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 δ- l-α-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. δ- l-α-Aminoadipoyl- l-cysteinyl- d-2-amino-3,3-dideuteriobutyrate ( lld-AC d 2Ab) has the same configuration as the natural substrate lld-ACV at each of its three stereocentres; its epimer δ- l-α-aminoadipoyl- l-cysteinyl- l-2-amino-3,3-dideuteriobutyrate ( lll-AC d 2Ab) 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 δ- l-α-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-AC d 2Ab and IPNS:Fe(II): lll-AC d 2Ab complexes offer a possible rationale for the previously observed inhibitory effects of lll-ACV on IPNS activity.