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 tu.-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. (c) 2010 Elsevier Inc. All rights reserved.