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 (ACV) to isopenicillin N. Based on crystallographic evidence from the Aspergillus nidulans IPNS crystal structure complexed with the substrate ACV (Roach et al. (1997) Nature 387, 827-830), we were able to provide mutational evidence for the critical involvement of the conserved R-X-S motif in ACV binding in IPNS. The crystal structure further implicated arginine-87 in the binding of the aminoadipyl portion of ACV. Thus, in this study, the site-directed mutagenesis of the corresponding arginine-89 in Cephalosporium acremonium 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 activity was detected in all the mutants obtained when enzyme bioassays were performed. Furthermore, the solubility of the mutants was considerably lower than the wild-type cIPNS after expression at 37 degrees C, but could be recovered when the expression temperature was lowered to 25 degrees C. This suggests that arginine-89 could be critical for the activity of cIPNS due to its involvement in ACV binding and the solubility of wild-type enzyme.
Loke
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P
Department of Microbiology, Faculty of Medicine, National University of Singapore, 10 Kent Ridge Crescent, Singapore, Singapore.
Sim
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T
D000164:Acremonium / Q000201:enzymology*; D000595:Amino Acid Sequence; D001120:Arginine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D013379:Substrate Specificity
D001120:Arginine; D010088:Oxidoreductases; C038370:isopenicillin N synthetase