Alternative oxidation by isopenicillin N synthase observed by X-ray diffraction

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Background: Isopenicillin N synthase (IPNS) catalyses formation of bicyclic isopenicillin N, precursor to all penicillin and cephalosporin antibiotics, from the linear tripeptide δ-( L-α-aminoadipoyl)- L-cysteinyl- D-valine. IPNS is a non-haem iron(II)-dependent enzyme which utilises the full oxidising potential of molecular oxygen in catalysing the bicyclisation reaction. The reaction mechanism is believed to involve initial formation of the β-lactam ring (via a thioaldehyde intermediate) to give an iron(IV)-oxo species, which then mediates closure of the 5-membered thiazolidine ring. Results: Here we report experiments employing time-resolved crystallography to observe turnover of an isosteric substrate analogue designed to intercept the catalytic pathway at an early stage. Reaction in the crystalline enzyme–substrate complex was initiated by the application of high-pressure oxygen, and subsequent flash freezing allowed an oxygenated product to be trapped, bound at the iron centre. A mechanism for formation of the observed thiocarboxylate product is proposed. Conclusions: In the absence of its natural reaction partner (the N–H proton of the L-cysteinyl- D-valine amide bond), the proposed hydroperoxide intermediate appears to attack the putative thioaldehyde species directly. These results shed light on the events preceding β-lactam closure in the IPNS reaction cycle, and enhance our understanding of the mechanism for reaction of the enzyme with its natural substrate.