosphatidylinositol-anchored protein of capillary endothelial cells, shuttles lipoprotein lipase (LPL) from subendothelial spaces to the capillary lumen. An absence of GPIHBP1 prevents the entry of LPL into capillaries, blocking LPL-mediated triglyceride hydrolysis and leading to markedly elevated triglyceride levels in the plasma (i.e., chylomicronemia). Earlier studies have established that chylomicronemia can be caused by LPL mutations that interfere with catalytic activity. We hypothesized that some cases of chylomicronemia might be caused by LPL mutations that interfere with LPL's ability to bind to GPIHBP1. Any such mutation would provide insights into LPL sequences required for GPIHBP1 binding. Here, we report that two LPL missense mutations initially identified in patients with chylomicronemia, C418Y and E421K, abolish LPL's ability to bind to GPIHBP1 without interfering with LPL catalytic activity or binding to heparin. Both mutations abolish LPL transport across endothelial cells by GPIHBP1. These findings suggest that sequences downstream from LPL's principal heparin-binding domain (amino acids 403-407) are important for GPIHBP1 binding. In support of this idea, a chicken LPL (cLPL)-specific monoclonal antibody, xCAL 1-11 (epitope, cLPL amino acids 416-435), blocks cLPL binding to GPIHBP1 but not to heparin. Also, changing cLPL residues 421 to 425, 426 to 430, and 431 to 435 to alanine blocks cLPL binding to GPIHBP1 without inhibiting catalytic activity. Together, these data define a mechanism by which LPL mutations could elicit disease and provide insights into LPL sequences required for binding to GPIHBP1.
The lipolytic processing of triglyceride-rich lipoproteins by lipoprotein lipase (LPL) is the central event in plasma lipid metabolism, providing lipids for storage in adipose tissue and fuel for vital organs such as the heart. LPL is synthesized and secreted by myocytes and adipocytes, but then finds its way into the lumen of capillaries, where it hydrolyzes lipoprotein triglycerides. The mechanism by which LPL reaches the lumen of capillaries has remained an unresolved problem of plasma lipid metabolism. Here, we show that GPIHBP1 is responsible for the transport of LPL into capillaries. In Gpihbp1-deficient mice, LPL is mislocalized to the interstitial spaces surrounding myocytes and adipocytes. Also, we show that GPIHBP1 is located at the basolateral surface of capillary endothelial cells and actively transports LPL across endothelial cells. Our experiments define the function of GPIHBP1 in triglyceride metabolism and provide a mechanism for the transport of LPL into capillaries.
The association of polymorphisms affecting lipid metabolism with the risk of myocardial infarction (MI) in type 2 diabetes mellitus was investigated. The Genetics, Outcomes and Lipids in type 2 Diabetes (GOLD) Study is a prospective, multicenter study, conducted on 990 patients presenting diabetes and MI (n=386), or diabetes without previous manifestation of stroke, peripheral or coronary arterial disease (n=604), recruited from 27 institutions in Brazil. APO A1 (A/G -75 and C/T +83) and APO C3 (C/G 3'UTR) non-coding sequences, CETP (Taq 1B), LPL (D9N), APO E (epsilon2, epsilon3, epsilon4,), PON-1 (Q192R), and two LCAT variants Arg(147)-->Trp and Tyr(171)-->Stop were tested by PCR-RFLP. There was a higher prevalence of LPL DN genotype (19% vs.12%, p=0.03) and a higher frequency of the N allele (11% vs. 7%) among subjects with MI when compared to controls, with an odds ratio of MI for carriers of 9N allele of 2.46 (95% CI=1.79-3.39, p<0.0001). This association was present in men and women, in non-smokers and in hypertensive patients. A logistic regression model including gender, duration of diabetes, systolic blood pressure, HDL-C, left ventricle hypertrophy and D9N polymorphism showed that the latter still remained significantly associated with MI (OR=1.50, 95% CI=1.02-2.25, p=0.049). These findings suggest that D9N polymorphism can be a useful risk marker for myocardial infarction and that further potential candidate genes should be screened for exploratory analysis and for future therapeutic intervention in diabetes.
High-density lipoprotein (HDL) cholesterol levels are inversely associated with risk of atherosclerotic cardiovascular disease. At least 50% of the variation in HDL cholesterol levels is genetically determined, but the genes responsible for variation in HDL levels have not been fully elucidated. Lipoprotein lipase (LPL) and hepatic lipase (HL), two members of the triacylglyerol (TG) lipase family, both influence HDL metabolism and the HL (LIPC) locus has been associated with variation in HDL cholesterol levels in humans. We describe here the cloning and in vivo functional analysis of a new member of the TG lipase family. In contrast to other family members, this new lipase is synthesized by endothelial cells in vitro and thus has been termed endothelial lipase (encoded by the LIPG gene). EL is expressed in vivo in organs including liver, lung, kidney and placenta, but not in skeletal muscle. In contrast to LPL and HL, EL has a lid of only 19 residues. EL has substantial phospholipase activity, but less triglyceride lipase activity. Overexpression of EL in mice reduced plasma concentrations of HDL cholesterol and its major protein apolipoprotein A-I. The endothelial expression, enzymatic profile and in vivo effects of EL suggest that it may have a role in lipoprotein metabolism and vascular biology.
Title: Lipoprotein lipase. Molecular model based on the pancreatic lipase x-ray structure: consequences for heparin binding and catalysis van Tilbeurgh H, Roussel A, Lalouel JM, Cambillau C Ref: Journal of Biological Chemistry, 269:4626, 1994 : PubMed
Lipoprotein lipase and pancreatic lipase have about 30% sequence identity, suggesting a similar tertiary fold. Three-dimensional models of lipoprotein lipase were constructed, based upon two recently determined x-ray crystal structures of pancreatic lipase, in which the active site was in an open and closed conformation, respectively. These models allow us to propose a few hypotheses on the structural determinants of lipoprotein lipase which are responsible for heparin binding, dimer formation, and phospholipase activity. The folding of the protein assembles a number of positive charge clusters at the back of the molecule, opposite the active site. These clusters probably form the heparin binding site, as confirmed by recent site-directed mutagenesis experiments. The active sites of lipoprotein lipase and pancreatic lipase look very similar, except for the lid (a surface loop covering the catalytic serine in the inactive state). A different open (active) conformation of the lid in both enzymes may be responsible for their differing substrate specificities. Predictions of the nature of the lipoprotein lipase dimer remain elusive, although our model enabled us to propose a few possibilities.
Lipoprotein lipase (LPL) plays a crucial role in the regulation of lipoprotein metabolism by hydrolysing the core triglycerides of circulating chylomicrons and VLDL. Human, bovine, mouse, and guinea pig complementary DNA clones have recently been isolated and the organization of the human LPL gene is now known to comprise 10 exons spanning approximately 30 kb. Here we report a similar mutation on 21 alleles from 13 unrelated affected probands with LPL deficiency of French Canadian, English, Polish, German, Dutch, and East Indian ancestry. We show that an identical missense mutation within exon 5, resulting in an amino acid substitution of glutamic acid for glycine at position 188, is responsible for LPL deficiency in 21 of 88 LPL alleles assessed. This mutation alters an Ava II restriction site in exon 5 and will allow a rapid screening test for this mutation in patients with LPL deficiency. This mutation has occurred on the same haplotype in all the unrelated affected persons suggesting a common origin. The amino acid substitution lies within the longest segment of homology for LPL in different species and results in a protein that is catalytically defective.
Lipoprotein lipase is a key enzyme of lipid metabolism that acts to hydrolyze triglycerides, providing free fatty acids for cells and affecting the maturation of circulating lipoproteins. It has been proposed that the enzyme plays a role in the development of obesity and atherosclerosis. The human enzyme has been difficult to purify and its protein sequence was heretofore undetermined. A complementary DNA for human lipoprotein lipase that codes for a mature protein of 448 amino acids has now been cloned and sequenced. Analysis of the sequence indicates that human lipoprotein lipase, hepatic lipase, and pancreatic lipase are members of a gene family. Two distinct species of lipoprotein lipase messenger RNA that arise from alternative sites of 3'-terminal polyadenylation were detected in several different tissues.
Lysosomal phospholipase A2 (LPLA2) and lecithin:cholesterol acyltransferase (LCAT), Lecithin-cholesterol acyltransferase, belong to this family of key lipid-metabolizing enzymes responsible for lung surfactant catabolism and for reverse cholesterol transport, respectively. Whereas LPLA2 is predicted to underlie the development of drug-induced phospholipidosis, somatic mutations in LCAT cause fish eye disease and familial LCAT deficiency. LACT also known as phosphatidylcholine-sterol acyltransferase (EC), is involved in extracellular metabolism of plasma lipoproteins, including cholesterol. It esterifies the free cholesterol transported in plasma lipoproteins, and is activated by apolipoprotein A-I. Defects in LACT cause Norum and Fish eye diseases. This family correspond to group XV phospholipase A2. For bacterial enzymes this family correspond to family XIV of the upgraded classification of Arpigny and Jaeger (1999)
Lecithin:cholesterol acyltransferase (LCAT) and LCAT-activating compounds are being investigated as treatments for coronary heart disease (CHD) and familial LCAT deficiency (FLD). Herein we report the crystal structure of human LCAT in complex with a potent piperidinylpyrazolopyridine activator and an acyl intermediate-like inhibitor, revealing LCAT in an active conformation. Unlike other LCAT activators, the piperidinylpyrazolopyridine activator binds exclusively to the membrane-binding domain (MBD). Functional studies indicate that the compound does not modulate the affinity of LCAT for HDL, but instead stabilizes residues in the MBD and facilitates channeling of substrates into the active site. By demonstrating that these activators increase the activity of an FLD variant, we show that compounds targeting the MBD have therapeutic potential. Our data better define the substrate binding site of LCAT and pave the way for rational design of LCAT agonists and improved biotherapeutics for augmenting or restoring reverse cholesterol transport in CHD and FLD patients.
Title: A thermostable esterase from Thermoanaerobacter tengcongensis opening up a new family of bacterial lipolytic enzymes Rao L, Xue Y, Zhou C, Tao J, Li G, Lu JR, Ma Y Ref: Biochimica & Biophysica Acta, 1814:1695, 2011 : PubMed
An unidentified alpha/beta hydrolase gene lipA3 from thermostable eubacterium species Thermoanaerobacter tengcongensis MB4 was cloned and heterologously expressed by Escherichia coli BL21(DE3)pLysS. The purified recombinant enzyme EstA3 turned out to be a monomeric thermostable esterase with optimal activity at 70degC and pH 9.5. The enzyme showed lipolytic activity towards a wide range of ester substrates including p-nitrophenyl esters and triacylglycerides, with the highest activity being observed for p-nitrophenyl caproate at 150 U/mg and for Triacetin at 126U/mg, respectively. Phylogenetic analysis revealed that EstA3 did not show homology to any identified bacterial lipolytic hydrolases. Sequence alignment showed that there was a common pentapeptide CHSMG with a cysteine replacing the first glycine in most esterase and lipase conserved motif GXSXG. The catalytic triad of EstA3 is Ser92, Asp269 and His292, which was confirmed by site directed mutagenesis. Based on the enzymatic properties and sequence alignment we concluded that the esterase EstA3 represented a novel bacterial lipolytic enzyme group and in chronological order this group was assigned as Family XIV.
Title: Schizosaccharomyces pombe cells deficient in triacylglycerols synthesis undergo apoptosis upon entry into the stationary phase. Zhang Q, Chieu HK, Low CP, Zhang S, Heng CK, Yang H Ref: Journal of Biological Chemistry, 278:47145", 2003 : PubMed
Triacylglycerols (TAG) are important energy storage molecules for nearly all eukaryotic organisms. In this study, we found that two gene products (Plh1p and Dga1p) are responsible for the terminal step of TAG synthesis in the fission yeast Schizosaccharomyces pombe through two different mechanisms: Plh1p is a phospholipid diacylglycerol acyltransferase, whereas Dga1p is an acyl-CoA:diacylglycerol acyltransferase. Cells with both dga1+ and plh1+ deleted (DKO cells) lost viability upon entry into the stationary phase and demonstrated prominent apoptotic markers. Exponentially growing DKO cells also underwent dramatic apoptosis when briefly treated with diacylglycerols (DAGs) or free fatty acids. We provide strong evidence suggesting that DAG, not sphingolipids, mediates fatty acids-induced lipoapoptosis in yeast. Lastly, we show that generation of reactive oxygen species is essential to lipoapoptosis.
Title: Deficiency of lecithin:cholesterol acyltransferase due to compound heterozygosity of two novel mutations (Gly33Arg and 30 bp ins) in the LCAT gene Wiebusch H, Cullen P, Owen JS, Collins D, Sharp PS, Funke H, Assmann G Ref: Hum Mol Genet, 4:143, 1995 : PubMed
The presence of lecithin:cholesterol acyltransferase (LCAT) deficiency in six probands from five families originating from four different countries was confirmed by the absence or near absence of LCAT activity. Also, other invariate symptoms of LCAT deficiency, a significant increase of unesterified cholesterol in plasma lipoproteins and the reduction of plasma HDL-cholesterol to levels below one-tenth of normal, were present in all probands. In the probands from two families, no mass was detectable, while in others reduced amounts of LCAT mass indicated the presence of a functionally inactive protein. Sequence analysis identified homozygous missense or nonsense mutations in four probands. Two probands from one family both were found to be compound heterozygotes for a missense mutation and for a single base insertion causing a reading frame-shift. Subsequent family analyses were carried out using mutagenic primers for carrier identification. LCAT activity and LCAT mass in 23 genotypic heterozygotes were approximately half normal and clearly distinct from those of 20 unaffected family members. In the homozygous patients no obvious relationship between residual LCAT activity and the clinical phenotype was seen. The observation that the molecular defects in LCAT deficiency are dispersed in different regions of the enzyme suggests the existence of several functionally important structural domains in this enzyme.
The enzyme, lecithin cholesterol acyltransferase (LCAT), is responsible for the esterification of plasma cholesterol mediating the transfer of an acyl group from lecithin to the 3-hydroxy group of cholesterol. Deficiency of the enzyme is a well-known syndrome with a widespread geographic occurrence. We have cloned an allele from a patient homozygous for the LCAT deficiency. The only change that we could detect is a C to T transition in the fourth exon of the gene; this causes a substitution of Arg for Trp at position 147 of the mature protein. The functional significance of such a substitution with respect to the enzyme defect was demonstrated by transfecting the mutated LCAT gene in the cell line COS-1.
Title: Inhibitory effect of normal high density lipoproteins on lecithin:cholesterol acyltransferase activity in fish eye disease plasma Holmquist L, Carlson LA Ref: Acta Med Scand, 222:15, 1987 : PubMed
The lecithin:cholesterol acyltransferase (LCAT) activity of lipoprotein depleted normal and fish eye disease (FED) plasma was assayed in a modified Glomset-Wright incubation system where the enzyme was allowed to act on three different normal lipoprotein substrates consisting of an authentic mixture of very low (VLDL), low (LDL) and high (HDL) density lipoproteins to assay total LCAT activity, HDL to assay alpha-LCAT activity and combined VLDL and LDL to assay beta-LCAT activity, respectively. However, using normal plasma depleted of HDL, leaving its combined VLDL and LDL as enzyme substrate, resulted in a more than twofold increase in the LCAT activity of FED plasma from the two patients compared to the activity obtained with HDL present in the incubation mixture, indicating an inhibitory effect of HDL on the beta-LCAT activity present in FED plasma. This inhibitory effect of normal HDL could also be demonstrated by autoincubation of FED plasma mixed with isolated HDL2 or HDL3. Both these HDL subfractions had a pronounced inhibitory effect on the cholesteryl ester formation in FED plasma. The present study thus clearly demonstrates that normal HDL inhibits the beta-LCAT activity present in FED plasma, esterifying the free cholesterol of combined VLDL and LDL, derived from controls as well as from the two FED patients.
Title: Alpha-lecithin:cholesterol acyltransferase deficiency. Lack of both phospholipase A2 and acyltransferase activities characteristic of high density lipoprotein lecithin:cholesterol acyltransferase in fish eye disease Holmquist L, Carlson LA Ref: Acta Med Scand, 222:23, 1987 : PubMed
The phospholipase A2 and acyltransferase activities characteristic of human plasma lecithin: cholesterol acyltransferase have been evaluated in incubation mixtures of lipoprotein depleted plasma of fish eye disease patients and autologous HDL or homologous normal HDL3. Both enzyme activities were strongly reduced as compared to those of normal controls. These findings further support the claim that fish eye disease plasma has a specific lack of high density lipoprotein lecithin:cholesterol acyltransferase (alpha-LCAT deficiency), although the cholesterol esterification of combined VLDL and LDL in such plasma proceeds at a normal rate.
Pancreatic, hepatic and gastric/lingual lipase are closely related to each other and to lipoprotein lipase (EC: 3.1.1.34), which hydrolyses triglycerides of chylomicrons and very low density lipoproteins (VLDL). Pancreatic lipase (triacylglycerol acylhydrolase, EC: 3.1.1.3) plays a key role in dietary fat absorption by hydrolysing dietary long chain triacyl-glycerol to free fatty acids and monoacylglycerols in the intestinal lumen. The activity of lipase is stimulated by colipase in the presence of bile acids. Congenital pancreatic lipase deficiency is a rare, monoenzymatic form of exocrine pancreatic failure. Patients have oily/greasy stools from infancy or early childhood and the absence of discernable pancreatic disease. The pancreatic lipase-related protein show no significant catalytic activity on any of the substrates tested di and tri-glycerides phospholipids. Introducing the double mutation Val 178 Ala and Ala 180 Pro into the human pancreatic RP1 HPLRP1 gene yielded an enzyme is kinetically active on triglycerides. The guinea pig pancreatic lipase-related protein 2 (GPLRP2) differs from classical pancreatic lipases in that it displays both lipase and phospholipase A1 activities; classical pancreatic lipases have no phospholipase activity. human-PNLIPRP2 adopt in solution an open lid conformation which creates a large cavity capable of accommodating the galactose polar head of galactolipids (galactolipase)
Title: Comparative Study of the Molecular Characterization, Evolution, and Structure Modeling of Digestive Lipase Genes Reveals the Different Evolutionary Selection Between Mammals and Fishes Tang SL, Liang XF, He S, Li L, Alam MS, Wu J Ref: Front Genet, 13:909091, 2022 : PubMed
Vertebrates need suitable lipases to digest lipids for the requirement of energy and essential nutrients; however, the main digestive lipase genes of fishes have certain controversies. In this study, two types of digestive lipase genes (pancreatic lipase (pl) and bile salt-activated lipase (bsal)) were identified in mammals and fishes. The neighborhood genes and key active sites of the two lipase genes were conserved in mammals and fishes. Three copies of PL genes were found in mammals, but only one copy of the pl gene was found in most of the fish species, and the pl gene was even completely absent in some fish species (e.g., zebrafish, medaka, and common carp). Additionally, the hydrophobic amino acid residues (Ile and Leu) which are important to pancreatic lipase activity were also absent in most of the fish species. The PL was the main digestive lipase gene in mammals, but the pl gene seemed not to be the main digestive lipase gene in fish due to the absence of the pl gene sequence and the important amino acid residues. In contrast, the bsal gene existed in all fish species, even two to five copies of bsal genes were found in most of the fishes, but only one copy of the BSAL gene was found in mammals. The amino acid residues of bile salt-binding sites and the three-dimensional (3D) structure modeling of Bsal proteins were conserved in most of the fish species, so bsal might be the main digestive lipase gene in fish. The phylogenetic analysis also indicated that pl or bsal showed an independent evolution between mammals and fishes. Therefore, we inferred that the evolutionary selection of the main digestive lipase genes diverged into two types between mammals and fishes. These findings will provide valuable evidence for the study of lipid digestion in fish.
Title: Pancreatic lipase inhibitors: The road voyaged and successes Kumar A, Chauhan S Ref: Life Sciences, :119115, 2021 : PubMed
Human pancreatic lipase (triacylglycerol acyl hydrolase EC3.1.1.3) is the most widely studied member of the human lipase superfamily related to carboxyl esterase. It is secreted from the acinar cell of pancreas and has strong preference for triacylglycerides over cholesterol esters, phospholipids, and galactolipids. Apart from the hydrolysis of triacylglycerides, pancreatic lipase may cause the hydrolysis of retinyl esters in vivo. So, it is very much evidenced that pancreatic lipase with its cofactor colipase has prominent role in efficient digestion of dietary fat. Hence, the modulation of human pancreatic lipase may represent a new insight in the discovery of a number of therapeutics that can inhibit the absorption of fat in body and can be used in obesity and other related metabolic disorders. Even, the only Food and drug administration (FDA) approved antiobesity drug, orlistat, is also an inhibitor of pancreatic lipase. This review summarizes studies about structure, mechanistic approach of pancreatic lipase enzyme while emphasizing on the various synthetic pancreatic lipase inhibitors with their structure activity relationship (SAR).
Title: Structure and Function of Pancreatic Lipase-Related Protein 2 and Its Relationship With Pathological States Zhu G, Fang Q, Zhu F, Huang D, Yang C Ref: Front Genet, 12:693538, 2021 : PubMed
Pancreatic lipase is critical for the digestion and absorption of dietary fats. The most abundant lipolytic enzymes secreted by the pancreas are pancreatic triglyceride lipase (PTL or PNLIP) and its family members, pancreatic lipase-related protein 1 (PNLIPRP1or PLRP1) and pancreatic lipase-related protein 2 (PNLIPRP2 or PLRP2). Unlike the family's other members, PNLIPRP2 plays an elemental role in lipid digestion, especially for newborns. Therefore, if genetic factors cause gene mutation, or other factors lead to non-expression, it may have an effect on fat digestion and absorption, on the susceptibility to pancreas and intestinal pathogens. In this review, we will summarize what is known about the structure and function of PNLIPRP2 and the levels of PNLIPRP2 and associated various pathological states.
Title: The beta5-Loop and Lid Domain Contribute to the Substrate Specificity of Pancreatic Lipase-related Protein 2 (PNLIPRP2) Xiao X, Lowe ME Ref: Journal of Biological Chemistry, 290:28847, 2015 : PubMed
Pancreatic triglyceride lipase (PNLIP) is essential for dietary fat digestion in children and adults, whereas a homolog, pancreatic lipase-related protein 2 (PNLIPRP2), is critical in newborns. The two lipases are structurally similar, yet they have different substrate specificities. PNLIP only cleaves neutral fats. PNLIPRP2 cleaves neutral and polar fats. To test the hypothesis that the differences in activity between PNLIP and PNLIPRP2 are governed by surface loops around the active site, we created multiple chimeras of both lipases by exchanging the surface loops singly or in combination. The chimeras were expressed, purified, and tested for activity against various substrates. The structural determinants of PNLIPRP2 galactolipase activity were contained in the N-terminal domain. Of the surface loops tested, the lid domain and the beta5-loop influenced activity against triglycerides and galactolipids. Any chimera on PNLIP with the PNLIPRP2 lid domain or beta5-loop had decreased triglyceride lipase activity similar to that of PNLIPRP2. The corresponding chimeras of PNLIPRP2 did not increase activity against neutral lipids. Galactolipase activity was abolished by the PNLIP beta5-loop and decreased by the PNLIP lid domain. The source of the beta9-loop had minimal effect on activity. We conclude that the lid domain and beta5-loop contribute to substrate specificity but do not completely account for the differing activities of PNLIP and PNLIPRP2. Other regions in the N-terminal domain must contribute to the galactolipase activity of PNLIPRP2 through direct interactions with the substrate or by altering the conformation of the residues surrounding the hydrophilic cavity in PNLIPRP2.
Title: Inhibition of phospholipase A1, lipase and galactolipase activities of pancreatic lipase-related protein 2 by methyl arachidonyl fluorophosphonate (MAFP) Amara S, Delorme V, Record M, Carriere F Ref: Biochimica & Biophysica Acta, 1821:1379, 2012 : PubMed
Methyl arachidonyl fluorophosphonate (MAFP) is a known inhibitor of cytosolic phospholipase A2 and some other serine enzymes. MAFP was found here to be an irreversible inhibitor of human pancreatic lipase-related protein 2 (HPLRP2), an enzyme displaying lipase, phospholipase A1 and galactolipase activities. In the presence of MAFP, mass spectrometry analysis of HPLRP2 revealed a mass increase of 351Da, suggesting a covalent binding of MAFP to the active site serine residue. When HPLRP2 was pre-incubated with MAFP before measuring residual activity, a direct inhibition of HPLRP2 occurred, confirming that HPLRP2 has an active site freely accessible to solvent and differs from most lipases in solution. HPLRP2 activities on tributyrin (TC4), phosphatidylcholine (PC) and monogalactosyl dioctanoylglycerol (C8-MGDG) were equally inhibited under these conditions. Bile salts were not required to trigger the inhibition, but they significantly increased the rate of HPLRP2 inhibition, probably because of MAFP micellar solubilization. Since HPLRP2 is active on various substrates that self-organize differently in the presence of water, HPLRP2 inhibition by MAFP was tested in the presence of these substrates after adding MAFP in the course of the lipolysis reaction. In this case, the rates of inhibition of lipase, phospholipase A1 and galactolipase activities were not equivalent (triglycerides>PC>MGDG), suggesting different enzyme/inhibitor partitioning between the aqueous phase and lipid aggregates. The inhibition by MAFP of a well identified phospholipase A1 (HPLRP2), present in pancreatic juice and also in human monocytes, indicates that MAFP cannot be used for discriminating phospholipase A2 from A1 activities at the cellular level.
Access to the active site of pancreatic lipase (PL) is controlled by a surface loop, the lid, which normally undergoes conformational changes only upon addition of lipids or amphiphiles. Structures of PL with their lids in the open and functional conformation have required cocrystallization with amphiphiles. Here we report two crystal structures of wild-type and unglycosylated human pancreatic lipase-related protein 2 (HPLRP2) with the lid in an open conformation in the absence of amphiphiles. These structures solved independently are strikingly similar, with some residues of the lid being poorly defined in the electron-density map. The open conformation of the lid is however different from that previously observed in classical liganded PL, suggesting different kinetic properties for HPLRP2. Here we show that the HPLRP2 is directly inhibited by E600, does not present interfacial activation, and acts preferentially on substrates forming monomers or small aggregates (micelles) dispersed in solution like monoglycerides, phospholipids and galactolipids, whereas classical PL displays reverse properties and a high specificity for unsoluble substrates like triglycerides and diglycerides forming oil-in-water interfaces. These biochemical properties imply that the lid of HPLRP2 is likely to spontaneously adopt in solution the open conformation observed in the crystal structure. This open conformation generates a large cavity capable of accommodating the digalactose polar head of galactolipids, similar to that previously observed in the active site of the guinea pig PLRP2, but absent from the classical PL. Most of the structural and kinetic properties of HPLRP2 were found to be different from those of rat PLRP2, the structure of which was previously obtained with the lid in a closed conformation. Our findings illustrate the essential role of the lid in determining the substrate specificity and the mechanism of action of lipases.
Human pancreatic lipase-related protein 2 (HPLRP2) was found to be expressed in the pancreas, but its biochemical properties were not investigated in detail. A recombinant HPLRP2 was produced in insect cells and the yeast Pichia pastoris and purified by cation exchange chromatography. Its substrate specificity was investigated using pH-stat and monomolecular film techniques and various lipid substrates (triglycerides, diglycerides, phospholipids, and galactolipids). Lipase activity of HPLRP2 on trioctanoin was inhibited by bile salts and poorly restored by adding colipase. In vivo, HPLRP2 therefore seems unlikely to show any lipase activity on dietary fat. In human pancreatic lipase (HPL), residues R256, D257, Y267, and K268 are involved in the stabilization of the open conformation of the lid domain, which interacts with colipase. These residues are not conserved in HPLRP2. When the corresponding mutations (R256G, D257G, Y267F, and K268E) are introduced into HPL, the effects of colipase are drastically reduced in the presence of bile salts. This may explain why colipase has such weak effects on HPLRP2. HPLRP2 displayed a very low level of activity on phospholipid micelles and monomolecular films. Its activity on monogalactosyldiglyceride monomolecular film, which was much higher, was similar to the activity of guinea pig pancreatic lipase related-protein 2, which shows the highest galactolipase activity ever measured. The physiological role of HPLRP2 suggested by the present results is the digestion of galactolipids, the most abundant lipids occurring in plant cells, and therefore, in the vegetables that are part of the human diet.
Both classical pancreatic lipase (DPL) and pancreatic lipase-related protein 1 (DPLRP1) have been found to be secreted by dog exocrine pancreas. These two proteins were purified to homogeneity from canine pancreatic juice and no significant catalytic activity was observed with dog PLRP1 on any of the substrates tested: di- and tri-glycerides, phospholipids, etc. DPLRP1 was crystallized and its structure solved by molecular replacement and refined at a resolution of 2.10 A. Its structure is similar to that of the classical PL structures in the absence of any inhibitors or micelles. The lid domain that controls the access to the active site was found to have a closed conformation. An amino-acid substitution (Ala 178 Val) in the DPLRP1 may result in a steric clash with one of the acyl chains observed in the structures of a C11 alkyl phosphonate inhibitor, a transition state analogue, bound to the classical PL. This substitution was suspected of being responsible for the absence of DPLRP1 activity. The presence of Val and Ala residues in positions 178 and 180, respectively, are characteristic of all the known PLRP1, whereas Ala and Pro residues are always present in the same positions in all the other members of the PL gene family. Introducing the double mutation Val 178 Ala and Ala 180 Pro into the human pancreatic RP1 (HPLRP1) gene yielded a well expressed and folded enzyme in insect cells. This enzyme is kinetically active on triglycerides. Our findings on DPLRP1 and HPLRP1 are therefore likely to apply to all the RP1 lipases.
BACKGROUND: The guinea pig pancreatic lipase-related protein 2 (GPLRP2) differs from classical pancreatic lipases in that it displays both lipase and phospholipase A1 activities; classical pancreatic lipases have no phospholipase activity. The sequence of GPLRP2 is 63 % identical to that of human pancreatic lipase (HPL), but the so-called lid domain, is much reduced in GPLRP2. A phospholipase A1 from hornet venom (Dolml PLA1) is very similar to HPL and GPLRP2 but is devoid of lipase activity; Dolml PLA1 also contains a reduced lid domain and lacks a region termed the beta9 loop, which is located in the vicinity of the HPL and GPLRP2 active sites. The structure determination of a chimera of GPLRP2 and HPL and domain building of Dolml PLA1 were undertaken to gain a better understanding of the structural parameters responsible for the differences in lipase versus phospholipase activity among these structurally related enzymes. RESULTS: The crystal structure of a chimeric mutant of GPLRP2, consisting of the catalytic domain of GPLRP2 and the C-terminal domain of HPL, has been solved and refined to 2.1 A resolution. This enzyme belongs to the alpha/beta hydrolase fold family and shows high structural homology with classical pancreatic lipases. The active site is closely related to those of serine esterases, except for an unusual geometry of the catalytic triad. Due to the reduced size of the lid domain, the catalytic serine is fully accessible to solvent. Part of the beta9 loop, which stabilizes the lid domain in the closed conformation of the classical HPL, is totally exposed to the solvent and is not visible in the electron-density map. CONCLUSIONS: The structures of the related enzymes, GPLRP2 and HPL and the model of Dolml PLA1, provide insights into the role played by the loops located above the active site in controlling substrate selectivity towards triglycerides or phospholipids. In GPLRP2, the lid domain is reduced in size compared to HPL, and hydrophilic residues are exposed to solvent. GPLRP2 is thus able to accommodate the polar head of phospholipids. The beta9 loop is still present in GPLRP2, making it possible for this enzyme to still accommodate triglycerides. In Dolml PLA1, the beta9 loop is absent, and this enzyme is unable to process triglycerides retaining only the phospholipase A1 activity.
Title: Two novel human pancreatic lipase related proteins, hPLRP1 and hPLRP2. Differences in colipase dependence and in lipase activity Giller T, Buchwald P, Blum-Kaelin D, Hunziker W Ref: Journal of Biological Chemistry, 267:16509, 1992 : PubMed
We have isolated cDNAs coding for two novel human pancreatic lipase (hPL)-related human proteins, referred to as hPL-related proteins 1 and 2 (hPLRP1 and hPLRP2) and for hPL. The two novel proteins show an amino acid sequence identity to hPL of 68 and 65% for hPLRP1 and 2, respectively. All three proteins are secreted into the medium after transfection of COS cells with the corresponding cDNAs. The size of the three expressed proteins is similar and ranges between 45 and 50 kDa. The expressed hPLRP2 shows a lipolytic activity that is, however, in contrast to that of hPL only marginally dependent on the presence of colipase, whereas hPLRP1 shows no activity in this assay. A Northern analysis of normal human pancreas mRNA shows that the expression levels of hPLRP1 and hPLRP2 are about 4-fold and 24-fold lower, respectively, than that of hPL. hPLRP2 is, additionally, most closely related to a lipase reported to be expressed in mouse T-cells. A comparison of the sequences of the three proteins with sequences described as pancreatic lipases of other animal species shows three subfamilies of closer kinship. This suggests that the two novel proteins also exist in other species and that some of the sequences reported to be pancreatic lipase might more likely be the orthologues of hPLRP1 or hPLRP2 in those species.
By hydrolyzing the dietary triacylglycerols, pancreatic lipase causes catalysis in heterogeneous medium. In vivo, lipase action cannot take place without colipase due to the presence of bile salts. The cofactor enables lipase anchoring to the water-lipid interface. The lipase-colipase system furnishes an excellent example of specific interactions (protein-protein and protein-lipid). The studies of lipase catalytic properties brought to light the importance of certain parameters related to the 'quality of the interface'. The structure-function relationship analyses revealed a certain number of functional amino acid residues in lipase and colipase involved either in the catalytic site of the enzyme or in the recognition sites (lipase-colipase and protein-interface). Comparisons of the sequences of lipases derived from different sources display interesting similarities in certain cases.
A 5 1/2-year-old boy is reported with congenital lipase deficiency and the presence of colipase. He presented with greasy-oily stools since infancy, but growth and development have been normal. No other cause for exocrine pancreatic insufficiency could be found. Intraluminal (jejunal) fat digestion was defective, but some hydrolytic products of dietary long-chain triglyceride were present. The di- and monoglycerides were probably generated by pregastric lipases, although this was not measured directly. Amylase activity was depressed to some extent, a finding which could not be explained. Our studies do not clarify the issue of whether or not the absence of pancreatic lipase is explained as an inherited defect of lipase synthesis, or if it was acquired in utero or in the early postnatal period.
Phospholipase A(1) (PLA(1)) is an enzyme that hydrolyzes phospholipids and produces 2-acyl-lysophospholipids and fatty acids and is conserved in a wide range of organisms. Hypotrichosis, or woolly hair with or without hypotrichosis hypotrichosis (deficiency of hair growth), can be caused by homozygous or compound heterozygous mutation in the LIPH (human-LIPH) gene on chromosome 3q27 (not hepatic lipase: human-LIPC). Other mammalian enzymes that exhibit PLA(1) activity in vitro are hepatic lipase HL endothelial lipase EL and pancreatic lipase-related protein 2 PLRP2 and belong to alpha/beta hydrolase superfamily.
Lysophospholipids are potent hormone-like signalling biological lipids that regulate many important biological processes in mammals (including humans). Lysophosphatidic acid and sphingosine-1-phosphate represent the best studied examples for this lipid class, and their metabolic enzymes and/or cognate receptors are currently under clinical investigation for treatment of various neurological and autoimmune diseases in humans. Over the past two decades, the lysophsophatidylserines (lyso-PSs) have emerged as yet another biologically important lysophospholipid, and deregulation in its metabolism has been linked to various human pathophysiological conditions. Despite its recent emergence, an exhaustive review summarizing recent advances on lyso-PSs and the biological pathways that this bioactive lysophospholipid regulates has been lacking. To address this, here, we summarize studies that led to the discovery of lyso-PS as a potent signalling biomolecule, and discuss the structure, its detection in biological systems, and the biodistribution of this lysophospholipid in various mammalian systems. Further, we describe in detail the enzymatic pathways that are involved in the biosynthesis and degradation of this lipid and the putative lyso-PS receptors reported in the literature. Finally, we discuss the various biological pathways directly regulated by lyso-PSs in mammals and prospect new questions for this still emerging biomedically important signalling lysophospholipid.
Title: Structure and function of extracellular phospholipase A1 belonging to the pancreatic lipase gene family Aoki J, Inoue A, Makide K, Saiki N, Arai H Ref: Biochimie, 89:197, 2007 : PubMed
Phospholipase A1 (PLA1) is an enzyme that hydrolyzes phospholipids and produces 2-acyl-lysophospholipids and fatty acids and is conserved in a wide range of organisms. Mammals have several enzymes that exhibit PLA1 activity in vitro. The extracellular PLA1s include phosphatidylserine (PS)-specific PLA1 (PS-PLA1), membrane-associated phosphatidic acid (PA)-selective PLA1s (mPA-PLA1alpha and mPA-PLA1beta), hepatic lipase (HL), endothelial lipase (EL) and pancreatic lipase-related protein 2 (PLRP2), all of which belong to the pancreatic lipase gene family. The former three PLA1s differ from other members in their substrate specificities, structural features and gene organizations, and form a subfamily in the pancreatic lipase gene family. PS-PLA1, mPA-PLA1alpha and mPA-PLA1beta exhibit only PLA1 activity, while HL, EL and PLRP2 show triacylglycerol-hydrolyzing activity in addition to PLA1 activity. The tertiary structures of lipases have two surface loops, the lid and the beta9 loop. The lid and the beta9 loop cover the active site in its closed conformation. An alignment of amino acid sequences of the pancreatic lipase gene family members revealed two molecular characteristics of PLA1s in the two surface loops. First, lipase members exhibiting PLA1 activity (PS-PLA1, mPA-PLA1alpha and mPA-PLA1beta, EL, guinea pig PLRP2 and PLA1 from hornet venom (DolmI)) have short lids. Second, PS-PLA1, mPA-PLA1alpha, mPA-PLA1beta and DolmI, which exhibit only PLA(1) activity, have short beta9 loops. Thus, the two surface loops appear to be involved in the ligand recognition. PS-PLA1 and mPA-PLA1s specifically hydrolyze PS and PA, respectively, producing their corresponding lysophospholipids. Lysophosphatidylserine and lysophosphatidic acid have been defined as lipid mediators with multiple biological functions. Thus, these PLA1s have a role in the production of these lysophospholipid mediators.
Title: An alternative splicing form of phosphatidylserine-specific phospholipase A1 that exhibits lysophosphatidylserine-specific lysophospholipase activity in humans Nagai Y, Aoki J, Sato T, Amano K, Matsuda Y, Arai H and Ref: Journal of Biological Chemistry, 274:11053, 1999 : PubMed
Phosphatidylserine-specific phospholipase A1 (PS-PLA1), which acts specifically on phosphatidylserine (PS) and 1-acyl-2-lysophosphatidylserine (lyso-PS) to hydrolyze fatty acids at the sn-1 position of these phospholipids, was first identified in rat platelets (Sato, T., Aoki, J., Nagai, Y., Dohmae, N., Takio, K., Doi, T., Arai, H., and Inoue, K. (1997) J. Biol. Chem. 272, 2192-2198). In this study we isolated and sequenced cDNA clones encoding human PS-PLA1, which showed 80% homology with rat PS-PLA1 at the amino acid level. In addition to an mRNA encoding a 456-amino acid product (PS-PLA1), an mRNA with four extra bases inserted at the boundary of the exon-intron junction was detected in human tissues and various human cell lines. This mRNA is most probably produced via an alternative use of the 5'-splicing site (two consensus sequences for RNA splicing occur at the boundary of the exon-intron junction) and encodes a 376-amino acid product (PS-PLA1DeltaC) that lacks two-thirds of the C-terminal domain of PS-PLA1. Unlike PS-PLA1, PS-PLA1DeltaC hydrolyzed exclusively lyso-PS but not PS appreciably. Any other phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), and their lyso derivatives were not hydrolyzed at all. These data demonstrated that PS-PLA1DeltaC exhibits lyso-PS-specific lysophospholipase activity and that the C-terminal domain of PS-PLA1 is responsible for recognizing diacylphospholipids. In addition, human PS-PLA1 gene was mapped to chromosome 3q13.13-13.2 and was unexpectedly identical to the nmd gene, which is highly expressed in nonmetastatic melanoma cell lines but poorly expressed in metastatic cell lines (van Groningen, J. J., Bloemers, H. P., and Swart, G. W. (1995) Cancer Res. 55, 6237-6243).
Enhanced Disease Susceptibility 1 (EDS1), an essential component of R gene-mediated disease resistance. The Arabidopsis EDS1 (arath-eds1) and PAD4 (arath-F22O6.190) genes encode lipase-like proteins that function in resistance (R) gene-mediated and basal plant disease resistance. EDS1 can dimerize and interact with PAD4. EDS1 (arath-eds1) and PAD4 (arath-F22O6.190) genes encode lipase-like proteins that function in resistance (R) gene-mediated and basal plant disease resistance. EDS1 can dimerize and interact with PAD4 or with SAG101 (arath-At5g14930). This family is extracted from the Lipase3 gene family. The C-terminal domain known as the EP domain and its interface consists of hydrophobic interactions, salt bridges, and an extensive hydrogen bonding network. Plants utilise intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors to detect pathogen effectors and activate local and systemic defence. NRG1 and ADR1 'helper' NLRs (RNLs) cooperate with enhanced disease susceptibility 1 (EDS1), senescence-associated gene 101 (SAG101) and phytoalexin-deficient 4 (PAD4) lipase-like proteins to mediate signalling from TIR domain NLR receptors (TNLs). Two distinct modules (NRG1/EDS1/SAG101 and ADR1/EDS1/PAD4) mediate TNL receptor defence signalling. (In the seed alignment and the HMM alignement the EP domain (not alpha/beta hydrolase domain) is excluded)
Arabidopsis pathogen effector-triggered immunity (ETI) is controlled by a family of three lipase-like proteins EDS1, PAD4 and SAG101 and two sub-families of HET-S/LOB-B (HeLo)-domain "helper" NLRs, ADR1s and NRG1s. EDS1-PAD4 dimers cooperate with ADR1s, and EDS1-SAG101 dimers with NRG1s, in two separate defense-promoting modules. EDS1-PAD4-ADR1 and EDS1-SAG101-NRG1 complexes were detected in immune-activated leaf extracts but the molecular determinants for specific complex formation and function remain unknown. EDS1 signaling is mediated by a C-terminal EP domain (EPD) surface surrounding a cavity formed by the heterodimer. Here we investigated whether the EPDs of PAD4 and SAG101 contribute to EDS1 dimer functions. Using a structure-guided approach, we undertook a comprehensive mutational analysis of Arabidopsis PAD4. We identify two conserved residues (Arg314 and Lys380) lining the PAD4 EPD cavity that are essential for EDS1-PAD4 mediated pathogen resistance, but are dispensible for PAD4 mediated restriction of green peach aphid infestation. Positionally equivalent Met304 and Arg373 at the SAG101 EPD cavity are required for EDS1-SAG101 promotion of ETI-related cell death. In a PAD4 and SAG101 interactome analysis of ETI-activated tissues, PAD4(R314A) and SAG101(M304R) EPD variants maintain interaction with EDS1 but lose association, respectively, with helper NLRs ADR1-L1 and NRG1.1, and other immune-related proteins. Our data reveal a fundamental contribution of similar but non-identical PAD4 and SAG101 EPD surfaces to specific EDS1 dimer protein interactions and pathogen immunity.
Plants utilise intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors to detect pathogen effectors and activate local and systemic defence. NRG1 and ADR1 "helper" NLRs (RNLs) cooperate with enhanced disease susceptibility 1 (EDS1), senescence-associated gene 101 (SAG101) and phytoalexin-deficient 4 (PAD4) lipase-like proteins to mediate signalling from TIR domain NLR receptors (TNLs). The mechanism of RNL/EDS1 family protein cooperation is not understood. Here, we present genetic and molecular evidence for exclusive EDS1/SAG101/NRG1 and EDS1/PAD4/ADR1 co-functions in TNL immunity. Using immunoprecipitation and mass spectrometry, we show effector recognition-dependent interaction of NRG1 with EDS1 and SAG101, but not PAD4. An EDS1-SAG101 complex interacts with NRG1, and EDS1-PAD4 with ADR1, in an immune-activated state. NRG1 requires an intact nucleotide-binding P-loop motif, and EDS1 a functional EP domain and its partner SAG101, for induced association and immunity. Thus, two distinct modules (NRG1/EDS1/SAG101 and ADR1/EDS1/PAD4) mediate TNL receptor defence signalling.
Title: Origins and Immunity Networking Functions of EDS1 Family Proteins Lapin D, Bhandari DD, Parker JE Ref: Annu Rev Phytopathol, :, 2020 : PubMed
The EDS1 family of structurally unique lipase-like proteins EDS1, SAG101, and PAD4 evolved in seed plants, on top of existing phytohormone and nucleotide-binding-leucine-rich-repeat (NLR) networks, to regulate immunity pathways against host-adapted biotrophic pathogens. Exclusive heterodimers between EDS1 and SAG101 or PAD4 create essential surfaces for resistance signaling. Phylogenomic information, together with functional studies in Arabidopsis and tobacco, identify a coevolved module between the EDS1-SAG101 heterodimer and coiled-coil (CC) HET-S and LOP-B (CCHELO) domain helper NLRs that is recruited by intracellular Toll-interleukin1-receptor (TIR) domain NLR receptors to confer host cell death and pathogen immunity. EDS1-PAD4 heterodimers have a different and broader activity in basal immunity that transcriptionally reinforces local and systemic defenses triggered by various NLRs. Here, we consider EDS1 family protein functions across seed plant lineages in the context of networking with receptor and helper NLRs and downstream resistance machineries. The different modes of action and pathway connectivities of EDS1 family members go some way to explaining their central role in biotic stress resilience. Expected final online publication date for the Annual Review of Phytopathology, Volume 58 is August 25, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Plant nucleotide binding/leucine-rich repeat (NLR) immune receptors are activated by pathogen effectors to trigger host defenses and cell death. Toll-interleukin 1 receptor domain NLRs (TNLs) converge on the ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) family of lipase-like proteins for all resistance outputs. In Arabidopsis (Arabidopsis thaliana) TNL-mediated immunity, AtEDS1 heterodimers with PHYTOALEXIN DEFICIENT4 (AtPAD4) transcriptionally induced basal defenses. AtEDS1 uses the same surface to interact with PAD4-related SENESCENCE-ASSOCIATED GENE101 (AtSAG101), but the role of AtEDS1-AtSAG101 heterodimers remains unclear. We show that AtEDS1-AtSAG101 functions together with N REQUIRED GENE1 (AtNRG1) coiled-coil domain helper NLRs as a coevolved TNL cell death-signaling module. AtEDS1-AtSAG101-AtNRG1 cell death activity is transferable to the Solanaceous species Nicotiana benthamiana and cannot be substituted by AtEDS1-AtPAD4 with AtNRG1 or AtEDS1-AtSAG101 with endogenous NbNRG1. Analysis of EDS1-family evolutionary rate variation and heterodimer structure-guided phenotyping of AtEDS1 variants and AtPAD4-AtSAG101 chimeras identify closely aligned a-helical coil surfaces in the AtEDS1-AtSAG101 partner C-terminal domains that are necessary for reconstituted TNL cell death signaling. Our data suggest that TNL-triggered cell death and pathogen growth restriction are determined by distinctive features of EDS1-SAG101 and EDS1-PAD4 complexes and that these signaling machineries coevolved with other components within plant species or clades to regulate downstream pathways in TNL immunity.
Title: Comparative genomic analysis of the Lipase3 gene family in five plant species reveals distinct evolutionary origins Wang D, Zhang L, Hu J, Gao D, Liu X, Sha Y Ref: Genetica, 146:179, 2018 : PubMed
Lipases are physiologically important and ubiquitous enzymes that share a conserved domain and are classified into eight different families based on their amino acid sequences and fundamental biological properties. The Lipase3 family of lipases was reported to possess a canonical fold typical of alpha/beta hydrolases and a typical catalytic triad, suggesting a distinct evolutionary origin for this family. Genes in the Lipase3 family do not have the same functions, but maintain the conserved Lipase3 domain. There have been extensive studies of Lipase3 structures and functions, but little is known about their evolutionary histories. In this study, all lipases within five plant species were identified, and their phylogenetic relationships and genetic properties were analyzed and used to group them into distinct evolutionary families. Each identified lipase family contained at least one dicot and monocot Lipase3 protein, indicating that the gene family was established before the split of dicots and monocots. Similar intron/exon numbers and predicted protein sequence lengths were found within individual groups. Twenty-four tandem Lipase3 gene duplications were identified, implying that the distinctive function of Lipase3 genes appears to be a consequence of translocation and neofunctionalization after gene duplication. The functional genes EDS1, PAD4, and SAG101 that are reportedly involved in pathogen response were all located in the same group. The nucleotide diversity (Dxy) and the ratio of nonsynonymous to synonymous nucleotide substitutions rates (Ka/Ks) of the three genes were significantly greater than the average across the genomes. We further observed evidence for selection maintaining diversity on three genes in the Toll-Interleukin-1 receptor type of nucleotide binding/leucine-rich repeat immune receptor (TIR-NBS LRR) immunity-response signaling pathway, indicating that they could be vulnerable to pathogen effectors.
Erwinia amylovora causes fire blight in rosaceous plants. In nonhost Arabidopsis thaliana, E. amylovora triggers necrotic symptoms associated with transient bacterial multiplication, suggesting either that A. thaliana lacks a susceptibility factor or that it actively restricts E. amylovora growth. Inhibiting plant protein synthesis at the time of infection led to an increase in necrosis and bacterial multiplication and reduced callose deposition, indicating that A. thaliana requires active protein synthesis to restrict E. amylovora growth. Analysis of the callose synthase-deficient pmr4-1 mutant indicated that lack of callose deposition alone did not lead to increased sensitivity to E. amylovora. Transcriptome analysis revealed that approximately 20% of the genes induced following E. amylovora infection are related to defense and signaling. Analysis of mutants affected in NDR1 and EDS1, two main components of the defense-gene activation observed, revealed that E. amylovora multiplied ten times more in the eds1-2 mutant than in the wild type but not in the ndr1-1 mutant. Analysis of mutants affected in three WRKY transcription factors showing EDS1-dependent activation identified WRKY46 and WRKY54 as positive regulators and WRKY70 as a negative regulator of defense against E. amylovora. Altogether, we show that EDS1 is a positive regulator of nonhost resistance against E. amylovora in A. thaliana and hypothesize that it controls the production of several effective defenses against E. amylovora through the action of WRKY46 and WRKY54, while WRKY70 acts as a negative regulator.
Title: Crystallization and preliminary crystallographic analysis of Arabidopsis thaliana EDS1, a key component of plant immunity, in complex with its signalling partner SAG101 Wagner S, Rietz S, Parker JE, Niefind K Ref: Acta Crystallographica Sect F Struct Biol Cryst Commun, 67:245, 2011 : PubMed
In plants, the nucleocytoplasmic protein EDS1 (Enhanced disease susceptibility1) is an important regulator of innate immunity, coordinating host-cell defence and cell-death programs in response to pathogen attack. Arabidopsis thaliana EDS1 stabilizes and signals together with its partners PAD4 (Phytoalexin deficient4) and SAG101 (Senescence-associated gene101). Characterization of EDS1 molecular configurations in vitro and in vivo points to the formation of structurally and spatially distinct EDS1 homomeric dimers and EDS1 heteromeric complexes with either PAD4 or SAG101 as necessary components of the immune response. EDS1, PAD4 and SAG101 constitute a plant-specific protein family with a unique `EP' (EDS1-PAD4-specific) domain at their C-termini and an N-terminal domain resembling enzymes with an alpha/beta-hydrolase fold. Here, the expression, purification and crystallization of a functional EDS1 complex formed by EDS1 and SAG101 from Arabidopsis thaliana are reported. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 101.8, b = 115.9, c = 122.8 A, and diffracted to 3.5 A resolution.
Title: A functional EDS1 ortholog is differentially regulated in powdery mildew resistant and susceptible grapevines and complements an Arabidopsis eds1 mutant Gao F, Shu X, Ali MB, Howard S, Li N, Winterhagen P, Qiu W, Gassmann W Ref: Planta, 231:1037, 2010 : PubMed
Vitis vinifera (grapevine) is the most economically important deciduous fruit crop, but cultivated grapevine varieties lack adequate innate immunity to a range of devastating diseases. To identify genetic resources for grapevine innate immunity and understand pathogen defense pathways in a woody perennial plant, we focus in this study on orthologs of the central Arabidopsis thaliana defense regulator ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1). The family of EDS1-like genes is expanded in grapevine, and members of this family were previously found to be constitutively upregulated in the resistant variety 'Norton' of the North American grapevine species Vitis aestivalis, while they were induced by Erysiphe necator, the causal agent of grapevine powdery mildew (PM), in the susceptible V. vinifera variety 'Cabernet Sauvignon'. Here, we determine the responsiveness of individual EDS1-like genes in grapevine to PM and salicylic acid, and find that EDS1-like paralogs are differentially regulated in 'Cabernet Sauvignon', while two are constitutively upregulated in 'Norton'. Sequencing of VvEDS1 and VaEDS1 cDNA and genomic clones revealed high conservation in the protein-encoding sequence and some divergence of the promoter sequence in the two grapevine varieties. Complementation of the Arabidopsis eds1-1 mutant showed that the EDS1-like gene with highest predicted amino acid sequence similarity to AtEDS1 from either grapevine varieties is a functional ortholog of AtEDS1. Together, our analyses show that differential susceptibility to PM is correlated with differences in EDS1 expression, not differences in EDS1 function, between resistant 'Norton' and susceptible 'Cabernet Sauvignon'.
An important layer of plant innate immunity to host-adapted pathogens is conferred by intracellular nucleotide-binding/oligomerization domain-leucine rich repeat (NB-LRR) receptors recognizing specific microbial effectors. Signaling from activated receptors of the TIR (Toll/Interleukin-1 Receptor)-NB-LRR class converges on the nucleo-cytoplasmic immune regulator EDS1 (Enhanced Disease Susceptibility1). In this report we show that a receptor-stimulated increase in accumulation of nuclear EDS1 precedes or coincides with the EDS1-dependent induction and repression of defense-related genes. EDS1 is capable of nuclear transport receptor-mediated shuttling between the cytoplasm and nucleus. By enhancing EDS1 export from inside nuclei (through attachment of an additional nuclear export sequence (NES)) or conditionally releasing EDS1 to the nucleus (by fusion to a glucocorticoid receptor (GR)) in transgenic Arabidopsis we establish that the EDS1 nuclear pool is essential for resistance to biotrophic and hemi-biotrophic pathogens and for transcriptional reprogramming. Evidence points to post-transcriptional processes regulating receptor-triggered accumulation of EDS1 in nuclei. Changes in nuclear EDS1 levels become equilibrated with the cytoplasmic EDS1 pool and cytoplasmic EDS1 is needed for complete resistance and restriction of host cell death at infection sites. We propose that coordinated nuclear and cytoplasmic activities of EDS1 enable the plant to mount an appropriately balanced immune response to pathogen attack.
Title: Plant immunity: the EDS1 regulatory node Wiermer M, Feys BJ, Parker JE Ref: Curr Opin Plant Biol, 8:383, 2005 : PubMed
ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) and its interacting partner, PHYTOALEXIN DEFICIENT 4 (PAD4), constitute a regulatory hub that is essential for basal resistance to invasive biotrophic and hemi-biotrophic pathogens. EDS1 and PAD4 are also recruited by Toll-Interleukin-1 receptor (TIR)-type nucleotide binding-leucine rich repeat (NB-LRR) proteins to signal isolate-specific pathogen recognition. Recent work points to a fundamental role of EDS1 and PAD4 in transducing redox signals in response to certain biotic and abiotic stresses. These intracellular proteins are important activators of salicylic acid (SA) signaling and also mediate antagonism between the jasmonic acid (JA) and ethylene (ET) defense response pathways. EDS1 forms several molecularly and spatially distinct complexes with PAD4 and a newly discovered in vivo signaling partner, SENESCENCE ASSOCIATED GENE 101 (SAG101). Together, EDS1, PAD4 and SAG101 provide a major barrier to infection by both host-adapted and non-host pathogens.
Title: EDS1, an essential component of R gene-mediated disease resistance in Arabidopsis has homology to eukaryotic lipases Falk A, Feys BJ, Frost LN, Jones JDG, Daniels MJ, Parker JE Ref: Proceedings of the National Academy of Sciences of the United States of America, 96:3292, 1999 : PubMed
A major class of plant disease resistance (R) genes encodes leucine-rich-repeat proteins that possess a nucleotide binding site and amino-terminal similarity to the cytoplasmic domains of the Drosophila Toll and human IL-1 receptors. In Arabidopsis thaliana, EDS1 is indispensable for the function of these R genes. The EDS1 gene was cloned by targeted transposon tagging and found to encode a protein that has similarity in its amino-terminal portion to the catalytic site of eukaryotic lipases. Thus, hydrolase activity, possibly on a lipid-based substrate, is anticipated to be central to EDS1 function. The predicted EDS1 carboxyl terminus has no significant sequence homologies, although analysis of eight defective eds1 alleles reveals it to be essential for EDS1 function. Two plant defense pathways have been defined previously that depend on salicylic acid, a phenolic compound, or jasmonic acid, a lipid-derived molecule. We examined the expression of EDS1 mRNA and marker mRNAs (PR1 and PDF1.2, respectively) for these two pathways in wild-type and eds1 mutant plants after different challenges. The results suggest that EDS1 functions upstream of salicylic acid-dependent PR1 mRNA accumulation and is not required for jasmonic acid-induced PDF1.2 mRNA expression.
Family close to Lipase_3. The DEFECTIVE IN ANTHER DEHISCIENCE gene encodes a novel phospholipase A1 catalyzing the initial step of jasmonic acid biosynthesis, which synchronizes pollen maturation, anther dehiscence, and flower opening in Arabidopsis. AtDSEL, an Arabidopsis cytosolic DAD1-like acylhydrolase, is involved in negative regulation of storage oil mobilization during seedling establishment. This family is close to Plant_lipase_EDS1-like and belongs to Lipase_3 family
Phospholipase is an enzyme that hydrolyzes various phospholipid substrates at specific ester bonds and plays important roles such as membrane remodeling, as digestive enzymes, and the regulation of cellular mechanism. Phospholipase proteins are divided into following the four major groups according to the ester bonds they cleave off: phospholipase A1 (PLA1), phospholipase A2 (PLA2), phospholipase C (PLC), and phospholipase D (PLD). Among the four phospholipase groups, PLA1 has been less studied than the other phospholipases. Here, we report the first molecular structures of plant PLA1s: AtDSEL and CaPLA1 derived from Arabidopsis thaliana and Capsicum annuum, respectively. AtDSEL and CaPLA1 are novel PLA1s in that they form homodimers since PLAs are generally in the form of a monomer. The dimerization domain at the C-terminal of the AtDSEL and CaPLA1 makes hydrophobic interactions between each monomer, respectively. The C-terminal domain is also present in PLA1s of other plants, but not in PLAs of mammals and fungi. An activity assay of AtDSEL toward various lipid substrates demonstrates that AtDSEL is specialized for the cleavage of sn-1 acyl chains. This report reveals a new domain that exists only in plant PLA1s and suggests that the domain is essential for homodimerization.
Title: Comparative genomic analysis of the Lipase3 gene family in five plant species reveals distinct evolutionary origins Wang D, Zhang L, Hu J, Gao D, Liu X, Sha Y Ref: Genetica, 146:179, 2018 : PubMed
Lipases are physiologically important and ubiquitous enzymes that share a conserved domain and are classified into eight different families based on their amino acid sequences and fundamental biological properties. The Lipase3 family of lipases was reported to possess a canonical fold typical of alpha/beta hydrolases and a typical catalytic triad, suggesting a distinct evolutionary origin for this family. Genes in the Lipase3 family do not have the same functions, but maintain the conserved Lipase3 domain. There have been extensive studies of Lipase3 structures and functions, but little is known about their evolutionary histories. In this study, all lipases within five plant species were identified, and their phylogenetic relationships and genetic properties were analyzed and used to group them into distinct evolutionary families. Each identified lipase family contained at least one dicot and monocot Lipase3 protein, indicating that the gene family was established before the split of dicots and monocots. Similar intron/exon numbers and predicted protein sequence lengths were found within individual groups. Twenty-four tandem Lipase3 gene duplications were identified, implying that the distinctive function of Lipase3 genes appears to be a consequence of translocation and neofunctionalization after gene duplication. The functional genes EDS1, PAD4, and SAG101 that are reportedly involved in pathogen response were all located in the same group. The nucleotide diversity (Dxy) and the ratio of nonsynonymous to synonymous nucleotide substitutions rates (Ka/Ks) of the three genes were significantly greater than the average across the genomes. We further observed evidence for selection maintaining diversity on three genes in the Toll-Interleukin-1 receptor type of nucleotide binding/leucine-rich repeat immune receptor (TIR-NBS LRR) immunity-response signaling pathway, indicating that they could be vulnerable to pathogen effectors.
Title: AtDSEL, an Arabidopsis cytosolic DAD1-like acylhydrolase, is involved in negative regulation of storage oil mobilization during seedling establishment Kim EY, Seo YS, Kim WT Ref: J Plant Physiol, 168:1705, 2011 : PubMed
Mobilization of seed storage reserves is essential for seed germination and seedling establishment. Here, we report that AtDSEL, an Arabidopsis thalianaDAD1-like Seedling Establishment-related Lipase, is involved in the mobilization of storage oils for early seedling establishment. AtDSEL is a cytosolic member of the DAD1-like acylhydrolase family encoded by At4g18550. Bacterially expressed AtDSEL preferentially hydrolyzed 1,3-diacylglycerol and 1-monoacylglycerol, suggesting that AtDSEL is an sn-1-specific lipase. AtDSEL-overexpressing transgenic Arabidopsis plants (35S:AtDSEL) were defective in post-germinative seedling growth in medium without an exogenous carbon source. This phenotype was rescued by the addition of sucrose to the growth medium. In contrast, loss-of-function mutant plants (atdsel-1 and atdsel-2) had a mildly fast-growing phenotype regardless of the presence of an exogenous carbon source. Electron microscopy revealed that 5-day-old 35S:AtDSEL cotyledons retained numerous peroxisomes and oil bodies, which were exhausted in wild-type and mutant cotyledons. The impaired seedling establishment of 35S:AtDSEL was not rescued by the addition of an exogenous fatty acid source, and 35S:AtDSEL seedling growth was insensitive to 2,4-dichlorophenoxybutyric acid, indicating that beta-oxidation was blocked in AtDSEL-overexpressers. These results suggest that AtDSEL is involved in the negative regulation of seedling establishment by inhibiting the breakdown of storage oils.
Title: Phospholipid-derived signaling mediated by phospholipase A in plants Ryu SB Ref: Trends Plant Sci, 9:229, 2004 : PubMed
Title: The DEFECTIVE IN ANTHER DEHISCIENCE gene encodes a novel phospholipase A1 catalyzing the initial step of jasmonic acid biosynthesis, which synchronizes pollen maturation, anther dehiscence, and flower opening in Arabidopsis Ishiguro S, Kawai-Oda A, Ueda J, Nishida I, Okada K Ref: Plant Cell, 13:2191, 2001 : PubMed
The Arabidopsis mutant defective in anther dehiscence1 (dad1) shows defects in anther dehiscence, pollen maturation, and flower opening. The defects were rescued by the exogenous application of jasmonic acid (JA) or linolenic acid, which is consistent with the reduced accumulation of JA in the dad1 flower buds. We identified the DAD1 gene by T-DNA tagging, which is characteristic to a putative N-terminal transit peptide and a conserved motif found in lipase active sites. DAD1 protein expressed in Escherichia coli hydrolyzed phospholipids in an sn-1-specific manner, and DAD1-green fluorescent protein fusion protein expressed in leaf epidermal cells localized predominantly in chloroplasts. These results indicate that the DAD1 protein is a chloroplastic phospholipase A1 that catalyzes the initial step of JA biosynthesis. DAD1 promoter::beta-glucuronidase analysis revealed that the expression of DAD1 is restricted in the stamen filaments. A model is presented in which JA synthesized in the filaments regulates the water transport in stamens and petals.
This family differs substantially from the cutinase acetyl-xylan esterase family (cutinase monofunctional). Several cutinases from the genus Thermobifida act on biodegradable plastics such as synthetic polyesters. Not all cutinases can degrade polyester plastics. Aerial plant organs are protected by a cuticle composed of an insoluble polymeric structural compound, cutin, which is a polyester composed of hydroxy and hydroxyepoxy fatty acids. Cutinases are lipases with a specificity for p-nitrophenyl acyl esters with short chain acyl group. This family was extracted from the Bacterial_lipase family which is close to PAF-Acetylhydrolase family. Streptomyces exfoliatus lipase (1JFR) Pseudomonas mendocina lipase (2FX5) are included in this family. This family correspond to family III of the classification of Arpigny et al 1999. Polyethylene terephthalate degrading hydrolase/PET-hydrolase/PET Hydrolase. Two enzymes in Ideonella sakaiensis (for example) act on PET (Poly ethylene terephthalate): idesa-peth from Polyesterase-lipase-cutinase family and idesa-mheth which acts on extremity of PET (Exo-PETase Function PET hydrolase PET-Hydrolase) and on MHET the product of hydrolysis of PET. MHETase belongs to the Tannase family
Petroleum-based plastics are durable and accumulate in all ecological niches. Knowledge on enzymatic degradation is sparse. Today, less than 50 verified plastics-active enzymes are known. First examples of enzymes acting on the polymers polyethylene terephthalate (PET) and polyurethane (PUR) have been reported together with a detailed biochemical and structural description. Furthermore, very few polyamide (PA) oligomer active enzymes are known. In this article, the current known enzymes acting on the synthetic polymers PET and PUR are briefly summarized, their published activity data were collected and integrated into a comprehensive open access database. The Plastics-Active Enzymes Database (PAZy) represents an inventory of known and experimentally verified enzymes that act on synthetic fossil fuel-based polymers. Almost 3000 homologs of PET-active enzymes were identified by profile hidden Markov models. Over 2000 homologs of PUR-active enzymes were identified by BLAST. Based on multiple sequence alignments, conservation analysis identified the most conserved amino acids, and sequence motifs for PET- and PUR-active enzymes were derived.
We report a bioinformatic workflow and subsequent discovery of a new polyethylene terephthalate (PET) hydrolase, which we named MG8, from the human saliva metagenome. MG8 has robust PET plastic degradation activities under different temperature and salinity conditions, outperforming several naturally occurring and engineered hydrolases in degrading PET. Moreover, we genetically encoded 2,3-diaminopropionic acid (DAP) in place of the catalytic serine residue of MG8, thereby converting a PET hydrolase into a covalent binder for bio-functionalization of PET. We show that MG8(DAP), in conjunction with a split green fluorescent protein system, can be used to attach protein cargos to PET as well as other polyester plastics. The discovery of a highly active PET hydrolase from the human metagenome-currently an underexplored resource for industrial enzyme discovery-as well as the repurposing of such an enzyme into a plastic functionalization tool, should facilitate ongoing efforts to degrade and maximize reusability of PET.
Title: Novel Pet-Degrading Enzymes: Structure-Function from a Computational Perspective Berselli A, Ramos MJ, Menziani MC Ref: Chembiochem, 22:2032, 2021 : PubMed
The bacterium strain Ideonella sakaiensis 201-F6 is able to hydrolyze low-crystallinity PET films at 30 degrees C due to two enzymes named PETase and MHETase. Since its discovery, many efforts have been dedicated to elucidating the structure and features of those two enzymes, and various authors have highlighted the necessity to optimize both the substrate binding site and the global structure in order to enhance the stability and catalytic activity of these PET biocatalysts so as to make them more suitable for industrial applications. In this review, the strategies adopted by different research groups to investigate the structure and functionality of both PETase and MHETase in depth are described, emphasizing the advantages provided by the use of computational methods to complement and drive experiments. Subsequently, the modifications implemented with protein engineering are discussed. The versatility of the enzymes secreted by I. sakaiensis enables the prediction that they will find several applications in the disposal of PET debris, encouraging a prioritization of efforts in this prolific research field.
Poly(ethylene terephthalate) (PET) is the most abundant polyester plastic and a major contributor to plastic pollution. IsPETase, from the PET-assimilating bacterium Ideonella sakaiensis, is a unique PET-hydrolytic enzyme that shares high sequence identity to canonical cutinases, but shows substrate preference towards PET and exhibits higher PET-hydrolytic activity at ambient temperature. Structural analyses suggest that IsPETase harbours a substrate-binding residue, W185, with a wobbling conformation and a highly flexible W185-locating beta6-beta7 loop. Here, we show that these features result from the presence of S214 and I218 in IsPETase, whose equivalents are strictly His and Phe, respectively, in all other homologous enzymes. We found that mutating His/Phe residues to Ser/Ile could enhance the PET-hydrolytic activity of several IsPETase-like enzymes. In conclusion, the Ser/Ile mutations should provide an important strategy to improve the activity of potential PET-hydrolytic enzymes with properties that may be useful for various applications.
Title: Yeast cell surface display of bacterial PET hydrolase as a sustainable biocatalyst for the degradation of polyethylene terephthalate Chen Z, Xiao Y, Weber G, Wei R, Wang Z Ref: Methods Enzymol, 648:457, 2021 : PubMed
Enzymatic hydrolysis of polyethylene terephthalate (PET) is considered to be an environmentally friendly method for the recycling of plastic waste. Recently, a bacterial enzyme named IsPETase was found in Ideonella sakaiensis with the ability to degrade amorphous PET at ambient temperature suggesting its possible use in recycling of PET. However, applying the purified IsPETase in large-scale PET recycling has limitations, i.e., a complicated production process, high cost of single-use, and instability of the enzyme. Yeast cell surface display has proven to be an effectual alternative for improving enzyme degradation efficiency and realizing industrial applications. This chapter deals with the construction and application of a whole-cell biocatalyst by displaying IsPETase on the surface of yeast (Pichia pastoris) cells.
Title: Enhancing PET hydrolytic enzyme activity by fusion of the cellulose-binding domain of cellobiohydrolase I from Trichoderma reesei Dai L, Qu Y, Huang JW, Hu Y, Hu H, Li S, Chen CC, Guo RT Ref: J Biotechnol, 334:47, 2021 : PubMed
The large amounts of polyethylene terephthalate (PET) that enter and accumulate in the environment have posed a serious threat to global ecosystems and human health. A PET hydrolase from PET-assimilating bacterium Ideonella sakaiensis (IsPETase) that exhibits superior PET hydrolytic activity at mild conditions is attracting enormous attention in development of plastic biodegrading strategies. In order to enhance the PET hydrolysis capacity of IsPETase, we selected several polymer-binding domains that can adhere to a hydrophobic polymer surface and fused these to a previously engineered IsPETase(S121E/D186H/R280A) (IsPETase(EHA)) variant. We found that fusing a cellulose-binding domain (CBM) of cellobiohydrolase I from Trichoderma reesei onto the C-terminus of IsPETase(EHA) showed a stimulatory effect on enzymatic hydrolysis of PET. Compared to the parental enzyme, IsPETase(EHA)_CBM exhibited 71.5 % and 44.5 % higher hydrolytic activity at 30 degC and 40 degC, respectively. The catalytic activity of IsPETase(EHA)_CBM was increased by 86 % when the protein concentration was increased from 2.5 microg/mL to 20microg/mL. These findings suggest that the fusion of polymer-binding module to IsPETase is a promising strategy to stimulate the enzymatic hydrolysis of PET.
Title: Structural analysis of PET-degrading enzymes PETase and MHETase from Ideonella sakaiensis Graf LG, Michels EAP, Yew Y, Liu W, Palm GJ, Weber G Ref: Methods Enzymol, 648:337, 2021 : PubMed
The concept of biocatalytic PET degradation for industrial recycling processes had made a big step when the bacterium Ideonella sakaiensis was discovered to break PET down to its building blocks at ambient temperature. This process involves two enzymes: cleavage of ester bonds in PET by PETase and in MHET, the resulting intermediate, by MHETase. To understand and further improve this unique capability, structural analysis of the involved enzymes was aimed at from early on. We describe a repertoire of methods to this end, including protein expression and purification, crystallization of apo and substrate-bound enzymes, and modeling of PETase complexed with a ligand.
Poly(ethylene terephthalate) (PET) is the world's most abundant polyester plastic, and its ongoing accumulation in nature is causing a global environmental problem. Currently, the main recycling processes utilize thermomechanical or chemical means, resulting in the deterioration of the mechanical properties of PET. Consequently, polluting de novo synthesis remains preferred, creating the need for more efficient and bio-sustainable ways to hydrolyze the polymer. Recently, a PETase enzyme from the bacterium Ideonella sakaiensis was shown to facilitate PET biodegradation, albeit at slow rate. Engineering of more efficient PETases is required for industrial relevance, but progress is currently hampered by the dependency on intracellular expression in Escherichia coli. To create a more efficient screening platform in E. coli, we explore different surface display anchors for fast and easy assaying of PETase activity. We show that PETases can be functionally displayed on the bacterial cell surface, enabling screening of enzyme activity on PET microparticles - both while anchored to the cell and following solubilization of the enzymes.
Title: Structural Insights into Carboxylic Polyester-Degrading Enzymes and Their Functional Depolymerizing Neighbors Leitao AL, Enguita FJ Ref: Int J Mol Sci, 22:, 2021 : PubMed
Esters are organic compounds widely represented in cellular structures and metabolism, originated by the condensation of organic acids and alcohols. Esterification reactions are also used by chemical industries for the production of synthetic plastic polymers. Polyester plastics are an increasing source of environmental pollution due to their intrinsic stability and limited recycling efforts. Bioremediation of polyesters based on the use of specific microbial enzymes is an interesting alternative to the current methods for the valorization of used plastics. Microbial esterases are promising catalysts for the biodegradation of polyesters that can be engineered to improve their biochemical properties. In this work, we analyzed the structure-activity relationships in microbial esterases, with special focus on the recently described plastic-degrading enzymes isolated from marine microorganisms and their structural homologs. Our analysis, based on structure-alignment, molecular docking, coevolution of amino acids and surface electrostatics determined the specific characteristics of some polyester hydrolases that could be related with their efficiency in the degradation of aromatic polyesters, such as phthalates.
Title: Emerging Roles of PETase and MHETase in the Biodegradation of Plastic Wastes Maity W, Maity S, Bera S, Roy A Ref: Appl Biochem Biotechnol, 193:2699, 2021 : PubMed
Polyethylene terephthalate (PET) is extensively used in plastic products, and its accumulation in the environment has become a global concern. Being a non-degradable pollutant, a tremendous quantity of PET-bearing plastic materials have already accumulated in the environment, posing severe challenges towards the existence of various endangered species and consequently threatening the ecosystem and biodiversity. While conventional recycling and remediation methodologies so far have been ineffective in formulating a "green" degradation protocol, the bioremediation strategies-though nascent-are exhibiting greater promises towards achieving the target. Very recently, a novel bacterial strain called Ideonella sakaiensis 201-F6 has been discovered that produces a couple of unique enzymes, polyethylene terephthalate hydrolase and mono(2-hydroxyethyl) terephthalic acid hydrolase, enabling the bacteria to utilize PET as their sole carbon source. With a detailed understanding of the protein structure of these enzymes, possibilities for their optimization as PET degrading agents have started to emerge. In both proteins, several amino acids have been identified that are not only instrumental for catalysis but also provide avenues for the applications of genetic engineering strategies to improve the catalytic efficiencies of the enzymes. In this review, we focused on such unique structural features of these two enzymes and discussed their potential as molecular tools that can essentially become instrumental towards the development of sustainable bioremediation strategies. Degradation PET by wild type and genetically engineered PETase and MHETase. Effect of the MHETase-PETase chimeric protein and PETase expressed on the surface of yeast cells on PET degradation is also shown.
Title: Positive Charge Introduction on the Surface of Thermostabilized PET Hydrolase Facilitates PET Binding and Degradation Nakamura A, Kobayashi N, Koga N, Iino R Ref: ACS Catal, 11:8550, 2021 : PubMed
A thermostable enzyme PET2, found in a metagenome library, has been engineered to improve its hydrolytic activity against polyethylene terephthalate (PET). The PET2 wild-type (WT) showed a melting temperature of 69.0 C and produced water-soluble reaction products at a rate of 0.40 min-1 (2.4 microM products from 0.1 microM enzyme after 60 min reaction) from an amorphous PET film at 60 C. Mutations for surface charge modification, backbone stabilization, and formation of additional disulfide bond were introduced into the PET2 WT, and the best mutant (PET2 7M) showed a melting temperature of 75.7 C and hydrolytic activity of 1.3 min-1 (7.8 micrM products from 0.1 microM enzyme after 60 min reaction at 60 C). X-ray crystal structures of PET2 mutants showed that introduced arginine and lysine residues oriented to the solvent, similar to a PET hydrolase from Ideonella sakaiensis 201-F6. Single-molecule fluorescence imaging revealed that these positively charged surface residues increased binding rate constant of PET2 7M to PET surface 2.7 times, compared with PET2 WT, and resulted in higher activity. Optimal temperature for amorphous PET hydrolysis by PET2 7M (68 C) was 8 C higher than that by PET2 WT (60 C), and hydrolytic activity of PET2 7M at the optimal temperature (2.7 min-1, 16.2 microM products from 0.1 microM enzyme after 60 min reaction) was 6.8 times higher than that of PET2 WT (0.40 min-1). Furthermore, PET2 7M generated reaction products with a constant rate for at least 24 h at 68 C, indicating long-term thermal stability at the optimal temperature.
Title: Structural basis for Ca(2+)-dependent catalysis of a cutinase-like enzyme and its engineering: application to enzymatic PET depolymerization Oda M Ref: Biophys Physicobiol, 18:168, 2021 : PubMed
A cutinase-like enzyme from Saccharomonospora viridis AHK190, Cut190, can depolymerize polyethylene terephthalate (PET). As high activity at approximately 70 degreesC is required for PET depolymerization, structure-based protein engineering of Cut190 was carried out. Crystal structure information of the Cut190 mutants was used for protein engineering and for evaluating the molecular basis of activity and thermal stability. A variety of biophysical methods were employed to unveil the mechanisms underlying the unique features of Cut190, which included the regulation of its activity and thermal stability by Ca(2+). Ca(2+) association and dissociation can change the enzyme conformation to regulate catalytic activity. Weak metal-ion binding would be required for the naive conformational change of Cut190, while maintaining its fluctuation, to "switch" the enzyme on and off. The activity of Cut190 is regulated by the weak Ca(2+) binding to the specific site, Site 1, while thermal stability is mainly regulated by binding to another Site 2, where a disulfide bond could be introduced to increase the stability. Recent results on the structure-activity relationship of engineered Cut190 are reviewed, including the application for PET depolymerization by enzymes.
Title: Implications for the PET decomposition mechanism through similarity and dissimilarity between PETases from Rhizobacter gummiphilus and Ideonella sakaiensis Sagong HY, Son HF, Seo H, Hong H, Lee D, Kim KJ Ref: J Hazard Mater, :, 2021 : PubMed
The development of a superb polyethylene terephthalate (PET) hydrolyzing enzyme requires an accurate understanding of the PET decomposition mechanism. However, studies on PET degrading enzymes, including the PET hydrolase from Ideonella sakaiensis (IsPETase), have not provided sufficient knowledge of the molecular mechanisms for the hardly accessible substrate. Here, we report a novel PET hydrolase from Rhizobacter gummiphilus (RgPETase), which has a hydrolyzing activity similar to IsPETase toward microcrystalline PET but distinct behavior toward low crystallinity PET film. Structural analysis of RgPETase reveals that the enzyme shares the key structural features of IsPETase for high PET hydrolysis activity but has distinguished structures at the surface-exposed regions. RgPETase shows a unique conformation of the wobbling tryptophan containing loop (WW-loop) and change of the electrostatic surface charge on the loop dramatically affects the PET-degrading activity. We further show that effect of the electrostatic surface charge to the activity varies depending on locations. This work provides valuable information underlying the uncovered PET decomposition mechanism.
Recently, a bacterium strain of Ideonella sakaiensis was identified with the uncommon ability to degrade the poly(ethylene terephthalate) (PET). The PETase from I. sakaiensis strain 201-F6 (IsPETase) catalyzes the hydrolysis of PET converting it to mono(2-hydroxyethyl) terephthalic acid (MHET), bis(2-hydroxyethyl)-TPA (BHET), and terephthalic acid (TPA). Despite the potential of this enzyme for mitigation or elimination of environmental contaminants, one of the limitations of the use of IsPETase for PET degradation is the fact that it acts only at moderate temperature due to its low thermal stability. Besides, molecular details of the main interactions of PET in the active site of IsPETase remain unclear. Herein, molecular docking and molecular dynamics (MD) simulations were applied to analyze structural changes of IsPETase induced by PET binding. Results from the essential dynamics revealed that the beta1-beta2 connecting loop is very flexible. This loop is located far from the active site of IsPETase and we suggest that it can be considered for mutagenesis to increase the thermal stability of IsPETase. The free energy landscape (FEL) demonstrates that the main change in the transition between the unbound to the bound state is associated with the beta7-alpha5 connecting loop, where the catalytic residue Asp206 is located. Overall, the present study provides insights into the molecular binding mechanism of PET into the IsPETase structure and a computational strategy for mapping flexible regions of this enzyme, which can be useful for the engineering of more efficient enzymes for recycling plastic polymers using biological systems.
Title: Microbial Polyethylene Terephthalate Hydrolases: Current and Future Perspectives Carr CM, Clarke DJ, Dobson ADW Ref: Front Microbiol, 11:571265, 2020 : PubMed
Plastic has rapidly transformed our world, with many aspects of human life now relying on a variety of plastic materials. Biological plastic degradation, which employs microorganisms and their degradative enzymes, has emerged as one way to address the unforeseen consequences of the waste streams that have resulted from mass plastic production. The focus of this review is microbial hydrolase enzymes which have been found to act on polyethylene terephthalate (PET) plastic. The best characterized examples are discussed together with the use of genomic and protein engineering technologies to obtain PET hydrolase enzymes for different applications. In addition, the obstacles which are currently limiting the development of efficient PET bioprocessing are presented. By continuing to study the possible mechanisms and the structural elements of key enzymes involved in microbial PET hydrolysis, and by assessing the ability of PET hydrolase enzymes to work under practical conditions, this research will help inform large-scale waste management operations. Finally, the contribution of microbial PET hydrolases in creating a potential circular PET economy will be explored. This review combines the current knowledge on enzymatic PET processing with proposed strategies for optimization and use, to help clarify the next steps in addressing pollution by PET and other plastics.
Title: PMBD: a Comprehensive Plastics Microbial Biodegradation Database Gan Z, Zhang H Ref: Database (Oxford), 2019:bav119, 2019 : PubMed
Since the invention over a hundred years ago, plastics have been used in many applications, and they are involved in every aspect of our lives. The extensive usage of plastics results in a tremendous amount of waste, which has become a severe burden on the environment. Several degradation approaches exist in nature to cope with ever-increasing plastic waste. Among these approaches, biodegradation by microorganisms has emerged as a natural way, which is favored by many environmentally conscious societies. To facilitate the study on biodegradation of plastics, we developed an online resource, Plastics Microbial Biodegradation Database (PMBD), to gather and present the information about microbial biodegradation of plastics. In this database, 949 microorganisms-plastics relationships and 79 genes involved in the biodegradation of plastics were manually collected and confirmed through literature searching. In addition, more than 8000 automatically annotated enzyme sequences, which were predicted to be involved in the plastics biodegradation, were extracted from the TrEMBL section of the UniProt database. The PMBD database is presented with a website at http://pmbd.genome-mining.cn/home. Data may be accessed through browsing or searching. Also included on the website are a sequence alignment tool and a function prediction tool.
The discovery of Ideonella sakaiensis, a plastic-degrading bacterium, creates possibilities for a sustainable "bioeconomy" for recycling plastic waste.
Poly(ethylene terephthalate) (PET) is one of the most abundantly produced synthetic polymers and is accumulating in the environment at a staggering rate as discarded packaging and textiles. The properties that make PET so useful also endow it with an alarming resistance to biodegradation, likely lasting centuries in the environment. Our collective reliance on PET and other plastics means that this buildup will continue unless solutions are found. Recently, a newly discovered bacterium, Ideonella sakaiensis 201-F6, was shown to exhibit the rare ability to grow on PET as a major carbon and energy source. Central to its PET biodegradation capability is a secreted PETase (PET-digesting enzyme). Here, we present a 0.92 A resolution X-ray crystal structure of PETase, which reveals features common to both cutinases and lipases. PETase retains the ancestral alpha/beta-hydrolase fold but exhibits a more open active-site cleft than homologous cutinases. By narrowing the binding cleft via mutation of two active-site residues to conserved amino acids in cutinases, we surprisingly observe improved PET degradation, suggesting that PETase is not fully optimized for crystalline PET degradation, despite presumably evolving in a PET-rich environment. Additionally, we show that PETase degrades another semiaromatic polyester, polyethylene-2,5-furandicarboxylate (PEF), which is an emerging, bioderived PET replacement with improved barrier properties. In contrast, PETase does not degrade aliphatic polyesters, suggesting that it is generally an aromatic polyesterase. These findings suggest that additional protein engineering to increase PETase performance is realistic and highlight the need for further developments of structure/activity relationships for biodegradation of synthetic polyesters.
Title: Structural studies reveal the molecular mechanism of PETase Chen CC, Han X, Ko TP, Liu W, Guo RT Ref: Febs J, 285:3717, 2018 : PubMed
Poly(ethylene terephthalate) (PET) is a class of plastic material widely used in modern society, but large amounts of PET waste cause severe environmental problems. Obtained from a PET-consuming bacterium Ideonella sakaiensis, the enzyme PETase exhibits superb hydrolytic activity and substrate preference toward PET. Here, we summarize some recent advances in the crystallographic analysis of PETase. These reports uncover structural features of PETase that are involved in its catalytic activity. In comparison to homologous enzymes, PETase contains an additional disulfide bond as well as an extended beta8-alpha6 loop. More importantly, the crystal structures of PETase in complex with substrate and product analogs provide critical information for understanding the mechanism of action of PETase. In particular, the wobbling conformation of W156 is closely related to the binding of substrate and product. These new findings are of great importance for further in-depth research and engineering development of PETase, and should advance the implementation of plastic biodegradation strategy.
Polyethylene terephthalate (PET) is one of the most-consumed synthetic polymers, with an annual production of 50 million tons. Unfortunately, PET accumulates as waste and is highly resistant to biodegradation. Recently, fungal and bacterial thermophilic hydrolases were found to catalyze PET hydrolysis with optimal activities at high temperatures. Strikingly, an enzyme from Ideonella sakaiensis, termed PETase, was described to efficiently degrade PET at room temperature, but the molecular basis of its activity is not currently understood. Here, a crystal structure of PETase was determined at 2.02 resolution and employed in molecular dynamics simulations showing that the active site of PETase has higher flexibility at room temperature than its thermophilic counterparts. This flexibility is controlled by a novel disulfide bond in its active site, with its removal leading to destabilization of the catalytic triad and reduction of the hydrolase activity. Molecular docking of a model substrate predicts that PET binds to PETase in a unique and energetically favorable conformation facilitated by several residue substitutions within its active site when compared to other enzymes. These computational predictions are in excellent agreement with recent mutagenesis and PET film degradation analyses. Finally, we rationalize the increased catalytic activity of PETase at room temperature through molecular dynamics simulations of enzyme-ligand complexes for PETase and other thermophilic PET-degrading enzymes at 298, 323, and 353 K. Our results reveal that both the binding pose and residue substitutions within PETase favor proximity between the catalytic residues and the labile carbonyl of the substrate at room temperature, suggesting a more favorable hydrolytic reaction. These results are valuable for enabling detailed evolutionary analysis of PET-degrading enzymes and for rational design endeavors aiming at increasing the efficiency of PETase and similar enzymes toward plastic degradation.
Plastics, including poly(ethylene terephthalate) (PET), possess many desirable characteristics and thus are widely used in daily life. However, non-biodegradability, once thought to be an advantage offered by plastics, is causing major environmental problem. Recently, a PET-degrading bacterium, Ideonella sakaiensis, was identified and suggested for possible use in degradation and/or recycling of PET. However, the molecular mechanism of PET degradation is not known. Here we report the crystal structure of I. sakaiensis PETase (IsPETase) at 1.5 A resolution. IsPETase has a Ser-His-Asp catalytic triad at its active site and contains an optimal substrate binding site to accommodate four monohydroxyethyl terephthalate (MHET) moieties of PET. Based on structural and site-directed mutagenesis experiments, the detailed process of PET degradation into MHET, terephthalic acid, and ethylene glycol is suggested. Moreover, other PETase candidates potentially having high PET-degrading activities are suggested based on phylogenetic tree analysis of 69 PETase-like proteins.
A cutinase-type polyesterase from Saccharomonospora viridis AHK190 (Cut190) has been shown to degrade the inner block of polyethylene terephthalate. A unique feature of Cut190 is that its function and stability are regulated by Ca(2+) binding. Our previous crystal structure analysis of Cut190S226P showed that one Ca(2+) binds to the enzyme, which induces large conformational changes in several loop regions to stabilize an open conformation [Miyakawa, T., et al. (2015) Appl. Microbiol. Biotechnol. 99, 4297]. In this study, to analyze the substrate recognition mechanism of Cut190, we determined the crystal structure of the inactive form of a Cut190 mutant, Cut190*S176A, in complex with calcium ions and/or substrates. We found that three calcium ions bind to Cut190*S176A, which is supported by analysis using native mass spectrometry experiments and 3D Reference Interaction Site Model calculations. The complex structures with the two substrates, monoethyl succinate and monoethyl adipate (engaged and open forms), presumably correspond to the pre- and post-reaction states, as the ester bond is close to the active site and pointing outward from the active site, respectively, for the two complexes. Ca(2+) binding induces the pocket to open, enabling the substrate to access the pocket more easily. Molecular dynamics simulations suggest that a post-reaction state in the engaged form presumably exists between the experimentally observed forms, indicating that the substrate would be cleaved in the engaged form and then requires the enzyme to change to the open form to release the product, a process that Ca(2+) can greatly accelerate.
PET hydrolase (PETase), which hydrolyzes polyethylene terephthalate (PET) into soluble building blocks, provides an attractive avenue for the bioconversion of plastics. Here we present the structures of a novel PETase from the PET-consuming microbe Ideonella sakaiensis in complex with substrate and product analogs. Through structural analyses, mutagenesis, and activity measurements, a substrate-binding mode is proposed, and several features critical for catalysis are elucidated.
We have investigated the structures of two native cutinases from Thermobifida cellulosilytica, namely Thc_Cut1 and Thc_Cut2 as well as of two variants, Thc_Cut2_DM (Thc_Cut2_ Arg29Asn_Ala30Val) and Thc_Cut2_TM (Thc_Cut2_Arg19Ser_Arg29Asn_Ala30Val). The four enzymes showed different activities towards the aliphatic polyester poly(lactic acid) (PLLA). The crystal structures of the four enzymes were successfully solved and in combination with Small Angle X-Ray Scattering (SAXS) the structural features responsible for the selectivity difference were elucidated. Analysis of the crystal structures did not indicate significant conformational differences among the different cutinases. However, the distinctive SAXS scattering data collected from the enzymes in solution indicated a remarkable surface charge difference. The difference in the electrostatic and hydrophobic surface properties could explain potential alternative binding modes of the four cutinases on PLLA explaining their distinct activities. Biotechnol. Bioeng. 2017;114: 2481-2488. (c) 2017 Wiley Periodicals, Inc.
Title: Structural basis for the Ca(2+)-enhanced thermostability and activity of PET-degrading cutinase-like enzyme from Saccharomonospora viridis AHK190 Miyakawa T, Mizushima H, Ohtsuka J, Oda M, Kawai F, Tanokura M Ref: Applied Microbiology & Biotechnology, 99:4297, 2015 : PubMed
A cutinase-like enzyme from Saccharomonospora viridis AHK190, Cut190, hydrolyzes the inner block of polyethylene terephthalate (PET); this enzyme is a member of the lipase family, which contains an alpha/beta hydrolase fold and a Ser-His-Asp catalytic triad. The thermostability and activity of Cut190 are enhanced by high concentrations of calcium ions, which is essential for the efficient enzymatic hydrolysis of amorphous PET. Although Ca(2+)-induced thermostabilization and activation of enzymes have been well explored in alpha-amylases, the mechanism for PET-degrading cutinase-like enzymes remains poorly understood. We focused on the mechanisms by which Ca(2+) enhances these properties, and we determined the crystal structures of a Cut190 S226P mutant (Cut190(S226P)) in the Ca(2+)-bound and free states at 1.75 and 1.45 A resolution, respectively. Based on the crystallographic data, a Ca(2+) ion was coordinated by four residues within loop regions (the Ca(2+) site) and two water molecules in a tetragonal bipyramidal array. Furthermore, the binding of Ca(2+) to Cut190(S226P) induced large conformational changes in three loops, which were accompanied by the formation of additional interactions. The binding of Ca(2+) not only stabilized a region that is flexible in the Ca(2+)-free state but also modified the substrate-binding groove by stabilizing an open conformation that allows the substrate to bind easily. Thus, our study explains the structural basis of Ca(2+)-enhanced thermostability and activity in PET-degrading cutinase-like enzyme for the first time and found that the inactive state of Cut190(S226P) is activated by a conformational change in the active-site sealing residue, F106.
Title: Structural and functional studies on a thermostable polyethylene terephthalate degrading hydrolase from Thermobifida fusca Roth C, Wei R, Oeser T, Then J, Follner C, Zimmermann W, Strater N Ref: Applied Microbiology & Biotechnology, 98:7815, 2014 : PubMed
Bacterial cutinases are promising catalysts for the modification and degradation of the widely used plastic polyethylene terephthalate (PET). The improvement of the enzyme for industrial purposes is limited due to the lack of structural information for cutinases of bacterial origin. We have crystallized and structurally characterized a cutinase from Thermobifida fusca KW3 (TfCut2) in free as well as in inhibitor-bound form. Together with our analysis of the thermal stability and modelling studies, we suggest possible reasons for the outstanding thermostability in comparison to the less thermostable homolog from Thermobifida alba AHK119 and propose a model for the binding of the enzyme towards its polymeric substrate. The TfCut2 structure is the basis for the rational design of catalytically more efficient enzyme variants for the hydrolysis of PET and other synthetic polyesters.
Thermophilic actinomycetes produce enzymes capable of hydrolyzing synthetic polyesters such as polyethylene terephthalate (PET). In addition to carboxylesterases, which have hydrolytic activity predominantly against PET oligomers, esterases related to cutinases also hydrolyze synthetic polymers. The production of these enzymes by actinomycetes as well as their recombinant expression in heterologous hosts is described and their catalytic activity against polyester substrates is compared. Assays to analyze the enzymatic hydrolysis of synthetic polyesters are evaluated, and a kinetic model describing the enzymatic heterogeneous hydrolysis process is discussed. Structure-function and structure-stability relationships of actinomycete polyester hydrolases are compared based on molecular dynamics simulations and recently solved protein structures. In addition, recent progress in enhancing their activity and thermal stability by random or site-directed mutagenesis is presented.
We determined the crystal structure of a cutinase from Thermobifida alba AHK119 (Est119) at a resolution of 1.76A. The overall structure of Est119 displays a typical alpha/beta-hydrolase fold consisting of a central twisted beta-sheet of nine beta-strands that are flanked by nine alpha-helices on both sides. The refined model contains two monomers in the asymmetric unit that form a dimer interface; a polyethylene glycol fragment is bound in the interface. Polyethylene glycol-binding site on the protein may suggest a glycol-binding site. A putative polymer-recognizing groove is observed to continue through the catalytic pocket. Water molecules are bound to hydrophilic amino acids along the groove, indicating the alternating pattern of polar and hydrophobic residues.
Title: Isolation of a novel cutinase homolog with polyethylene terephthalate-degrading activity from leaf-branch compost by using a metagenomic approach Sulaiman S, Yamato S, Kanaya E, Kim JJ, Koga Y, Takano K, Kanaya S Ref: Applied Environmental Microbiology, 78:1556, 2012 : PubMed
The gene encoding a cutinase homolog, LC-cutinase, was cloned from a fosmid library of a leaf-branch compost metagenome by functional screening using tributyrin agar plates. LC-cutinase shows the highest amino acid sequence identity of 59.7% to Thermomonospora curvata lipase. It also shows the 57.4% identity to Thermobifida fusca cutinase. When LC-cutinase without a putative signal peptide was secreted to the periplasm of Escherichia coli cells with the assistance of the pelB leader sequence, more than 50% of the recombinant protein, termed LC-cutinase*, was excreted into the extracellular medium. It was purified and characterized. LC-cutinase* hydrolyzed various fatty acid monoesters with acyl chain lengths of 2 to 18, with a preference for short-chain substrates (C(4) substrate at most) most optimally at pH 8.5 and 50 degrees C, but could not hydrolyze olive oil. It lost activity with half-lives of 40 min at 70 degrees C and 7 min at 80 degrees C. LC-cutinase* had an ability to degrade poly(epsilon-caprolactone) and polyethylene terephthalate (PET). The specific PET-degrading activity of LC-cutinase* was determined to be 12 mg/h/mg of enzyme (2.7 mg/h/mukat of pNP-butyrate-degrading activity) at pH 8.0 and 50 degrees C. This activity is higher than those of the bacterial and fungal cutinases reported thus far, suggesting that LC-cutinase* not only serves as a good model for understanding the molecular mechanism of PET-degrading enzyme but also is potentially applicable for surface modification and degradation of PET.
Neutral lipases are ubiquitous and diverse enzymes. The molecular architecture of the structurally characterized lipases is similar, often despite a lack of detectable homology at the sequence level. Some of the microbial lipases are evolutionarily related to physiologically important mammalian enzymes. For example, limited sequence similarities were recently noted for the Streptomyces exfoliatus lipase (SeL) and two mammalian platelet-activating factor acetylhydrolases (PAF-AHs). The determination of the crystal structure of SeL allowed us to explore the structure-function relationships in this novel family of homologous hydrolases.
RESULTS:
The crystal structure of SeL was determined by multiple isomorphous replacement and refined using data to 1.9 A resolution. The molecule exhibits the canonical tertiary fold of an alpha/beta hydrolase. The putative nucleophilic residue, Ser131, is located within a nucleophilic elbow and is hydrogen bonded to His209, which in turn interacts with Asp177. These three residues create a triad that closely resembles the catalytic triads found in the active sites of other neutral lipases. The mainchain amides of Met132 and Phe63 are perfectly positioned to create an oxyanion hole. Unexpectedly, there are no secondary structure elements that could render the active site inaccessible to solvent, like the lids that are commonly found in neutral lipases.
CONCLUSIONS:
The crystal structure of SeL reinforces the notion that it is a homologue of the mammalian PAF-AHs. We have used the catalytic triad in SeL to model the active site of the PAF-AHs. Our model is consistent with the site-directed mutagenesis studies of plasma PAF-AH, which implicate Ser273, His351 and Asp296 in the active site. Our study therefore provides direct support for the hypothesis that the plasma and isoform II PAF-AHs are triad-containing alpha/beta hydrolases.
Family extracted from Lipase_3. (from InterPro) This family includes triacylglycerol lipase OBL1 from Arabidopsis thaliana and Nicotiana tabacum, which are homologues of the acid lipase from castor bean (RcOBL1). OBL1 is an acid GXSXG - lipase localized to lipid droplets that can hydrolyse a range of triacylglycerols without a clear preference for acyl-chains. It can also cleave 1,2-diacylglycerol, 1,3-diacylglycerol and 1-monoacylglycerol, but not phosphatidylcholine, phosphatidylethanolamine, or sterol esters. It is required for pollen tube growth. Triacylglycerol hydrolysis by OBL1 may provide acyl groups for the synthesis of membrane lipids in growing pollen tubes
Title: Characterization of the enzymatic activity and physiological function of the lipid droplet-associated triacylglycerol lipase AtOBL1 Muller AO, Ischebeck T Ref: New Phytol, 217:1062, 2018 : PubMed
Similar to seeds, pollen tubes contain lipid droplets that store triacylglycerol (TAG), but the fate of this TAG as well as the enzymes involved in its breakdown are unknown. Therefore, two potential TAG lipases from tobacco and Arabidopsis, NtOBL1 (Oil body lipase 1) and AtOBL1, were investigated, especially with respect to their importance for pollen tube growth. We expressed NtOBL1 and AtOBL1 as fluorescent fusion proteins to study their localization by confocal microscopy. Furthermore, we overexpressed AtOBL1 in Nicotiana benthamiana leaves to characterize it enzymatically. The obl1 mutant was studied in respect to its pollen tube growth in vivo and its seed germination. Both NtOBL1 and AtOBL1 localized to lipid droplets. AtOBL1 was abundant in pollen tubes and seedlings, and acted as a lipase on TAG, diacylglycerol and 1-monoacylglycerol at a pH optimum of 5.5. The obl1 mutant was hampered in pollen tube growth, whereas seedling establishment was not affected under optimal conditions, even though AtOBL1 accounted for a major lipase activity in seeds. TAG could be a direct precursor for the synthesis of membrane lipids in pollen tubes and proteins of the OBL family involved in the flux of acyl groups.
These proteins sometimes referred as vitellogenins are specific to higher dipters and are not evolutionnary related to the vitellogenins accumulated in oocytes of most oviparous animals
Title: Cyclorraphan yolk proteins and lepidopteran minor yolk proteins originate from two unrelated lipase families Hens K, Lemey P, Macours N, Francis C, Huybrechts R Ref: Insect Molecular Biology, 13:615, 2004 : PubMed
Vitellogenins, cyclorraphan yolk proteins and lepidopteran minor yolk proteins are three classes of female-specific proteins that serve as an embryonic nutritional store. Similarity to vertebrate lipid-binding proteins was established for vitellogenins and yolk proteins, vitellogenins being related to apolipoprotein B and yolk proteins to lipases. Recently, similarity between yolk proteins and minor yolk proteins was reported and it was suggested that yolk proteins are more related to minor yolk proteins than to vertebrate lipases. In this study, we cloned five additional yolk proteins from the grey fleshfly Neobellieria bullata, formerly known as Sarcophaga bullata. We used this sequence data, combined with sequence data retrieved from the NCBI protein database to evaluate the yolk protein-lipase and the yolk protein-minor yolk protein relationship. We found no similarity between yolk proteins and minor yolk proteins, but we showed that yolk proteins are related to a family of lipases containing vertebrate hepatic and pancreatic lipases while minor yolk proteins are related to a family of lipases containing vertebrate gastric and lingual lipases. The fact that three different classes of yolk storage proteins show similarity to three different classes of vertebrate lipid-binding proteins strongly suggests that this lipid-binding feature is important for insect yolk storage proteins.
Title: The major yolk proteins of higher Diptera are homologs of a class of minor yolk proteins in lepidoptera Sappington TW Ref: Journal of Molecular Evolution, 55:470, 2002 : PubMed
In most oviparous animals, including insects, vitellogenin (Vg) is the major yolk protein precursor. However, in the higher Diptera (cyclorrhaphan flies), a class of proteins homologous to lipoprotein lipases called yolk polypeptides (YP) are accumulated by oocytes instead of Vg, which is not produced at all. Lepidopterans (moths) produce Vg as the major yolk protein precursor, but also manufacture a class of minor yolk proteins referred to as egg-specific proteins (ESP) or YP2s. Although the lepidopteran ESP/YP2s are related to lipoprotein lipases, previous attempts to directly demonstrate their homology with higher-dipteran YPs were unsuccessful. In this paper, a multiple alignment of amino acid sequences was constructed using a shared lipid binding motif as an anchor, to demonstrate that lepidopteran ESP/YP2s, higher-dipteran YPs, and lipoprotein lipases are indeed homologous. Phylogenetic analyses of the aligned sequences were performed using both distance-based and parsimony strategies. It is apparent that the higher dipterans did not requisition a lipoprotein lipase to replace Vg as a yolk protein precursor, but instead utilize a class of proteins with an evolutionary history of use as minor constituents of yolk in other insects.
Title: Cloning and characterization of three Musca domestica yolk protein genes White NM, Bownes M Ref: Insect Molecular Biology, 6:329, 1997 : PubMed
The yolk protein (yp) genes encode the major nutritional polypeptides deposited in developing oocytes for subsequent utilization during embryogenesis, and represent a highly conserved family of genes in higher Diptera. Originally isolated from Drosophila melanogaster, they are expressed in a temporal-, tissue- and sex-specific manner in all species in which they have been identified. We report here the isolation of cDNAs encoding three independent yolk proteins from the common housefly, Musca domestica. Expression of the three M. domestica yp genes is analysed both by Northern and in-situ hybridization. We discuss in an evolutionary context both the significance of the expression patterns, and regions of apparent polypeptide sequence divergence.
Title: Why is there sequence similarity between insect yolk proteins and vertebrate lipases? Bownes M Ref: J Lipid Res, 33:777, 1992 : PubMed
The major proteins stored in the yolk of developing oocytes are thought to provide a nutritional store for utilization during embryogenesis. They seem to fall into two major families of proteins. The first are called vitellogenins and are found in frog, chicken, nematode, fish, and some insects such as the boll weevil. The other group are called yolk proteins and are found in dipteran insects such as fruitfly, housefly, fleshfly, and blue-bottles. Both groups are the major proteins found in the oocyte and are female-specific proteins endocytosed from the serum or hemolymph. The yolk protein group were found to have sequence similarity to the triacylglycerol lipases and lipoprotein lipases of vertebrates, including rat, pig, and human. The yolk proteins do not have lipase activity, but the sequences conserved between yolk proteins and lipases surround the active site where there are interactions with lipids. The likely reason for the presence of this domain in the yolk proteins is to bind a steroid hormone in a storage form conjugated to lipids. This permits the storage of the hormone in an inactive form until the yolk proteins are degraded, when it can be released from its conjugate to induce developmental decisions in embryogenesis. They may also transport lipids into the oocyte for use in embryogenesis. Whilst the vitellogenin family of proteins do not share this homology with the lipases they do have similarity to the human serum protein, apolipoprotein B, which also has a role in binding lipids. These findings are discussed in relation to the evolution and functions of lipases, apolipoproteins, vitellogenins, and yolk proteins. Experiments aimed at isolating genes encoding lipases in insects and at further elucidating the function of the yolk proteins are suggested.
List of Gene_Locus for Avian-virus_vlip (7)
In addition to causing allergic reactions, vPLA1 can hydrolyze the sn-1 fatty acids in phospholipids and convert them into their corresponding lyso compounds. vPLA1 may disrupt the phospholipid packing of biological membranes, causing severe hemolysis and leading to cardiac dysfunction and death in animals