PIRSF017388 Members of this group are esterases, lipases, and (possibly) other hydrolases of the alpha/beta hydrolase fold. The Bacillus stearothermophilus member, Est, has been characterized as a carboxylesterase (EC 3.1.1.1) involved in the detoxification of xenobiotics, and the Bacillus sp. member has been characterized as monoacylglycerol lipase. This family correspond to family XIII of the classification of Arpigny and Jaeger 1999. Family XIII was introduced by Ewis et al.(2004) and Liu et al. (2004). The closest family is Monoglyceridelipase_lysophospholip. The family XIII contains LipS Chow et al.. This family is composed of two sub families with different set of salt-bridges important for thermostability Charbonneau et al. (due to simultaneous publication of new families this family was numbered XV but really is a subset of familly XIII. See notice of this paper in Plos One)
9 moreTitle: A novel alkaliphilic bacillus esterase belongs to the 13(th) bacterial lipolytic enzyme family Rao L, Xue Y, Zheng Y, Lu JR, Ma Y Ref: PLoS ONE, 8:e60645, 2013 : PubMed
BACKGROUND: Microbial derived lipolytic hydrolysts are an important class of biocatalysts because of their huge abundance and ability to display bioactivities under extreme conditions. In spite of recent advances, our understanding of these enzymes remains rudimentary. The aim of our research is to advance our understanding by seeking for more unusual lipid hydrolysts and revealing their molecular structure and bioactivities. METHODOLOGYPRINCIPAL FINDINGS: Bacillus. pseudofirmus OF4 is an extreme alkaliphile with tolerance of pH up to 11. In this work we successfully undertook a heterologous expression of a gene estof4 from the alkaliphilic B. pseudofirmus sp OF4. The recombinant protein called EstOF4 was purified into a homologous product by Ni-NTA affinity and gel filtration. The purified EstOF4 was active as dimer with the molecular weight of 64 KDa. It hydrolyzed a wide range of substrates including p-nitrophenyl esters (C2-C12) and triglycerides (C2-C6). Its optimal performance occurred at pH 8.5 and 50 degrees C towards p-nitrophenyl caproate and triacetin. Sequence alignment revealed that EstOF4 shared 71% identity to esterase Est30 from Geobacillus stearothermophilus with a typical lipase pentapeptide motif G91LS93LG95. A structural model developed from homology modeling revealed that EstOF4 possessed a typical esterase 6alpha/7beta hydrolase fold and a cap domain. Site-directed mutagenesis and inhibition studies confirmed the putative catalytic triad Ser93, Asp190 and His220. CONCLUSION: EstOF4 is a new bacterial esterase with a preference to short chain ester substrates. With a high sequence identity towards esterase Est30 and several others, EstOF4 was classified into the same bacterial lipolytic family, Family XIII. All the members in this family originate from the same bacterial genus, bacillus and display optimal activities from neutral pH to alkaline conditions with short and middle chain length substrates. However, with roughly 70% sequence identity, these enzymes showed hugely different thermal stabilities, indicating their diverse thermal adaptations via just changing a few amino acid residues.
Monoacylglycerol lipases (MGLs) play an important role in lipid catabolism across all kingdoms of life by catalyzing the release of free fatty acids from monoacylglycerols. The three-dimensional structures of human and a bacterial MGL were determined only recently as the first members of this lipase family. In addition to the alpha/beta-hydrolase core, they showed unexpected structural similarities even in the cap region. Nevertheless, the structural basis for substrate binding and conformational changes of MGLs is poorly understood. Here, we present a comprehensive study of five crystal structures of MGL from Bacillus sp. H257 in its free form and in complex with different substrate analogs and the natural substrate 1-lauroylglycerol. The occurrence of different conformations reveals a high degree of conformational plasticity of the cap region. We identify a specific residue, Ile-145, that might act as a gatekeeper restricting access to the binding site. Site-directed mutagenesis of Ile-145 leads to significantly reduced hydrolase activity. Bacterial MGLs in complex with 1-lauroylglycerol, myristoyl, palmitoyl, and stearoyl substrate analogs enable identification of the binding sites for the alkyl chain and the glycerol moiety of the natural ligand. They also provide snapshots of the hydrolytic reaction of a bacterial MGL at different stages. The alkyl chains are buried in a hydrophobic tunnel in an extended conformation. Binding of the glycerol moiety is mediated via Glu-156 and water molecules. Analysis of the structural features responsible for cap plasticity and the binding modes of the ligands suggests conservation of these features also in human MGL.
        
Title: Molecular cloning and characterization of two thermostable carboxyl esterases from Geobacillus stearothermophilus Ewis HE, Abdelal AT, Lu CD Ref: Gene, 329:187, 2004 : PubMed
Screening of the genomic libraries of Geobacillus stearothermophilus ATCC12980 and ATCC7954 for esterase/lipase activity led to the isolation of two positive clones. The results of subclonings and sequence analyses identified two genes, est30 and est55, encoding two different carboxylesterases, and genetic rearrangement in the est55 locus was revealed from genomic comparison. The est30 gene encodes a polypeptide of 248 amino acids with a calculated molecular mass of 28338 Da, and the est55 gene encodes a polypeptide of 499 amino acids with a calculated molecular mass of 54867 Da. Both enzymes were purified to near homogeneity from recombinant strains of Escherichia coli. The results of enzyme characterization showed that while both enzymes possess optimal activities with short chain acyl derivatives, Est55 has a broader pH tolerance (pH 8-9) and optimal temperature range (30-60 degrees C) than Est30. The activation energy of Est55 (35.7 kJ/mol) was found to be significantly lower than that of Est30 (101.9 kJ/mol). Both enzymes were stable at 60 degrees C for more than 2 h; at 70 degrees C, the half-life for thermal inactivation was 40 and 180 min for Est55 and Est30, respectively. With p-nitrophenyl caproate as the substrate and assayed at 60 degrees C, Est55 had K(m) and k(cat) values of 0.5 microM and 39758 s(-1) while Est30 exhibited values of 2.16 microM and 38 s(-1). Inhibition studies indicated that both Est30 and Est55 were strongly inhibited by phenylmethanesulfonyl fluoride, p-hydroxymercuribenzoate, and tosyl-l-phenylalanine, consistent with the proposed presence of Ser-His-Glu catalytic triad of the alpha/beta hydrolase family. The enzymatic properties of Est30 and Est55 reported here warrant the potential applications of these enzymes in biotechnological industries.
        
9 lessTitle: Role of key salt bridges in thermostability of G. thermodenitrificans EstGtA2: distinctive patterns within the new bacterial lipolytic enzyme family XV Charbonneau DM, Beauregard M Ref: PLoS ONE, 8:e76675, 2013 : PubMed
Bacterial lipolytic enzymes were originally classified into eight different families defined by Arpigny and Jaeger (families I-VIII). Recently, the discovery of new lipolytic enzymes allowed for extending the original classification to fourteen families (I-XIV). We previously reported that G. thermodenitrificans EstGtA2 (access no. AEN92268) belonged to a novel group of bacterial lipolytic enzymes. Here we propose a 15(th) family (family XV) and suggest criteria for the assignation of protein sequences to the N' subfamily. Five selected salt bridges, hallmarks of the N' subfamily (E3/R54, E12/R37, E66/R140, D124/K178 and D205/R220) were disrupted in EstGtA2 using a combinatorial alanine-scanning approach. A set of 14 (R/K-->A) mutants was produced, including five single, three double, three triple and three quadruple mutants. Despite a high tolerance to non-conservative mutations for folding, all the alanine substitutions were destabilizing (decreasing T m by 5 to 14 degrees C). A particular combination of four substitutions exceeded this tolerance and prevents the correct folding of EstGtA2, leading to enzyme inactivation. Although other mutants remain active at low temperatures, the accumulation of more than two mutations had a dramatic impact on EstGtA2 activity at high temperatures suggesting an important role of these conserved salt bridge-forming residues in thermostability of lipolytic enzymes from the N' subfamily. We also identified a particular interloop salt bridge in EstGtA2 (D194/H222), located at position i -2 and i -4 residues from the catalytic Asp and His respectively which is conserved in other related bacterial lipolytic enzymes (families IV and XIII) with high tolerance to mutations and charge reversal. We investigated the role of residue identity at position 222 in controlling stability-pH dependence in EstGtA2. The introduction of a His to Arg mutation led to increase thermostability under alkaline pH. Our results suggest primary targets for optimization of EstGtA2 for specific biotechnological purposes.
        
Title: A novel alkaliphilic bacillus esterase belongs to the 13(th) bacterial lipolytic enzyme family Rao L, Xue Y, Zheng Y, Lu JR, Ma Y Ref: PLoS ONE, 8:e60645, 2013 : PubMed
BACKGROUND: Microbial derived lipolytic hydrolysts are an important class of biocatalysts because of their huge abundance and ability to display bioactivities under extreme conditions. In spite of recent advances, our understanding of these enzymes remains rudimentary. The aim of our research is to advance our understanding by seeking for more unusual lipid hydrolysts and revealing their molecular structure and bioactivities. METHODOLOGYPRINCIPAL FINDINGS: Bacillus. pseudofirmus OF4 is an extreme alkaliphile with tolerance of pH up to 11. In this work we successfully undertook a heterologous expression of a gene estof4 from the alkaliphilic B. pseudofirmus sp OF4. The recombinant protein called EstOF4 was purified into a homologous product by Ni-NTA affinity and gel filtration. The purified EstOF4 was active as dimer with the molecular weight of 64 KDa. It hydrolyzed a wide range of substrates including p-nitrophenyl esters (C2-C12) and triglycerides (C2-C6). Its optimal performance occurred at pH 8.5 and 50 degrees C towards p-nitrophenyl caproate and triacetin. Sequence alignment revealed that EstOF4 shared 71% identity to esterase Est30 from Geobacillus stearothermophilus with a typical lipase pentapeptide motif G91LS93LG95. A structural model developed from homology modeling revealed that EstOF4 possessed a typical esterase 6alpha/7beta hydrolase fold and a cap domain. Site-directed mutagenesis and inhibition studies confirmed the putative catalytic triad Ser93, Asp190 and His220. CONCLUSION: EstOF4 is a new bacterial esterase with a preference to short chain ester substrates. With a high sequence identity towards esterase Est30 and several others, EstOF4 was classified into the same bacterial lipolytic family, Family XIII. All the members in this family originate from the same bacterial genus, bacillus and display optimal activities from neutral pH to alkaline conditions with short and middle chain length substrates. However, with roughly 70% sequence identity, these enzymes showed hugely different thermal stabilities, indicating their diverse thermal adaptations via just changing a few amino acid residues.
Monoacylglycerol lipases (MGLs) play an important role in lipid catabolism across all kingdoms of life by catalyzing the release of free fatty acids from monoacylglycerols. The three-dimensional structures of human and a bacterial MGL were determined only recently as the first members of this lipase family. In addition to the alpha/beta-hydrolase core, they showed unexpected structural similarities even in the cap region. Nevertheless, the structural basis for substrate binding and conformational changes of MGLs is poorly understood. Here, we present a comprehensive study of five crystal structures of MGL from Bacillus sp. H257 in its free form and in complex with different substrate analogs and the natural substrate 1-lauroylglycerol. The occurrence of different conformations reveals a high degree of conformational plasticity of the cap region. We identify a specific residue, Ile-145, that might act as a gatekeeper restricting access to the binding site. Site-directed mutagenesis of Ile-145 leads to significantly reduced hydrolase activity. Bacterial MGLs in complex with 1-lauroylglycerol, myristoyl, palmitoyl, and stearoyl substrate analogs enable identification of the binding sites for the alkyl chain and the glycerol moiety of the natural ligand. They also provide snapshots of the hydrolytic reaction of a bacterial MGL at different stages. The alkyl chains are buried in a hydrophobic tunnel in an extended conformation. Binding of the glycerol moiety is mediated via Glu-156 and water molecules. Analysis of the structural features responsible for cap plasticity and the binding modes of the ligands suggests conservation of these features also in human MGL.
Triacylglycerol lipases (EC 3.1.1.3) catalyze both hydrolysis and synthesis reactions with a broad spectrum of substrates rendering them especially suitable for many biotechnological applications. Most lipases used today originate from mesophilic organisms and are susceptible to thermal denaturation whereas only few possess high thermotolerance. Here, we report on the identification and characterization of two novel thermostable bacterial lipases identified by functional metagenomic screenings. Metagenomic libraries were constructed from enrichment cultures maintained at 65 to 75 degrees C and screened resulting in the identification of initially 10 clones with lipolytic activities. Subsequently, two ORFs were identified encoding lipases, LipS and LipT. Comparative sequence analyses suggested that both enzymes are members of novel lipase families. LipS is a 30.2 kDa protein and revealed a half-life of 48 h at 70 degrees C. The lipT gene encoded for a multimeric enzyme with a half-life of 3 h at 70 degrees C. LipS had an optimum temperature at 70 degrees C and LipT at 75 degrees C. Both enzymes catalyzed hydrolysis of long-chain (C(12) and C(14)) fatty acid esters and additionally hydrolyzed a number of industry-relevant substrates. LipS was highly specific for (R)-ibuprofen-phenyl ester with an enantiomeric excess (ee) of 99%. Furthermore, LipS was able to synthesize 1-propyl laurate and 1-tetradecyl myristate at 70 degrees C with rates similar to those of the lipase CalB from Candida antarctica. LipS represents the first example of a thermostable metagenome-derived lipase with significant synthesis activities. Its X-ray structure was solved with a resolution of 1.99 A revealing an unusually compact lid structure.
        
Title: Cloning, expression, purification and preliminary X-ray analysis of a putative metagenome-derived lipase Fersini F, Dall'Antonia Y, Chow J, Streit WR, Mueller-Dieckmann J Ref: Acta Crystallographica Sect F Struct Biol Cryst Commun, 68:923, 2012 : PubMed
LipS is a novel thermostable putative lipase that was isolated from a metagenomic library using functional screening methods. The corresponding gene shows high similarity to that encoding a putative but uncharacterized esterase from Symbiobacterium thermophilum IAM14863 (99% nucleotide-sequence similarity). Two different constructs of the recombinant lipase were crystallized. Crystals belonging to space group P4(2)2(1)2 diffracted X-ray radiation to 2.8 A resolution and crystals belonging to space group P4 diffracted to 2.0 A resolution. The most probable content of their asymmetric units were two molecules (P4(2)2(1)2) and four or five molecules (P4), respectively.
Monoacylglycerol lipases (MGLs) catalyse the hydrolysis of monoacylglycerol into free fatty acid and glycerol. MGLs have been identified throughout all genera of life and have adopted different substrate specificities depending on their physiological role. In humans, MGL plays an integral part in lipid metabolism affecting energy homeostasis, signalling processes and cancer cell progression. In bacteria, MGLs degrade short-chain monoacylglycerols which are otherwise toxic to the organism. We report the crystal structures of MGL from the bacterium Bacillus sp. H257 (bMGL) in its free form at 1.2 and in complex with phenylmethylsulfonyl fluoride at 1.8 resolution. In both structures, bMGL adopts an alpha/beta hydrolase fold with a cap in an open conformation. Access to the active site residues, which were unambiguously identified from the protein structure, is facilitated by two different channels. The larger channel constitutes the highly hydrophobic substrate binding pocket with enough room to accommodate monoacylglycerol. The other channel is rather small and resembles the proposed glycerol exit hole in human MGL. Molecular dynamics simulation of bMGL yielded open and closed states of the entrance channel and the glycerol exit hole. Despite differences in the number of residues, secondary structure elements, and low sequence identity in the cap region, this first structure of a bacterial MGL reveals striking structural conservation of the overall cap architecture in comparison with human MGL. Thus it provides insight into the structural conservation of the cap amongst MGLs throughout evolution and provides a framework for rationalising substrate specificities in each organism.
        
Title: A novel thermostable carboxylesterase from Geobacillus thermodenitrificans: evidence for a new carboxylesterase family Charbonneau DM, Meddeb-Mouelhi F, Beauregard M Ref: J Biochem, 148:299, 2010 : PubMed
A novel gene encoding an esterase from Geobacillus thermodenitrificans strain CMB-A2 was cloned, sequenced and functionally expressed in Escherichia coli M15. Sequence analysis revealed an open reading frame of 747 bp corresponding to a polypeptide of 249 amino acid residues (named EstGtA2). After purification, a specific activity of 2.58 U mg(-1) was detected using p-NP caprylate (C8) at 50 degrees C and pH 8.0 (optimal conditions). The enzyme catalyses the hydrolysis of triglycerides (tributyrin) and a variety of p-nitrophenyl esters with different fatty acyl chain length (C4-C16). The enzyme has potential for various industrial applications since it is characterized by its activity under a wide range of pH, from 25 to 65 degrees C. Using Geobacillus stearothermophilus Est30 esterase structure as template, a model of EstGtA2 was built using ESyPred3D. Analysis of this structural model allowed identifying putative sequence features that control EstGtA2 enzymatic properties. Based on sequence properties, multiple sequence comparisons and phylogenetic analyses, this enzyme appears to belong to a new family of carboxylesterases.
        
Title: Molecular cloning and characterization of two thermostable carboxyl esterases from Geobacillus stearothermophilus Ewis HE, Abdelal AT, Lu CD Ref: Gene, 329:187, 2004 : PubMed
Screening of the genomic libraries of Geobacillus stearothermophilus ATCC12980 and ATCC7954 for esterase/lipase activity led to the isolation of two positive clones. The results of subclonings and sequence analyses identified two genes, est30 and est55, encoding two different carboxylesterases, and genetic rearrangement in the est55 locus was revealed from genomic comparison. The est30 gene encodes a polypeptide of 248 amino acids with a calculated molecular mass of 28338 Da, and the est55 gene encodes a polypeptide of 499 amino acids with a calculated molecular mass of 54867 Da. Both enzymes were purified to near homogeneity from recombinant strains of Escherichia coli. The results of enzyme characterization showed that while both enzymes possess optimal activities with short chain acyl derivatives, Est55 has a broader pH tolerance (pH 8-9) and optimal temperature range (30-60 degrees C) than Est30. The activation energy of Est55 (35.7 kJ/mol) was found to be significantly lower than that of Est30 (101.9 kJ/mol). Both enzymes were stable at 60 degrees C for more than 2 h; at 70 degrees C, the half-life for thermal inactivation was 40 and 180 min for Est55 and Est30, respectively. With p-nitrophenyl caproate as the substrate and assayed at 60 degrees C, Est55 had K(m) and k(cat) values of 0.5 microM and 39758 s(-1) while Est30 exhibited values of 2.16 microM and 38 s(-1). Inhibition studies indicated that both Est30 and Est55 were strongly inhibited by phenylmethanesulfonyl fluoride, p-hydroxymercuribenzoate, and tosyl-l-phenylalanine, consistent with the proposed presence of Ser-His-Glu catalytic triad of the alpha/beta hydrolase family. The enzymatic properties of Est30 and Est55 reported here warrant the potential applications of these enzymes in biotechnological industries.
        
Title: Covalent reaction intermediate revealed in crystal structure of the Geobacillus stearothermophilus carboxylesterase Est30 Liu P, Wang YF, Ewis HE, Abdelal AT, Lu CD, Harrison RW, Weber IT Ref: Journal of Molecular Biology, 342:551, 2004 : PubMed
Est30 is a thermophilic carboxylesterase cloned from Geobacillus stearothermophilus that showed optimal hydrolysis of esters with short acyl chains at 70 degrees C. Est30 is a member of a new family of carboxylesterases with representatives in other Gram-positive bacteria. The crystal structure has been determined at 1.63A resolution using multiple anomalous dispersion data. The two-domain crystal structure showed a large domain with a modified alpha/beta hydrolase core including a seven, rather than an eight-stranded beta sheet, and a smaller cap domain comprising three alpha helices. The catalytic triad consists of residues Ser94, Asp193, and His223. A 100Da tetrahedral ligand was observed to be covalently bound to the side-chain of Ser94. The propyl acetate ligand represents the first tetrahedral intermediate in the reaction mechanism. Therefore, this Est30 crystal structure will help understand the mode of action of all enzymes in the serine hydrolase superfamily.
        
Title: Monoacylglycerol lipase from moderately thermophilic Bacillus sp. strain H-257: molecular cloning, sequencing, and expression in Escherichia coli of the gene Kitaura S, Suzuki K, Imamura S Ref: J Biochem, 129:397, 2001 : PubMed
Monoacylglycerol lipase [MGLP, EC 3.1.1.23] is produced intracellularly by the moderately thermophilic Bacillus sp. strain H-257. The gene encoding MGLP was cloned, sequenced, and expressed in Escherichia coli. A genomic library of Bacillus sp. strain H-257, prepared in the plasmid vector pACYC184, was screened with a 0.2-kbp DNA fragment amplified by the polymerase chain reaction (PCR) with oligonucleotide primers designed based on the amino acid sequence of a purified MGLP. The plasmid pMGLP31, identified by hybridization with the amplified DNA fragment, contained a 5.3-kbp insert from Bacillus sp. strain H-257 DNA. Sequence analysis of the MGLP gene revealed an open reading frame encoding MGLP consisting of 250 amino acids, with a calculated molecular mass of 27.4 kDa. The deduced amino acid sequence of MGLP contained the consensus pentapeptide (-Gly-Xaa-Ser-Xaa-Gly-), which is conserved among lipases, esterases, and serine proteases. The MGLP is homologous to a putative esterase/lipase from Streptomyces coelicolor (41.8% homology). When pMGLP31 was introduced into E. coli DH1, the transformants produced MGLP intracellularly as an active form to an approximately 13.8-fold greater extent than Bacillus sp. strain H-257. The purified recombinant MGLP was shown to be identical to the native enzyme in terms of chromatographic behavior, isoelectric point, and physicochemical and catalytic properties.
        
Title: Purification and characterization of a monoacylglycerol lipase from the moderately thermophilic Bacillus sp. H-257 Imamura S, Kitaura S Ref: J Biochem, 127:419, 2000 : PubMed
A thermostable monoacylglycerol lipase [MGLP, EC 3.1.1.23] was purified for the first time from a cell-free extract of the moderately thermophilic Bacillus sp. H-257. The enzyme was purified 3,028-fold to homogeneity by chromatography using Octyl-Sepharose CL-4B, Q-Sepharose FF, and Superose 12 columns. The molecular mass of the MGLP was estimated to be 25 kDa by gel filtration and 24 kDa by SDS-PAGE, suggesting a monomeric protein. The isoelectric point was determined to be 4.66 by isoelectric focusing. The MGLP retained its full activity upon incubation at 60 degrees C for 10 min (pH 7. 3), and was stable at pH 7-10. The optimal temperature for activity at pH 7.5 was 75 degrees C, and the maximum activity was observed from pH 6-8. This enzyme hydrolyzes monoacylglycerols, with the highest activity occurring with 1-monolauroylglycerol. Di- and triacylglycerols, on the other hand, are essentially inert as substrates for the enzyme. The K(m) values for the hydrolysis of 1-monolauroylglycerol, 1-monooleoylglycerol, and 2-monooleoylglycerol were determined to be 140, 83 and 59 mM, respectively. The enzyme was not inhibited by cholate, but was slightly inhibited by Triton X-100 and deoxycholate. The amino acid sequence of the N-terminal region of the enzyme (16 residues) was also determined.
        
Title: Molecular cloning and structure of the gene for esterase from a thermophilic bacterium, Bacillus stearothermophilus IFO 12550 Kugimiya W, Otani Y, Hashimoto Y Ref: Biosci Biotechnol Biochem, 56:2074, 1992 : PubMed