scholarly journals An esterase from Escherichia coli with a sequence similarity to hormone-sensitive lipase

1998 ◽  
Vol 332 (1) ◽  
pp. 75-80 ◽  
Author(s):  
Shigenori KANAYA ◽  
Tomoyoshi KOYANAGI ◽  
Eiko KANAYA
Keyword(s):  
Amino Acid ◽  
Chain Length ◽  
Sequence Similarity ◽  
Acyl Chain ◽  
Hydrolytic Activity ◽  
Catalytic Triad ◽  
Enzymic Activity ◽  
Hormone Sensitive Lipase ◽  
Acyl Chain Length ◽  
Hormone Sensitive

An esterase from Escherichia colithat is a member of the hormone-sensitive lipase (HSL) family was overproduced, purified and characterized. It is encoded by the ybaCgene and composed of 319 amino acid residues with an Mr of 36038. The enzymic activity was determined by using various p-nitrophenyl esters of fatty acids as a substrate at 25 °C and pH 7.1. The enzyme showed hydrolytic activity towards substrates with an acyl chain length of less than 8, whereas it showed little hydrolytic activity towards those with an acyl chain length of more than 10. In addition, it showed little hydrolytic activity towards trioleoylglycerol and cholesterol oleate. Determination of the kinetic parameters for the hydrolyses of the substrates from C2 to C8 indicates that C4 and C5 substrates are the most preferred. Close agreement between the Mr determined by SDS/PAGE (37000) and column chromatography (38000) suggests that the enzyme exists in a monomeric form. It is an acidic protein with a pI value of 4.1. The far-UV CD spectrum suggests that its helical content is 26.1%. Comparison of the amino acid sequence of this enzyme with those involved in the HSL family allows us to propose that Ser165, Asp262 and His292 constitute the catalytic triad of E. coliesterase.

scholarly journals New Thermophilic and Thermostable Esterase with Sequence Similarity to the Hormone-Sensitive Lipase Family, Cloned from a Metagenomic Library

2005 ◽  
Vol 71 (2) ◽  
pp. 817-825 ◽  
Author(s):  
Jin-Kyu Rhee ◽  
Dae-Gyun Ahn ◽  
Yeon-Gu Kim ◽  
Jong-Won Oh
Keyword(s):  
Amino Acid ◽  
Lipolytic Activity ◽  
Sequence Similarity ◽  
Acyl Chain ◽  
Metagenomic Library ◽  
Environmental Dna ◽  
Hormone Sensitive Lipase ◽  
Functional Screening ◽  
E Coli ◽  
Hormone Sensitive

ABSTRACT A gene coding for a thermostable esterase was isolated by functional screening of Escherichia coli cells that had been transformed with fosmid environmental DNA libraries constructed with metagenomes from thermal environmental samples. The gene conferring esterase activity on E. coli grown on tributyrin agar was composed of 936 bp, corresponding to 311 amino acid residues with a molecular mass of 34 kDa. The enzyme showed significant amino acid similarity (64%) to the enzyme from a hyperthermophilic archaeon, Pyrobaculum calidifontis. An amino acid sequence comparison with other esterases and lipases revealed that the enzyme should be classified as a new member of the hormone-sensitive lipase family. The recombinant esterase that was overexpressed and purified from E. coli was active above 30°C up to 95°C and had a high thermal stability. It displayed a high degree of activity in a pH range of 5.5 to 7.5, with an optimal pH of approximately 6.0. The best substrate for the enzyme among the p-nitrophenyl esters (C4 to C16) examined was p-nitrophenyl caproate (C6), and no lipolytic activity was observed with esters containing an acyl chain length of longer than 10 carbon atoms, indicating that the enzyme is an esterase and not a lipase.

scholarly journals Alteration of Chain Length Substrate Specificity of Aeromonas caviae R-Enantiomer-Specific Enoyl-Coenzyme A Hydratase through Site-Directed Mutagenesis

2003 ◽  
Vol 69 (8) ◽  
pp. 4830-4836 ◽  
Author(s):  
Takeharu Tsuge ◽  
Tamao Hisano ◽  
Seiichi Taguchi ◽  
Yoshiharu Doi
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Chain Length ◽  
Acyl Chain ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Acyl Chain Length ◽  
Aeromonas Caviae

ABSTRACT Aeromonas caviae R-specific enoyl-coenzyme A (enoyl-CoA) hydratase (PhaJAc) is capable of providing (R)-3-hydroxyacyl-CoA with a chain length of four to six carbon atoms from the fatty acid β-oxidation pathway for polyhydroxyalkanoate (PHA) synthesis. In this study, amino acid substitutions were introduced into PhaJAc by site-directed mutagenesis to investigate the feasibility of altering the specificity for the acyl chain length of the substrate. A crystallographic structure analysis of PhaJAc revealed that Ser-62, Leu-65, and Val-130 define the width and depth of the acyl-chain-binding pocket. Accordingly, we targeted these three residues for amino acid substitution. Nine single-mutation enzymes and two double-mutation enzymes were generated, and their hydratase activities were assayed in vitro by using trans-2-octenoyl-CoA (C8) as a substrate. Three of these mutant enzymes, L65A, L65G, and V130G, exhibited significantly high activities toward octenoyl-CoA than the wild-type enzyme exhibited. PHA formation from dodecanoate (C12) was examined by using the mutated PhaJAc as a monomer supplier in recombinant Escherichia coli LS5218 harboring a PHA synthase gene from Pseudomonas sp. strain 61-3 (phaC1 Ps). When L65A, L65G, or V130G was used individually, increased molar fractions of 3-hydroxyoctanoate (C8) and 3-hydroxydecanoate (C10) units were incorporated into PHA. These results revealed that Leu-65 and Val-130 affect the acyl chain length substrate specificity. Furthermore, comparative kinetic analyses of the wild-type enzyme and the L65A and V130G mutants were performed, and the mechanisms underlying changes in substrate specificity are discussed.

scholarly journals Overexpression and properties of a new thermophilic and thermostable esterase from Bacillus acidocaldarius with sequence similarity to hormone-sensitive lipase subfamily

1998 ◽  
Vol 332 (1) ◽  
pp. 203-212 ◽  
Author(s):  
Giuseppe MANCO ◽  
Elena ADINOLFI ◽  
Francesca M. PISANI ◽  
Gianluca OTTOLINA ◽  
Giacomo CARREA ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Active Form ◽  
Enantiomeric Excess ◽  
Biochemical Properties ◽  
Catalytic Triad ◽  
Hormone Sensitive Lipase ◽  
High Sequence Identity ◽  
Reading Frame ◽  
Hormone Sensitive ◽  
Overexpression System

We previously purified a new esterase from the thermoacidophilic eubacterium Bacillus acidocaldarius whose N-terminal sequence corresponds to an open reading frame (ORF3) reported to show homology with the mammalian hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. To compare the biochemical properties of this thermophilic enzyme with those of the homologous mesophilic and psychrophilic members of the HSL group, an overexpression system in Escherichia coli was established. The protein, expressed in soluble and active form at 10 mg/l E. coli culture, was purified to homogeneity and characterized biochemically. The enzyme, a 34 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-exanoate with Km and kcat values of 11±2 µM (mean±S.D., n = 3) and 6610±880 s-1 (mean±S.D., n = 3) respectively at 70 °C and pH 7.1. In spite of relatively high sequence identity with the mammalian HSLs, the psychrophilic MoraxellaTA144lipase 2 and the human liver arylacetamide deacetylase, no lipase or amidase activity was detected. A series of substrates were tested for enantioselectivity. Substantial enantioselectivity was observed only in the resolution of (±)-3-bromo-5-(hydroxymethyl)-Δ2-isoxazoline, where the (R)-product was obtained with an 84% enantiomeric excess at 36% conversion. The enzyme was also able to synthesize acetyl esters when tested in vinyl acetate and toluene. Inactivation by diethylpyrocarbonate, diethyl-p-nitrophenyl phosphate, di-isopropylphosphofluoridate (DFP) and physostigmine, as well as labelling with [3H]DFP, supported our previous suggestion of a catalytic triad made up of Ser-His-Asp. The activity–stability–temperature relationship is discussed in relation to those of the homologous members of the HSL group.

Synthesis of fluorescent C24-ceramide: Evidence for acyl chain length dependent differences in penetration of exogenous NBD-ceramides into human skin

2009 ◽  
Vol 19 (24) ◽  
pp. 6975-6977 ◽  
Author(s):  
Jakub Novotný ◽  
Kateřina Pospěchová ◽  
Alexandr Hrabálek ◽  
Robert Čáp ◽  
Kateřina Vávrová
Keyword(s):  
Human Skin ◽  
Chain Length ◽  
Acyl Chain ◽  
Acyl Chain Length
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