Crystallographic studies on the binding of selectively deuterated LLD- and LLL-substrate epimers by isopenicillin N synthase

Abstract

Isopenicillin N synthase (IPNS) is a non-heme iron(II) oxidase which catalyses the biosynthesis of isopenicillin N (IPN) from the tripeptide delta-l-alpha-aminoadipoyl-l-cysteinyl-d-valine (lld-ACV). Herein we report crystallographic studies to investigate the binding of a truncated lll-substrate in the active site of IPNS. Two epimeric tripeptides have been prepared by solution phase peptide synthesis and crystallised with the enzyme. delta-l-alpha-Aminoadipoyl-l-cysteinyl-d-2-amino-3,3-dideuteriobutyrate (lld-ACd(2)Ab) has the same configuration as the natural substrate lld-ACV at each of its three stereocentres; its epimer delta-l-alpha-aminoadipoyl-l-cysteinyl-l-2-amino-3,3-dideuteriobutyrate (lll-ACd(2)Ab) has the opposite configuration at its third amino acid. lll-ACV has previously been shown to inhibit IPNS turnover of its substrate lld-ACV; the all-protiated tripeptide delta-l-alpha-aminoadipoyl-l-cysteinyl-d-2-aminobutyrate (lld-ACAb) is a substrate for IPNS, being turned over to a mixture of penam and cepham products. Comparisons between the crystal structures of the IPNS:Fe(II):lld-ACd(2)Ab and IPNS:Fe(II):lll-ACd(2)Ab complexes offer a possible rationale for the previously observed inhibitory effects of lll-ACV on IPNS activity.