Saturation mutagenesis reveals the importance of residues alphaR145 and alphaF146 of penicillin acylase in the synthesis of beta-lactam antibiotics

Abstract

Penicillin acylase (PA) from Escherichia coli can catalyze the coupling of an acyl group to penicillin- and cephalosporin-derived beta-lactam nuclei, a conversion that can be used for the industrial synthesis of beta-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 alphaR145 and alphaF146 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 beta-lactam nucleophile instead of water, mutants alphaR145G, alphaR145S and alphaR145L 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 beta-lactam antibiotics.