Hermoso JA

References (14)

Title : Biochemical and Structural Characterization of a novel thermophilic esterase EstD11 provide catalytic insights for the HSL family - Miguel-Ruano_2021_Comput.Struct.Biotechnol.J_19_1214
Author(s) : Miguel-Ruano V , Rivera I , Rajkovic J , Knapik K , Torrado A , Otero JM , Beneventi E , Becerra M , Sanchez-Costa M , Hidalgo A , Berenguer J , Gonzalez-Siso MI , Cruces J , Rua ML , Hermoso JA
Ref : Comput Struct Biotechnol J , 19 :1214 , 2021
Abstract : A novel esterase, EstD11, has been discovered in a hot spring metagenomic library. It is a thermophilic and thermostable esterase with an optimum temperature of 60C. A detailed substrate preference analysis of EstD11 was done using a library of chromogenic ester substrate that revealed the broad substrate specificity of EstD11 with significant measurable activity against 16 substrates with varied chain length, steric hindrance, aromaticity and flexibility of the linker between the carboxyl and the alcohol moiety of the ester. The tridimensional structures of EstD11 and the inactive mutant have been determined at atomic resolutions. Structural and bioinformatic analysis, confirm that EstD11 belongs to the family IV, the hormone-sensitive lipase (HSL) family, from the alpha/beta-hydrolase superfamily. The canonical alpha/beta-hydrolase domain is completed by a cap domain, composed by two subdomains that can unmask of the active site to allow the substrate to enter. Eight crystallographic complexes were solved with different substrates and reaction products that allowed identification of the hot-spots in the active site underlying the specificity of the protein. Crystallization and/or incubation of EstD11 at high temperature provided unique information on cap dynamics and a first glimpse of enzymatic activity in vivo. Very interestingly, we have discovered a unique Met zipper lining the active site and the cap domains that could be essential in pivotal aspects as thermo-stability and substrate promiscuity in EstD11
ESTHER : Miguel-Ruano_2021_Comput.Struct.Biotechnol.J_19_1214
PubMedSearch : Miguel-Ruano_2021_Comput.Struct.Biotechnol.J_19_1214
PubMedID: 33680362
Gene_locus related to this paper: 9delt-EstD11

Title : Disulfide Engineered Lipase to Enhance the Catalytic Activity: A Structure-Based Approach on BTL2 - Godoy_2019_Int.J.Mol.Sci_20_
Author(s) : Godoy CA , Klett J , Di Geronimo B , Hermoso JA , Guisan JM , Carrasco-Lopez C
Ref : Int J Mol Sci , 20 : , 2019
Abstract : Enhancement, control, and tuning of hydrolytic activity and specificity of lipases are major goals for the industry. Thermoalkaliphilic lipases from the I.5 family, with their native advantages such as high thermostability and tolerance to alkaline pHs, are a target for biotechnological applications. Although several strategies have been applied to increase lipases activity, the enhancement through protein engineering without compromising other capabilities is still elusive. Lipases from the I.5 family suffer a unique and delicate double lid restructuration to transition from a closed and inactive state to their open and enzymatically active conformation. In order to increase the activity of the wild type Geobacillus thermocatenulatus lipase 2 (BTL2) we rationally designed, based on its tridimensional structure, a mutant (ccBTL2) capable of forming a disulfide bond to lock the open state. ccBTL2 was generated replacing A191 and F206 to cysteine residues while both wild type C64 and C295 were mutated to serine. A covalently immobilized ccBTL2 showed a 3.5-fold increment in esterase activity with 0.1% Triton X-100 (2336 IU mg(-1)) and up to 6.0-fold higher with 0.01% CTAB (778 IU mg(-1)), both in the presence of oxidizing sulfhydryl agents, when compared to BTL2. The remarkable and industrially desired features of BTL2 such as optimal alkaliphilic pH and high thermal stability were not affected. The designed disulfide bond also conferred reversibility to the enhancement, as the increment on activity observed for ccBTL2 was controlled by redox pretreatments. MD simulations suggested that the most stable conformation for ccBTL2 (with the disulfide bond formed) was, as we predicted, similar to the open and active conformation of this lipase.
ESTHER : Godoy_2019_Int.J.Mol.Sci_20_
PubMedSearch : Godoy_2019_Int.J.Mol.Sci_20_
PubMedID: 31652673
Gene_locus related to this paper: bactc-lipas

Title : Crystal structures of Ophiostoma piceae sterol esterase: Structural insights into activation mechanism and product release - Gutierrez-Fernandez_2014_J.Struct.Biol_187_215
Author(s) : Gutierrez-Fernandez J , Vaquero ME , Prieto A , Barriuso J , Martinez MJ , Hermoso JA
Ref : J Struct Biol , 187 :215 , 2014
Abstract : Sterol esterases are able to efficiently hydrolyze both sterol esters and triglycerides and to carry out synthesis reactions in the presence of organic solvents. Their high versatility makes them excellent candidates for biotechnological purposes. Sterol esterase from fungus Ophiostoma piceae (OPE) belongs to the family abH03.01 of the Candida rugosa lipase-like proteins. Crystal structures of OPE were solved in this study for the closed and open conformations. Enzyme activation involves a large displacement of the conserved lid, structural rearrangements of loop alpha16-alpha17, and formation of a dimer with a large opening. Three PEG 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. One of them is an internal tunnel, connecting the active center with the outer surface of the enzyme 30A far from the catalytic Ser220. Based on our structural and biochemical results we propose a mechanism by which a great variety of different substrates can be hydrolyzed in OPE paving the way for the construction of new variants to improve the catalytic properties of these enzymes and their biotechnological applications.
ESTHER : Gutierrez-Fernandez_2014_J.Struct.Biol_187_215
PubMedSearch : Gutierrez-Fernandez_2014_J.Struct.Biol_187_215
PubMedID: 25108239
Gene_locus related to this paper: 9pezi-q2tfw1

Title : Promiscuous enantioselective (-)-gamma-lactamase activity in the Pseudomonas fluorescens esterase I - Torres_2012_Org.Biomol.Chem_10_3388
Author(s) : Torres LL , Schliessmann A , Schmidt M , Silva-Martin N , Hermoso JA , Berenguer J , Bornscheuer UT , Hidalgo A
Ref : Org Biomol Chem , 10 :3388 , 2012
Abstract : A promiscuous but very enantioselective (-)-gamma-lactamase activity in the kinetic resolution of the Vince lactam (2-azabicyclo[2.2.1]hept-5-en-3-one) was detected in the Pseudomonas fluorescens esterase I (PFEI). The lactamase activity was increased 200-fold by the introduction of a point mutation and resulted as enantioselective as the Microbacterium sp. enzyme used industrially in this resolution. The structural and mechanistic determinants for the catalytic promiscuity and enantioselectivity were identified by molecular modeling, setting a ground stone to engineer further amidase-related activities from this esterase.
ESTHER : Torres_2012_Org.Biomol.Chem_10_3388
PubMedSearch : Torres_2012_Org.Biomol.Chem_10_3388
PubMedID: 22359066
Gene_locus related to this paper: psefl-este

Title : Semisynthetic peptide-lipase conjugates for improved biotransformations - Romero_2012_Chem.Commun.(Camb)_48_9053
Author(s) : Romero O , Filice M , de Las Rivas B , Carrasco-Lopez C , Klett J , Morreale A , Hermoso JA , Guisan JM , Abian O , Palomo JM
Ref : Chem Commun (Camb) , 48 :9053 , 2012
Abstract : An efficient chemoselective method for the creation of semisynthetic lipases by site-specific incorporation of tailor-made peptides on the lipase-lid site was developed. These new enzymes showed excellent improved specificity and regio- or enantioselectivity in different biotransformations.
ESTHER : Romero_2012_Chem.Commun.(Camb)_48_9053
PubMedSearch : Romero_2012_Chem.Commun.(Camb)_48_9053
PubMedID: 22858661

Title : An esterase from Thermus thermophilus HB27 with hyper-thermoalkalophilic properties: Purification, characterisation and structural modelling - Fucinos_2011_J.Mol.Catal.B.Enzym_70_127
Author(s) : Fucinos P , Pastrana L , Sanroman A , Longo MA , Hermoso JA , Rua ML
Ref : J Mol Catal B Enzym , 70 :127 , 2011
Abstract : A membrane-associated esterase (E34Tt) was detected in Thermus thermophilus HB27. The enzyme was purified to homogeneity in a three-step protocol. Detergent (CHAPS) above the CMC was found to be essential to solubilise the enzyme from cell membranes as well as for maintaining activity and stability. By using mass fingerprinting, peptides were found to share identity with the YP_004875 protein, which was annotated as putative esterase in the genome analysis of T. thermophilus HB27, although experimental evidence was lacking. No homology was detected with any known lipase or esterase. However, a comparison with the high-scored sequences from a BLASTp search identified the consensus sequence for lipases/esterases between amino acids 157 and 161 (Gly-Cys-Ser159-Ala-Gly). Further inhibition assays with E600 confirmed that Ser159 was involved in the catalytic mechanism. The monomeric enzyme had a molecular mass of 34 kDa and exhibited esterase activity with preference for medium chain-length esters (C10). E34Tt was noticeable for its high thermal stability; the optimal reaction temperature was higher than 80 C and the half-life of thermal inactivation at 85C was 135 min, which makes it even more thermostable than some hyperthermophilic esterases. These properties convert E34Tt into a very attractive enzyme for biotechnological purposes. A theoretical structural model was constructed using as template a prolyl oligopeptidase from Sus scrofa, and a putative catalytic triad (Ser159, Glu255 and His293) with high similarity to the template was identified.
ESTHER : Fucinos_2011_J.Mol.Catal.B.Enzym_70_127
PubMedSearch : Fucinos_2011_J.Mol.Catal.B.Enzym_70_127
Gene_locus related to this paper: thet2-q72j75

Title : Activation of bacterial thermoalkalophilic lipases is spurred by dramatic structural rearrangements - Carrasco-Lopez_2009_J.Biol.Chem_284_4365
Author(s) : Carrasco-Lopez C , Godoy C , de Las Rivas B , Fernandez-Lorente G , Palomo JM , Guisan JM , Fernandez-Lafuente R , Martinez-Ripoll M , Hermoso JA
Ref : Journal of Biological Chemistry , 284 :4365 , 2009
Abstract : 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.
ESTHER : Carrasco-Lopez_2009_J.Biol.Chem_284_4365
PubMedSearch : Carrasco-Lopez_2009_J.Biol.Chem_284_4365
PubMedID: 19056729
Gene_locus related to this paper: bactc-lipas

Title : Crystallization and preliminary X-ray diffraction studies of the BTL2 lipase from the extremophilic microorganism Bacillus thermocatenulatus - Carrasco-Lopez_2008_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_64_1043
Author(s) : Carrasco-Lopez C , Godoy C , de Las Rivas B , Fernandez-Lorente G , Palomo JM , Guisan JM , Fernandez-Lafuente R , Martinez-Ripoll M , Hermoso JA
Ref : Acta Crystallographica Sect F Struct Biol Cryst Commun , 64 :1043 , 2008
Abstract : Bacillus thermocatenulatus lipase 2 (BTL2) is a thermoalkalophilic lipase that has been reported as an enantioselective biocatalyst for diverse reactions and that heads a group of enzymes that share high resistance towards many inactivation agents (heat, organic solvents, pH etc.). This makes BTL2 an important research target because of its potential industrial applications. BTL2 was cloned and overexpressed in Escherichia coli, purified and concentrated for crystallization using the sitting-drop vapour-diffusion method at 291 K. Crystals grew from a mixture of 13% MPD and 0.2 M ammonium acetate in 0.05 M sodium citrate pH 5.5-5.6. The crystals, which belonged to the orthorhombic space group I222 with unit-cell parameters a = 73.07, b = 129.08, c = 127.49 A, allowed the collection of an X-ray data set to 2.2 A resolution.
ESTHER : Carrasco-Lopez_2008_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_64_1043
PubMedSearch : Carrasco-Lopez_2008_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_64_1043
PubMedID: 18997337
Gene_locus related to this paper: bactc-lipas

Title : Enhancement of the stability of a prolipase from Rhizopus oryzae toward aldehydes by saturation mutagenesis - Di Lorenzo_2007_Appl.Environ.Microbiol_73_7291
Author(s) : Di Lorenzo M , Hidalgo A , Molina R , Hermoso JA , Pirozzi D , Bornscheuer UT
Ref : Applied Environmental Microbiology , 73 :7291 , 2007
Abstract : A prolipase from Rhizopus oryzae (proROL) was engineered in order to increase its stability toward lipid oxidation products such as aldehydes with the aim of improving its performance in oleochemical industries. Out of 22 amino acid residues (15 Lys and 7 His) prone to react with aldehydes, 6 Lys and all His residues (except for the catalytic histidine) were chosen and subjected to saturation mutagenesis. In order to quickly and reliably identify stability mutants within the resulting libraries, active variants were prescreened by an activity staining method on agar plates. Active mutants were expressed in Escherichia coli Origami in a 96-well microtiterplate format, and a stability test using octanal as a model deactivating agent was performed. The most stable histidine mutant (H201S) conferred a stability increase of 60%, which was further enhanced to 100% by combination with a lysine mutant (H201S/K168I). This increase in stability was also confirmed for other aldehydes. Interestingly, the mutations did not affect specific activity, as this was still similar to the wild-type enzyme.
ESTHER : Di Lorenzo_2007_Appl.Environ.Microbiol_73_7291
PubMedSearch : Di Lorenzo_2007_Appl.Environ.Microbiol_73_7291
PubMedID: 17890336

Title : Probing the determinants of substrate specificity of a feruloyl esterase, AnFaeA, from Aspergillus niger - Faulds_2005_Febs.J_272_4362
Author(s) : Faulds CB , Molina R , Gonzalez R , Husband F , Juge N , Sanz-Aparicio J , Hermoso JA
Ref : Febs J , 272 :4362 , 2005
Abstract : Feruloyl esterases hydrolyse 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 glycoside hydrolases. Here we describe the crystal structure of inactive S133A mutant of type-A feruloyl esterase from Aspergillus niger (AnFaeA) in complex with a feruloylated trisaccharide substrate. Only the ferulic acid moiety of the substrate is visible in the electron density map, showing interactions through its OH and OCH(3) groups with the hydroxyl groups of Tyr80. The importance of aromatic and polar residues in the activity of AnFaeA was also evaluated using site-directed mutagenesis. Four mutant proteins were heterologously expressed in Pichia pastoris, and their kinetic properties determined against methyl esters of ferulic, sinapic, caffeic and p-coumaric acid. The k(cat) of Y80S, Y80V, W260S and W260V was drastically reduced compared to that of the wild-type enzyme. However, the replacement of Tyr80 and Trp260 with smaller residues broadened the substrate specificity of the enzyme, allowing the hydrolysis of methyl caffeate. The role of Tyr80 and Trp260 in AnFaeA are discussed in light of the three-dimensional structure.
ESTHER : Faulds_2005_Febs.J_272_4362
PubMedSearch : Faulds_2005_Febs.J_272_4362
PubMedID: 16128806
Gene_locus related to this paper: aspni-FAEA

Title : The crystal structure of feruloyl esterase A from Aspergillus niger suggests evolutive functional convergence in feruloyl esterase family - Hermoso_2004_J.Mol.Biol_338_495
Author(s) : Hermoso JA , Sanz-Aparicio J , Molina R , Juge N , Gonzalez R , Faulds CB
Ref : Journal of Molecular Biology , 338 :495 , 2004
Abstract : 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.
ESTHER : Hermoso_2004_J.Mol.Biol_338_495
PubMedSearch : Hermoso_2004_J.Mol.Biol_338_495
PubMedID: 15081808
Gene_locus related to this paper: aspni-FAEA

Title : Crystallization of a proteolyzed form of the horse pancreatic lipase-related protein 2: structural basis for the specific detergent requirement - Mancheno_2004_Acta.Crystallogr.D.Biol.Crystallogr_60_2107
Author(s) : Mancheno JM , Jayne S , Kerfelec B , Chapus C , Crenon I , Hermoso JA
Ref : Acta Crystallographica D Biol Crystallogr , 60 :2107 , 2004
Abstract : Horse pancreatic lipase-related proteins PLRP1 and PLRP2 are produced by the pancreas together with pancreatic lipase (PL). Sequence-comparison analyses reveal that the three proteins possess the same two-domain organization: an N-terminal catalytic domain and a C-terminal domain, which in PL is involved in colipase binding. Nevertheless, despite the high level of sequence identity found, they exhibit distinct enzymatic properties. The intrinsic sensitivity of the peptide bond between Ser245 and Thr246 within the flap region of PLRP2 to proteolytic cleavage probably complicates PLRP2 crystallization since, as shown here, this proteolyzed form of PLRP2 is only crystallized after specific detergent stabilization of this region. This has been performed by the hanging-drop vapour-diffusion method at 291 K and exclusively in the presence of N,N-dimethyldecylamine-beta-oxide (DDAO). However, most crystals (>95%) are highly twinned and diffract poorly (to approximately 7-5 A resolution). Diffraction-quality trigonal crystals have unit-cell parameters a = b = 128.4, c = 85.8 A and belong to space group P3(2)21. A 2.9 A native data set was collected at ESRF on beamline ID14-2 with an R(merge) of 12.7%. Preliminary structural analysis provides a structural basis for the specific roles of DDAO.
ESTHER : Mancheno_2004_Acta.Crystallogr.D.Biol.Crystallogr_60_2107
PubMedSearch : Mancheno_2004_Acta.Crystallogr.D.Biol.Crystallogr_60_2107
PubMedID: 15502342
Gene_locus related to this paper: horse-2plrp

Title : Structural insights into the lipase\/esterase behavior in the Candida rugosa lipases family: crystal structure of the lipase 2 isoenzyme at 1.97A resolution - Mancheno_2003_J.Mol.Biol_332_1059
Author(s) : Mancheno JM , Pernas MA , Martinez MJ , Ochoa B , Rua ML , Hermoso JA
Ref : Journal of Molecular Biology , 332 :1059 , 2003
Abstract : The yeast Candida rugosa produces several closely related extracellular lipases that differ in their substrate specificity. Here, we report the crystal structure of the isoenzyme lipase 2 at 1.97A resolution in its closed conformation. Lipase 2 shows a 79.4% amino acid sequence identity with lipase 1 and 82.2% with lipase 3, which makes it relevant to compare these three isoenzymes. Despite this high level of sequence identity, structural comparisons reveal several amino acid changes affecting the flap (residue 69), the substrate-binding pocket (residues 127, 132 and 450) and the mouth of the hydrophobic tunnel (residues 296 and 344), which may be responsible for the different substrate specificity and catalytic properties of this group of enzymes. Also, these comparisons reveal two distinct regions in the hydrophobic tunnel: a phenylalanyl-rich region and an aliphatic-rich region. Whereas this last region is essentially identical in the three isoenzymes, the phenylalanyl content in the first one is specific for each lipase, resulting in a different environment of the catalytic triad residues, which probably tunes finely their lipase/esterase character. The greater structural similarity observed between the monomeric form of lipase 3 and lipase 2 concerning the above-mentioned key residues led us to propose a significant esterase activity for this last protein. This enzymatic activity has been confirmed with biochemical experiments using cholesteryl [1-14C]oleate as substrate. Surprisingly, lipase 2 is a more efficient esterase than lipase 3, showing a twofold specific activity against cholesteryl [1-14C]oleate in our experimental conditions. These results show that subtle amino acid changes within a highly conserved protein fold may produce protein variants endowed with new enzymatic properties.
ESTHER : Mancheno_2003_J.Mol.Biol_332_1059
PubMedSearch : Mancheno_2003_J.Mol.Biol_332_1059
PubMedID: 14499609
Gene_locus related to this paper: canru-2lipa

Title : Crystallization and preliminary X-ray diffraction studies of two different crystal forms of the lipase 2 isoform from the yeast Candida rugosa - Mancheno_2003_Acta.Crystallogr.D.Biol.Crystallogr_59_499
Author(s) : Mancheno JJ , Pernas MM , Rua MM , Hermoso JA
Ref : Acta Crystallographica D Biol Crystallogr , 59 :499 , 2003
Abstract : The yeast Candida rugosa produces several closely related lipases which show a high degree of sequence identity (between 77 and 88% for pairs of proteins). Despite this high sequence identity, they exhibit markedly different substrate specificities, indicating that subtle structural differences may produce significant functional changes. Isoform 2 (lip2) has been crystallized using the hanging-drop vapour-diffusion method at 291 K. Diffraction-quality crystals have been obtained from two different experimental conditions (designated A and B, respectively). Type A crystals belong to space group P1 and have unit-cell parameters a = 62.15, b = 91.14, c = 108.46 A, alpha = 90.78, beta = 106.31, gamma = 86.91 degrees; type B crystals are monoclinic with a nearly hexagonal topology, with unit-cell parameters a = 116.11, b = 225.55, c = 116.06 A, beta = 119.89 degrees, and belong to space group P2(1). Diffraction data were collected to a resolution of 1.97 A at a synchrotron facility from type A crystals and to 2.65 A on an in-house rotating-anode generator from type B crystals. Whereas the triclinic crystal reveals monomeric lip2, the monoclinic crystal contains dimeric lip2.
ESTHER : Mancheno_2003_Acta.Crystallogr.D.Biol.Crystallogr_59_499
PubMedSearch : Mancheno_2003_Acta.Crystallogr.D.Biol.Crystallogr_59_499
PubMedID: 12595707
Gene_locus related to this paper: canru-2lipa