The reaction cycle of isopenicillin N synthase observed by X-ray diffraction

Isopenicillin N synthase (IPNS), a non-haem iron-dependent oxidase, catalyses the biosynthesis of isopenicillin N (IPN), the precursor of all penicillins and cephalosporins1. The key steps in this reaction are the two iron-dioxygen-mediated ring closures of the tripeptide δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine (ACV). It has been proposed that the four-membered β-lactam ring forms initially, associated with a highly oxidized iron(IV)-oxo (ferryl) moiety, which subsequently mediates closure of the five-membered thiazolidine ring2. Here we describe observation of the IPNS reaction in crystals by X-ray crystallography. IPNS·Fe2+·substrate crystals were grown anaerobically3,4, exposed to high pressures of oxygen to promote reaction and frozen, and their structures were elucidated by X-ray diffraction. Using the natural substrate ACV, this resulted in the IPNS·Fe2+·IPN product complex. With the substrate analogue, δ-(L-α-aminoadipoyl)-L-cysteinyl-L-S-methylcysteine (ACmC) in the crystal, the reaction cycle was interrupted at the monocyclic stage. These mono- and bicyclic structures support our hypothesis of a two-stage reaction sequence leading to penicillin. Furthermore, the formation of a monocyclic sulphoxide product from ACmC is most simply explained by the interception of a high-valency iron-oxo species.