Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.1.79 (hormone-sensitive lipase)
 2,163 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

EST2, a thermophilic carboxylesterase from Alicyclobacillus acidocaldarius, belonging to the HSL group of the esterase/lipase superfamily, has been crystallized for the first time. Ammonium sulfate was used as a precipitant and the crystallization proceeded at pH 7.8. The crystals belong to space group P41212 or its enantiomorph P43212, with unit-cell parameters a = b = 78.8, c = 106. 4 A. A complete data set has been collected at the synchrotron source Elettra in Trieste to 2.4 A resolution, using a single frozen crystal.
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PMID:Crystallization and preliminary X-ray diffraction studies of the carboxylesterase EST2 from Alicyclobacillus acidocaldarius. 1039 4

Escherichia coli esterase (EcE) is a member of the hormone-sensitive lipase family. We have analyzed the roles of the conserved residues in this enzyme (His103, Glu128, Gly163, Asp164, Ser165, Gly167, Asp262, Asp266 and His292) by site-directed mutagenesis. Among them, Gly163, Asp164, Ser165, and Gly167 are the components of a G-D/E-S-A-G motif. We showed that Ser165, Asp262, and His292 are the active-site residues of the enzyme. We also showed that none of the other residues, except for Asp164, is critical for the enzymatic activity. The mutation of Asp164 to Ala dramatically reduced the catalytic efficiency of the enzyme by the factor of 10(4) without seriously affecting the substrate binding. This residue is probably structurally important to make the conformation of the active-site functional.
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PMID:Identification of catalytically essential residues in Escherichia coli esterase by site-directed mutagenesis. 1043 19

The moderate thermophilic eubacterium Alicyclobacillus (formerly Bacillus) acidocaldarius expresses a thermostable carboxylesterase (esterase 2) belonging to the hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. Based on secondary structures predictions and a secondary structure-driven multiple sequence alignment with remote homologous protein of known three-dimensional (3D) structure, we previously hypothesized for this enzyme the alpha/beta-hydrolase fold typical of several lipases and esterases and identified Ser155, Asp252, and His282 as the putative members of the catalytic triad. In this paper we report the construction of a 3D model for this enzyme based on the structure of mouse acetylcholinesterase complexed with fasciculin. The model reveals the topological organization of the fold corroborating our predictions. As regarding the active-site residues, Ser155, Asp252, and His282 are located close to each other at hydrogen bond distances. Their catalytic role was here probed by biochemical and mutagenic studies. Moreover, on the basis of the secondary structure-driven multiple sequence alignment and the 3D structural model, a residue supposed important for catalysis, Gly84, was mutated to Ser. The activity of the mutated enzyme was drastically reduced. We propose that Gly84 is part of a putative "oxyanion hole" involved in the stabilization of the transition state similar to the C group of the esterase/lipase family.
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PMID:Homology modeling and active-site residues probing of the thermophilic Alicyclobacillus acidocaldarius esterase 2. 1049 80

The gene encoding an esterase (HDE) was cloned from an oil-degrading bacterium, strain HD-1. HDE is a member of the hormone-sensitive lipase family and composed of 317 amino acid residues with a molecular weight of 33,633. The HDE-encoding gene was expressed in Escherichia coli, and the recombinant protein was purified and characterized. Amino acid sequence analysis indicated that the methionine residue was removed from its NH(2)-terminus. The good agreement of the molecular weights estimated by SDS-PAGE (35,000) and gel filtration (38,000) suggests that it acts in a monomeric form. HDE showed hydrolytic activity towards p-nitrophenyl esters of fatty acids with an acyl chain length of 2 to 14 and tributyrin, whereas it showed little hydrolytic activity towards p-nitrophenyl oleate (C(18)), tricaprylin and triolein. Determination of the kinetic parameters for the hydrolyses of the p-nitrophenyl substrates from C(2) to C(14) indicated that HDE shows a relatively broad substrate specificity. However, comparison of the k(cat)/K(m) values indicated that the C(10)-C(14) substrates are the most preferred ones. Such a preference for substrates with long acyl chains may be a characteristic of HDE.
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PMID:Identification of the gene encoding esterase, a homolog of hormone-sensitive lipase, from an oil-degrading bacterium, strain HD-1. 1050 82

A new esterase gene from the hyperthermophilic archaeon Archaeoglobus fulgidus, reported to show homology with the mammalian hormone-sensitive lipase (HSL)-like group of the esterase/lipase family, was cloned by means of the polymerase chain reaction from the A. fulgidus genome. In order to compare the biochemical properties of this putative hyperthermophilic enzyme with those of the homologous, thermophilic member of HSL group, namely Alicyclobacillus (formerly Bacillus) acidocaldarius esterase 2 (EST2), an overexpression system in Escherichia coli was established. The recombinant protein, expressed in soluble and active form at 20 mg/liter of E. coli culture, was purified to homogeneity and characterized. The enzyme, a 35.5-kDa monomeric protein, was demonstrated to be a B"-type carboxylesterase (EC 3.1.1.1) on the basis of substrate specificity and the action of inhibitors. Among the p-nitrophenyl (PNP) esters tested the best substrate was PNP-hexanoate with K(m) and k(cat) values of 11 +/- 3 microM (mean +/- SD, n = 3) and 1014 +/- 38 s(-1) (mean +/- SD, n = 3), respectively, at 70 degrees C and pH 7.1. Inactivation by diethylpyrocarbonate, phenylmethylsulfonylfluoride, diisopropylfosfofluoridate (DFP), and physostigmine, as well as labeling with [(3)H]DFP, supported our previous suggestion of a catalytic triad made up of Ser(160)-His(285)-Asp(255). The sequence identity with the thermostable A. acidocaldarius EST2 was 42.5%. The enzyme proved to be much more stable than its Alicyclobacillus counterpart. The conformational dynamics of the two proteins were investigated by frequency-domain fluorometry and anisotropy decay and the activity/stability/temperature relationship was discussed.
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PMID:Cloning, overexpression, and properties of a new thermophilic and thermostable esterase with sequence similarity to hormone-sensitive lipase subfamily from the archaeon Archaeoglobus fulgidus. 1062 Mar 37

The hyperthermophilic Archaeon Archaeoglobus fulgidus has a gene (AF1763) which encodes a thermostable carboxylesterase belonging to the hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. Based on secondary structure predictions and a secondary structure-driven multiple sequence alignment with remote homologous proteins of known three-dimensional structure, we previously hypothesized for this enzyme the alpha/beta-hydrolase fold typical of several lipases and esterases and identified Ser160, Asp 255 and His285 as the putative members of the catalytic triad. In this paper we report the building of a 3D model for this enzyme based on the structure of the homologous brefeldin A esterase from Bacillus subtilis whose structure has been recently elucidated. The model reveals the topological organization of the fold corroborating our predictions. As regarding the active-site residues, Ser160, Asp255 and His285 are located close each other at hydrogen bond distances. The catalytic role of Ser160 as the nucleophilic member of the triad is demonstrated by the [(3)H]diisopropylphosphofluoridate (DFP) active-site labeling and sequencing of a radioactive peptide containing the signature sequence GDSAGG.
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PMID:Homology modeling and identification of serine 160 as nucleophile of the active site in a thermostable carboxylesterase from the archaeon Archaeoglobus fulgidus. 1077 61

EST2 is a novel thermophilic carboxylesterase, isolated and cloned from Alicyclobacillus (formerly Bacillus) acidocaldarius, which optimally hydrolyses esters with acyl chain lengths of six to eight carbon atoms at 70 degrees C. On the basis of the amino acid sequence homology, it has been classified as a member of the mammalian hormone-sensitive lipase (HSL) subfamily. The crystal structure of EST2, complexed with a sulphonyl derivative, has been determined at 2.6 A resolution by a multiple wavelength anomalous diffraction experiment on a seleno-methionine derivative. EST2 presents a canonical alpha/beta hydrolase core, shielded at the C-terminal side by a cap region built up of five helices. It contains the lipase-like catalytic triad, Ser155, His282 and Asp252, whereby the nucleophile is covalently modified. This allows an unambiguous view of the putative active site of EST2, detecting the oxyanion hole, in whose formation the amino acid sequence motif His81-Gly82-Gly83-Gly84 is involved, and the hydrophobic binding pocket for the acyl chain. The structural model here reported provides the first example of a transition state analogue of an esterase/lipase belonging to the HSL group, thus affording useful information for the design of medical inhibitors. Moreover, as the first X-ray structure of a thermophilic carboxylesterase, the comparison with its mesophilic homologue, the Brefeldin A esterase (BFAE) from Bacillus subtilis, allows the identification of putative determinants of thermal stability.
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PMID:A snapshot of a transition state analogue of a novel thermophilic esterase belonging to the subfamily of mammalian hormone-sensitive lipase. 1106 74

We have found that the hyperthermophilic archaeon Pyrobaculum calidifontis VA1 produced a thermostable esterase. We isolated and sequenced the esterase gene (est(Pc)) from strain VA1. est(Pc) consisted of 939 bp, corresponding to 313 amino acid residues with a molecular mass of 34,354 Da. As est(Pc) showed significant identity (30%) to mammalian hormone-sensitive lipases (HSLs), esterase of P. calidifontis (Est) could be regarded as a new member of the HSL family. Activity levels of the enzyme were comparable or higher than those of previously reported enzymes not only at high temperature (6,410 U/mg at 90 degrees C), but also at ambient temperature (1,050 U/mg at 30 degrees C). The enzyme displayed extremely high thermostability and was also stable after incubation with various water-miscible organic solvents at a concentration of 80%. The enzyme also exhibited activity in the presence of organic solvents. Est of P. calidifontis showed higher hydrolytic activity towards esters with short to medium chains, with p-nitrophenyl caproate (C(6)) the best substrate among the p-nitrophenyl esters examined. As for the alcoholic moiety, the enzyme displayed esterase activity towards esters with both straight- and branched-chain alcohols. Most surprisingly, we found that this Est enzyme hydrolyzed the tertiary alcohol ester tert-butyl acetate, a feature very rare among previously reported lipolytic enzymes. The extreme stability against heat and organic solvents, along with its activity towards a tertiary alcohol ester, indicates a high potential for the Est of P. calidifontis in future applications.
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PMID:Extremely stable and versatile carboxylesterase from a hyperthermophilic archaeon. 1214 92

Aes, a 36-kDa acetylesterase from Escherichia coli, belongs to the hormone-sensitive lipase family, and it is involved in the regulation of MalT, the transcriptional activator of the maltose regulon. The activity of MalT is depressed through a direct protein-protein interaction with Aes. Although the effect is clear-cut, the meaning of this interaction and the conditions that trigger it still remain elusive. To perform a comparative thermodynamic study between the mesophilic Aes protein and two homologous thermostable enzymes, Aes was overexpressed in E. coli and purified. At the last step of the purification procedure the enzyme was eluted from a Mono Q HR 5/5 column as a major form migrating, anomalously, at 56 kDa on a calibrated Superdex 75 column. A minor peak that contains the Aes protein and a polypeptide of 50 kDa was also detected. By a combined analysis of size-exclusion chromatography and surface-enhanced laser desorption ionization-time of flight mass spectrometry, it was possible to demonstrate the presence in this peak of a stable 87-kDa complex, containing the Aes protein itself and the 50-kDa polypeptide in a 1:1 ratio. The homodimeric molecular species of Aes and of the 50-kDa polypeptide were also detected. The esterase activity associated with the 87-kDa complex, when assayed with p-nitrophenyl butanoate as substrate, proved 6-fold higher than the activity of the major Aes form of 56 kDa. Amino-terminal sequencing highlighted that the 50-kDa partner of Aes in the complex was the alpha-galactosidase from E. coli. The E. coli cells harboring plasmid pT7-SCII-aes and, therefore, expressing Aes were hampered in their growth on a minimal medium containing raffinose as a sole carbon source. Because alpha-galactosidase is involved in the metabolism of raffinose, the above findings suggest a potential role of Aes in the regulation of carbohydrate metabolism in E. coli.
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PMID:The Aes protein and the monomeric alpha-galactosidase from Escherichia coli form a non-covalent complex. Implications for the regulation of carbohydrate metabolism. 1237 3

The acetyl-esterase Aes from Escherichia coli, which belongs to the HSL group of the esterase/lipase superfamily, has been crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 8000 as a precipitant and magnesium chloride as an additive. Crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 110.0, b = 190.6, c = 218.6 A. A complete data set has been collected to 2.5 A resolution at the Elettra synchrotron source, Trieste using a single frozen crystal. Packing density considerations agree with 10-16 monomers in the asymmetric unit, with a corresponding solvent content of 61-38%.
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PMID:Crystallization and preliminary X-ray diffraction studies of Aes acetyl-esterase from Escherichia coli. 1450 Nov 34


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