Isopenicillin N Synthase: Crystallographic Studies

Isopenicillin N synthase (IPNS) is a non‐heme iron oxidase (NHIO) that catalyses the cyclisation of tripeptide δ‐(l‐α‐aminoadipoyl)‐l‐cysteinyl‐d‐valine (ACV) to bicyclic isopenicillin N (IPN). Over the last 25 years, crystallography has shed considerable light on the mechanism of IPNS catalysis. The first crystal structure, for apo‐IPNS with Mn bound in place of Fe at the active site, reported in 1995, was also the first structure for a member of the wider NHIO family. This was followed by the anaerobic enzyme‐substrate complex IPNS−Fe−ACV (1997), this complex plus nitric oxide as a surrogate for co‐substrate dioxygen (1997), and an enzyme product complex (1999). Since then, crystallography has been used to probe many aspects of the IPNS reaction mechanism, by crystallising the protein with a diversity of substrate analogues and triggering the oxidative reaction by using elevated oxygen pressures to force the gaseous co‐substrate throughout protein crystals and maximise synchronicity of turnover in crystallo. In this way, X‐ray structures have been elucidated for a range of complexes closely related to and/or directly derived from key intermediates in the catalytic cycle, thereby answering numerous mechanistic questions that had arisen from solution‐phase experiments, and posing many new ones. The results of these crystallographic studies have, in turn, informed computational experiments that have brought further insight. These combined crystallographic and computational investigations augment and extend the results of earlier spectroscopic analyses and solution phase studies of IPNS turnover, to enrich our understanding of this important protein and the wider NHIO enzyme family. A thousand words: Isopenicillin N synthase drives the central step in penicillin biosynthesis, oxidative cyclisation of a linear tripeptide to form the bicyclic β‐lactam core. Crystal structures of the protein with its substrate, product, trapped reaction intermediates, and diverse substrate analogues paint a rich mechanistic picture of the enzyme in action and deepen our understanding of non‐heme iron enzyme catalysis.