Modification of the enantioselectivity of two homologous thermophilic carboxylesterases from Alicyclobacillus acidocaldarius and Archaeoglobus fulgidus by random mutagenesis and screening

Manco, Giuseppe; Carrea, Giacomo; Giosuè, Elena; Ottolina, Gianluca; Adamo, Giovanna; Rossi, Mosè

doi:10.1007/s00792-001-0261-4

Cited by 24 publications

(14 citation statements)

References 15 publications

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“…Biochemical investigation demonstrated that EST2 optimally hydrolyzes esters with acyl chain length of six or eight carbon atoms because of the small dimension of its acyl-binding pocket (6,8). Kinetic and structural studies allowed us to propose the mechanism depicted in Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Instantaneous 'bursts' of pNP measured after addition of wild type or M211S/R215L enzymes to the assay mixtures. Shown are the readings at 405 nm and at zero time of assay mixtures containing pNP-hexanoate (C 6 ) or pNP-dodecanoate (C 12 ) after the addition of the indicated amounts of wild type (empty circles for C 6 and empty squares for C 12 ) or M211S/R215L (filled circles for C 6 and filled squares for C 12 ). Data were corrected for the pNP release due to the onset of the catalytic activity during the 4 s required to mix the enzyme with substrate and to read the absorption. )…”

Section: Measurement Of All Individual Kinetic Constants For the Est2mentioning

confidence: 99%

“…For some of them a role in lipid catabolism or detoxification has been proposed (4,5). EST2 hydrolyzes monoacyl esters of different acyl chain length, triglycerides of short acyl chain, and different compounds of pharmacological and industrial interest (3,6). The est2 gene has been overexpressed in Escherichia coli, and the protein, a monomeric B-type carboxylesterase of about 34 kDa, was purified and characterized.…”

Section: Esterase 2 (Est2)mentioning

confidence: 99%

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A Substrate-induced Switch in the Reaction Mechanism of a Thermophilic Esterase

Simone

Mandrich

Menchise

et al. 2004

Journal of Biological Chemistry

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The reaction mechanism of the esterase 2 (EST2) from Alicyclobacillus acidocaldarius was studied at the kinetic and structural level to shed light on the mechanism of activity and substrate specificity increase previously observed in its double mutant M211S/R215L. In particular, the values of kinetic constants (k 1 , k ؊1 , k 2 , and k 3 ) along with activation energies (E 1 , E ؊1 , E 2 , and E 3 ) were measured for wild type and mutant enzyme. The previously suggested substrate-induced switch in the reaction mechanism from k cat ‫؍‬ k 3 with a short acyl chain substrate (p-nitrophenyl hexanoate) to k cat ‫؍‬ k 2 with a long acyl chain substrate (p-nitrophenyl dodecanoate) was validated. The inhibition afforded by an irreversible inhibitor (1-hexadecanesulfonyl chloride), structurally related to p-nitrophenyl dodecanoate, was studied by kinetic analysis. Moreover the three-dimensional structure of the double mutant bound to this inhibitor was determined, providing essential information on the enzyme mechanism. In fact, structural analysis explained the observed substrate-induced switch because of an inversion in the binding mode of the long acyl chain derivatives with respect to the acyland alcohol-binding sites. Esterase 2 (EST2)1 from the thermophilic eubacterium Alicyclobacillus acidocaldarius is a thermostable serine hydrolase belonging to the hormone-sensitive lipase family of the esterase/lipase superfamily (1-3). Members of this family are ubiquitous (see the ESTHER database), comprise true lipases and amidases other than carboxylesterases, and are still poorly characterized. For some of them a role in lipid catabolism or detoxification has been proposed (4, 5). EST2 hydrolyzes monoacyl esters of different acyl chain length, triglycerides of short acyl chain, and different compounds of pharmacological and industrial interest (3, 6). The est2 gene has been overexpressed in Escherichia coli, and the protein, a monomeric B-type carboxylesterase of about 34 kDa, was purified and characterized. The enzyme displays an optimal temperature at 70°C and maximal activity with p-nitrophenyl (pNP) esters having an acyl chain of six to eight carbon atoms (3). Biochemical and mutagenic studies have allowed the identification of residues of the catalytic triad and oxyanion hole (7).The EST2 crystal structure was recently solved by our group as a covalently bound Hepes adduct (8), thus making it possible to obtain an unambiguous view of the active site and to detect the binding pockets for the acyl and alcohol chains of the ester substrate. On the basis of these structural data, EST2 variants with preferential specificity toward monoacyl esters with acyl chain length greater than eight carbon atoms were designed and generated by site-directed and saturation mutagenesis (9). M211S and R215L mutants and M211S/R215L double mutant were obtained, all having the desired properties in a mixed propan-2-ol/water medium (9). In particular, a 6-fold increase of the specificity constant of the double mutant for pNP-dodecanoate was o...

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Section: Discussionmentioning

confidence: 99%

Section: Measurement Of All Individual Kinetic Constants For the Est2mentioning

confidence: 99%

Section: Esterase 2 (Est2)mentioning

confidence: 99%

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A Substrate-induced Switch in the Reaction Mechanism of a Thermophilic Esterase

Simone

Mandrich

Menchise

et al. 2004

Journal of Biological Chemistry

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“…However, since the sequence identity was relatively low (<12 %) with other active hydrolases, this provides a novel dl-menthyl benzoate esterase as a starting biocatalyst for directed evolution and rational design, which may provide solutions to remarkably enhance the catalytic activity, enantioselectivity, and/or thermostability [35][36][37][38][39]. All these inspiring results aroused our interest to engineer the enantioselectivity, activity, and operational stability of AbMBH for potential application in menthol industry.…”

Section: Discussionmentioning

confidence: 95%

Cloning and Characterization of an Enantioselective l-Menthyl Benzoate Hydrolase from Acinetobacter sp. ECU2040

Yin

Zheng

et al. 2015

Appl Biochem Biotechnol

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A new esterase gene abmbh, encoding a benzoate hydrolase which can enantioselectively hydrolyze l-menthyl benzoate to l-menthol, was recently identified from the genomic library of a soil isolate Acinetobacter sp. ECU2040. The abmbh gene contains a 1080-bp open reading frame encoding a protein of 360 amino acids with a calculated molecular mass of 40.7 kDa. The corresponding enzyme AbMBH was functionally expressed in Escherichia coli BL21 (DE3), purified, and characterized. The AbMBH displayed the maximum activity towards p-nitrophenyl butyrate at 50 °C, and an optimum pH of 8.5. A K M of 2.6 mM and a k cat of 0.26 s(-1) were observed towards dl-menthyl benzoate. The AbMBH exhibited a moderate enantioselectivity (E = 27.5) towards dl-menthyl benzoate. It can also catalyze the enantioselective hydrolysis of a variety of racemic menthyl esters, including dl-menthyl acetate, dl-menthyl chloroacetate, and dl-menthyl butyrate.

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“…inra.fr/ESTHER/) is the carboxylesterase-2 (EST2) isolated and cloned from the thermophilic eubacterium Alicyclobacillus acidocaldarius [for a review see Mandrich et al 2009]. In a previous study on EST2, it was shown that using organic solvents at low concentrations (4-5 %; v/v), such as acetonitrile (CH 3 CN), dimethylformamide (DMF), dimethylsulphoxide (DMSO), methanol (MeOH), ethylene glycol and diethyl ether, the catalytic activity increases (Mandrich et al 2012) and the presence of solvents affects the enantioselectivity, expressed in a model hydrolytic reaction, using (RS)-p-nitrophenyl-2-chloropropionate, as substrate (Manco et al 2002). The analysis of the kinetic properties has shown that the action of EST2 is restricted to solutions of short acyl chain esters (Manco et al 1998).…”

Section: Introductionmentioning

confidence: 99%

Enlarging the substrate portfolio of the thermophilic esterase EST2 from Alicyclobacillus acidocaldarius

et al. 2015

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The enzymatic regioselective hydrolysis of (a) acetylated mono- to tetrasaccharides of different nature, (b) of acetylated aryl glycosides and (c) of different acetylated nucleosides was studied enlarging the portfolio of substrates that can be employed by the thermophilic esterase EST2 from Alicyclobacillus acidocaldarius. The reactions were optimised to the extent that the amount of enzyme needed was lowered of two orders of magnitude with respect to the previously reported reactions, namely from 4000 to 40 U of enzyme per reaction. New additional solvents were screened and dramatic changes in regioselectivity were observed depending on the amount and type of solvent used. For example, in the presence of 10 % DMF, only two α-D-glucose products 6-OH and 4,6-OH (in a 76:24 ratio) were detected, whereas with 25 % DMF, at least four products of similar amount were observed. This versatility adds specific value to the biocatalyst making possible the design of biocatalytic reactions with different hydrophobic ester substrates. As an additional remarkable example, EST2 catalysed with a good yield and high regioselectivity the hydrolysis of p-nitrophenyl β-D-xylopyranoside triacetate producing only the monoacetylated derivative with acetyl group in 3-O-position, in 2 min. The results with nucleosides as substrates are particularly interesting. The peracetates of 3',5'-di-O-acetylthymidine are converted almost quantitatively (95 %) to the monoacetylated derivative possessing free secondary OH; this regioselectivity is complementary to hydrolysis/alcoholysis reactions catalysed by CAL-B lipase or to other microbial hydrolytic biocatalysts, generally giving products with free primary OH groups. A docking analysis was undertaken with all analysed substrates suggesting a structural interpretation of the results. In most of cases, the best pose of the selected substrate was in line with the observed regioselectivity.

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Modification of the enantioselectivity of two homologous thermophilic carboxylesterases from Alicyclobacillus acidocaldarius and Archaeoglobus fulgidus by random mutagenesis and screening

Cited by 24 publications

References 15 publications

A Substrate-induced Switch in the Reaction Mechanism of a Thermophilic Esterase

A Substrate-induced Switch in the Reaction Mechanism of a Thermophilic Esterase

Cloning and Characterization of an Enantioselective l-Menthyl Benzoate Hydrolase from Acinetobacter sp. ECU2040

Enlarging the substrate portfolio of the thermophilic esterase EST2 from Alicyclobacillus acidocaldarius

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