Quantitative characterization of the nucleophile reactivity in penicillin acylase-catalyzed acyl transfer reactions

Youshko, Maxim I; Chilov, Ghermes G.; Shcherbakova, Tatyana A.; Švedas, Vytas K.

doi:10.1016/s1570-9639(02)00413-2

Cited by 63 publications

(52 citation statements)

References 21 publications

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“…The ratio between the rate of synthesis, v Ps and rate of hydrolysis, v Ph , is an important parameter for evaluating the synthetic performance of PA. Since the initial value of this ratio, (v Ps /v Ph ) ini , or the so-called synthesis/hydrolysis ratio (Youshko et al, 2001(Youshko et al, , 2002a, is dependent both on the kinetic properties of the enzyme and the concentration of the nucleophilic acceptor (i.e. 6-APA), it can be used to compare different enzyme variants.…”

Section: Introductionmentioning

confidence: 99%

Saturation mutagenesis reveals the importance of residues αR145 and αF146 of penicillin acylase in the synthesis of β-lactam antibiotics

Jager

Шаповалова

Jekel

et al. 2008

Journal of Biotechnology

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Saturation mutagenesis reveals the importance of residues alpha R145 and alpha F146 of penicillin acylase in the synthesis of beta-lactam antibiotics Jager, Simon A. W.; Shapovalova, Irina V.; Jekel, Peter A.; Alkema, Wynand B. L.; Svedas, Vytas K.; Janssen, Dick B.; Švedas, Vytas K. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. AbstractPenicillin acylase (PA) from Escherichia coli can catalyze the coupling of an acyl group to penicillin-and cephalosporin-derived ␤-lactam nuclei, a conversion that can be used for the industrial synthesis of ␤-lactam antibiotics. The modest synthetic properties of the wild-type enzyme make it desirable to engineer improved mutants. Analysis of the crystal structure of PA has shown that residues ␣R145 and ␣F146 undergo extensive repositioning upon binding of large ligands to the active site, suggesting that these residues may be good targets for mutagenesis aimed at improving the catalytic performance of PA. Therefore, site-saturation mutagenesis was performed on both positions and a complete set of all 38 variants was subjected to rapid HPLC screening for improved ampicillin synthesis. Not less than 33 mutants showed improved synthesis, indicating the importance of the mutated residues in PA-catalyzed acyl transfer kinetics. In several mutants at low substrate concentrations, the maximum level of ampicillin production was increased up to 1.5-fold, and the ratio of the synthetic rate over the hydrolytic rate was increased 5-15-fold. Moreover, due to increased tendency of the acyl-enzyme intermediate to react with ␤-lactam nucleophile instead of water, mutants ␣R145G, ␣R145S and ␣R145L demonstrated an enhanced synthetic yield over wild-type PA at high substrate concentrations. This was accompanied by an increased conversion of 6-APA to ampicillin as well as a decreased undesirable hydrolysis of the acyl donor. Therefore, these mutants are interesting candidates for the enzymatic production of semi-synthetic ␤-lactam antibiotics.

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Section: Introductionmentioning

confidence: 99%

Saturation mutagenesis reveals the importance of residues αR145 and αF146 of penicillin acylase in the synthesis of β-lactam antibiotics

Jager

Шаповалова

Jekel

et al. 2008

Journal of Biotechnology

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“…All three reactions (synthesis of the antibiotic, hydrolysis of the ester, and hydrolysis of the antibiotic) will occur simultaneously, but after a certain period of time, a maximum in the antibiotic concentration is reached, at which time the reaction is stopped and the product harvested. The kinetic parameters of the enzyme govern the maximum level of antibiotic accumulation P S max in such a process (14). Of crucial importance is the ratio of the specificity constants of the enzyme toward the acyl donor and the antibiotic, ␣, which is defined as (k cat /K m ) product / (k cat /K m ) acyl donor .…”

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confidence: 99%

“…At a certain concentration of nucleophile, this ratio indicates the tendency of the acyl enzyme to undergo aminolysis rather than hydrolysis. For optimal antibiotic yield, V s /V h ini needs to be maximal (14,15). Kinetic work has shown that mutation of Tyr 206 to alanine decreases the unwanted hydrolytic activity toward cephalexin in the Acetobacter enzyme (6).…”

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confidence: 99%

Acetobacter turbidans α-Amino Acid Ester Hydrolase

Barends

Polderman-Tijmes²,

Jekel

et al. 2006

Journal of Biological Chemistry

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The ␣-amino acid ester hydrolase (AEH) from Acetobacter turbidans is a bacterial enzyme catalyzing the hydrolysis and synthesis of ␤-lactam antibiotics. The crystal structures of the native enzyme, both unliganded and in complex with the hydrolysis product D-phenylglycine are reported, as well as the structures of an inactive mutant (S205A) complexed with the substrate ampicillin, and an active site mutant (Y206A) with an increased tendency to catalyze antibiotic production rather than hydrolysis. The structure of the native enzyme shows an acyl binding pocket, in which D-phenylglycine binds, and an additional space that is large enough to accommodate the ␤-lactam moiety of an antibiotic. In the S205A mutant, ampicillin binds in this pocket in a non-productive manner, making extensive contacts with the side chain of Tyr 112 , which also participates in oxyanion hole formation. In the Y206A mutant, the Tyr 112 side chain has moved with its hydroxyl group toward the catalytic serine. Because this changes the properties of the ␤-lactam binding site, this could explain the increased ␤-lactam transferase activity of this mutant.Thirty years ago, several bacterial strains, such as Acetobacter turbidans and Xanthomonas citri, were identified that were able to efficiently produce semi-synthetic ␤-lactam antibiotics from ␤-lactam nuclei produced by fermentation, and synthetic acyl compounds with an ␣-amino group (1). Important antibiotics with such acyl chains include cephalexin, cephadroxil, ampicillin, and amoxicillin. Given the difficulties in preparing such antibiotics by chemical means (2), much effort has been put into harnessing the ␤-lactam antibiotic synthesizing activity of these bacteria for application in the industrial production of antibiotics. It appeared that this activity originated from enzymes preferentially hydrolyzing esters of ␣-amino acids, the ␣-amino acid ester hydrolases (AEHs) 2 (3). Because of its potential usefulness in antibiotic synthesis, the AEH from A. turbidans has been studied extensively, and it was the first of its family for which the gene was cloned and overexpressed (4). The sequence showed a GXSYXG active site motif (4), which is characteristic of serine hydrolases of the X-prolyl dipeptidyl aminopeptidase family (5). Labeling studies with a suicide inhibitor, sequence alignments, and site-directed mutagenesis identified a catalytic triad of Ser 205 , Asp 338 , and His 370 in what was proposed to be a catalytic domain with an ␣/␤-hydrolase fold (6).Recently, the crystal structure of the X. citri AEH was solved (7). This enzyme shares 63% sequence identity with the A. turbidans AEH. The structure showed a tetrameric arrangement of monomers consisting of three domains each: an ␣/␤-hydrolase domain at the N terminus, a helical cap domain, and a C-terminal jellyroll fold domain. The active site indeed contained a Ser-His-Asp catalytic triad, the constituents of which were found in their canonical positions in the ␣/␤-hydrolase domain. Furthermore, a putative oxyanion hole was found i...

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“…Penicillin G acylase (PGA) is used as a catalyst for the enzymatic semisyntheses of several penicillins and cephalosporins (11,(15)(16)(17)21). An immobilized derivative of PGA is usually employed to ensure suitable enzyme stability and consequently enzyme recovery and reuse (7,10).…”

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Improvement of Catalytic Properties of Escherichia coli Penicillin G Acylase Immobilized on Glyoxyl Agarose by Addition of a Six-Amino-Acid Tag

Scaramozzino

Estruch

Rossolillo

et al. 2005

Appl Environ Microbiol

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Quantitative characterization of the nucleophile reactivity in penicillin acylase-catalyzed acyl transfer reactions

Cited by 63 publications

References 21 publications

Saturation mutagenesis reveals the importance of residues αR145 and αF146 of penicillin acylase in the synthesis of β-lactam antibiotics

Saturation mutagenesis reveals the importance of residues αR145 and αF146 of penicillin acylase in the synthesis of β-lactam antibiotics

Acetobacter turbidans α-Amino Acid Ester Hydrolase

Improvement of Catalytic Properties of Escherichia coli Penicillin G Acylase Immobilized on Glyoxyl Agarose by Addition of a Six-Amino-Acid Tag

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