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

Abstract

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.

Use of Enzymes in the Production of Semi-Synthetic Penicillins and Cephalosporins: Drawbacks and Perspectives

“…For example, saturation site-directed mutagenesis of residues R145 and F146 of PGA from E. coli increased the synthetic rate over the hydrolytic rate 5-15-fold. This improved the synthetic yield by a 50% [145].…”

“…Thus, many different enzymes have been identified, each adequate for a specific kind of -lactamic antibiotic [143,144]. The use of mutagenesis has been also employed to improve the properties of already known enzymes, and some critical residues have been identified [145][146][147]. For example, saturation site-directed mutagenesis of residues R145 and F146 of PGA from E. coli increased the synthetic rate over the hydrolytic rate 5-15-fold.…”

Impact of polybasic alcohols on biocompatibility and selectivity of Penicillin G Acylase for kinetically controlled synthesis

“…Protein engineering or directed evolution studies have been used to understand the structure-function relationship and to improve PGA properties such as activity, substrate specificity and stability [7][8][9][10][11][12][13][14][15][16].…”

Protein Engineering of Penicillin Acylase

“…However, using available data, it was shown that improving the enzyme nucleophilic specificity causes an increase in β: so the effects of point mutations in the nucleophile-binding site are rather predictable. In work [89], the authors achieved a substantial increase in parameter β by mutations of αArg145, which interacts with the ampicillin carboxylic group in the closed conformation and can affect 6-APA binding, according to X-ray analysis. In order to study all the possible effects of a substitution of αArg145, this residue was subjected to saturating mutagenesis.…”