Site-directed mutagenesis of arginine-89 supports the role of its guanidino side-chain in substrate binding by Cephalosporium acremonium isopenicillin N synthase

Isopenicillin N synthase (IPNS) catalyses a key step in the penicillin and cephalosporin biosynthetic pathway which involves the oxidative cyclisation of the acyclic peptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (A CV) to isopenicillin N. Based on crystallographic evidence from the Aspergi llus nidulans IPNS crystal structure complexed with the substrate ACV (Roac h et al. (1997) Nature 387, 827-830), we were able to provide mutational ev idence for the critical involvement of the conserved R-X-S motif in ACV bin ding in IPNS. The crystal structure further implicated arginine-87 in the b inding of the aminoadipyl portion of ACV. Thus, in this study, the site-dir ected mutagenesis of the corresponding arginine-89 in Cephalosporium acremo nium IPNS (cIPNS) was performed to ascertain its role in cIPNS. Alteration of arginine-89 to five amino acids from different amino acid groups, namely lysine, serine, alanine, aspartate and leucine, was performed and no activ ity was detected in all the mutants obtained when enzyme bioassays were per formed. Furthermore, the solubility of the mutants was considerably lower t han the wild-type cIPNS after expression at 37 degrees C, but could be reco vered when the expression temperature was lowered to 25 degrees C. This sug gests that arginine-89 could be critical for the activity of cIPNS due to i ts involvement in ACV binding and the solubility of wild-type enzyme. (C) 1 999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.