Analysis of Glutamines in Catalysis inCephalosporium acremoniumIsopenicillin N Synthase by Site-Directed Mutagenesis

Abstract

Isopenicillin N synthase (IPNS), an important enzyme in the β-lactam antibiotic biosynthetic pathway, is responsible for the catalytic conversion of δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N. Three catalytic ligands essential for IPNS activity have already been determined. Based on anAspergillus nidulansIPNS crystal structure, the probable involvement of a fourth amino acid as a catalytic ligand was previously revealed. To continue the search for the fourth catalytic ligand, we report investigations on whether or not glutamines play a role in the catalytic action ofCephalosporium acremoniumIPNS (cIPNS). Three glutamine residues were targeted for modification based on the previous revelation of one (Q337) via crystal structure coordinates, the conservation of one (Q234) in isozyme alignment and the proximity of one (Q227) to the catalytic centre. Analysis of the biotransformed mutant enzymes showed retention of activity, thereby rejecting the involvement of a possible glutamine as a catalytic ligand in cIPNS catalysis.