Substrate specificity of isopenicillin N synthase

Article abstract of DOI:10.1021/jm00088a028

Highly purified isopenicillin N synthase (IPNS) from two sources (naturally occurring in Penicillium chrysogenum and that expressed in Escherichia coli via a cloned gene derived from Cephalosporium acremonium) have been isolated and utilized in vitro to test synthetic modifications of the natural substrate, (L-α-amino-δ-adipyl)-L-cysteinyl-D-valine (ACV). A very sensitive procedure utilizing the ability of β-lactams to induce the synthesis of β-lactamase was employed to determine whether an ACV analogue could serve as a substrate for IPNS. A wide variety of amino and carboxyl terminal tripeptide substitutions were examined and found to elicit positive β-lactamase induction profiles. However, none of these modifications were found to function as efficiently as a substrate as ACV. One of the β-lactam products which was formed from the reaction of IPNS and the tripeptide analogue was independently synthesized and evaluated for antibacterial activity. Modification of the L-cysteine residue in the second position of ACV resulted in tripeptides that were unable to serve as substrates. Conversion of the D-valine residue in the third position of ACV to an aromatic amino acid or to a highly electronegative residue such as trifluorovaline resulted in elimination of substrate activity and creation of an inhibitor of the enzyme.