Mutation of Residue βF71 of Escherichia coli Penicillin Acylase Results in Enhanced Enantioselectivity and Improved Catalytic Properties

Abstract

Residue phenylalanine 71 of the β-chain of penicillin acylase from E. coli is involved in substrate binding and chiral discrimination of its enantiomers. Different amino acid residues have been introduced at position βF71, and the mutants were studied with respect to their enantioselectivity and substrate specificity. Some mutants demonstrated remarkably improved catalytic activity. Moreover, mutation of βF71 residue allowed to enhance penicillin acylase enantioselectivity. The catalytic activity to the specific substrates was improved up to 36 times, most notably for K, R, and L mutants. Increased activity to a D-phenylglycine derivative - a valuable specificity improvement for biocatalytic synthesis of new penicillins and cephalosporins - was shown for βF71R and βF71L mutants. The synthetic capacity of penicillin acylase with 6-aminopenicillanic acid as an external nucleophile was especially sensitive to mutation of the β71 residue in contrast to the synthesis with 7-aminodeacetoxycephalosporanic acid.

Structure mediation in substrate binding and post‐translational processing of penicillin acylases: Information from mutant structures of Kluyvera citrophila penicillin G acylase

“…Mutational studies of residues α146F, β24F, and β71F have already been reported in Ec PGA . Various complexes of phenylacetic acid and (R)‐α‐methylphenylacetic acid with wild‐type and mutants of Ec PGA have confirmed the role of α146F and β24F in substrate selectivity and diastereoselectivity .…”

Protein Engineering of Penicillin Acylase

“…Work [ 90 ] represents a good example of employing rational design for improving Ec PA catalytic properties. The authors performed a structural analysis and molecular modeling of ligand binding in the active site of the enzyme.…”

Selectivity and kinetic modeling of penicillin G acylase variants for the synthesis of cephalexin under a broad range of substrate concentrations

“…While the yield of the reaction is obviously a major concern, the unwanted byproducts are often sparingly soluble in water; therefore, it is important to minimize byproduct formation to reduce the risk of unwanted byproduct precipitation, complicating downstream isolation and processing of the desired product. As a result, PGA has been the subject of research to enhance its selectivity and stereoselectivity for the synthesis of β‐lactam antibiotics (Alkema et al, 2004; Deaguero et al, 2012; Gabor & Janssen, 2004; Jager et al, 2008; Shapovalova et al, 2009).…”