Structure of isopenicillinN synthase complexed with substrate and the mechanism ofpenicillin formation

Abstract

The biosynthesis of penicillin and cephalosporin antibiotics in microorganisms requires the formation of the bicyclic nucleus of penicillin. Isopenicillin N synthase (IPNS), a non-haem iron-dependent oxidase, catalyses the reaction of a tripeptide, delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV), and dioxygen to form isopenicillin N and two water molecules. Mechanistic studies suggest the reaction is initiated by ligation of the substrate thiolate to the iron centre, and proceeds through an enzyme-bound monocyclic intermediate. Here we report the crystal structure of IPNS complexed to ferrous iron and ACV, determined to 1.3 A resolution. Based on the structure, we propose a mechanism for penicillin formation that involves ligation of ACV to the iron centre, creating a vacant iron coordination site into which dioxygen can bind. Subsequently, iron-dioxygen and iron-oxo species remove the requisite hydrogens from ACV without the direct assistance of protein residues. The crystal structure of the complex with the dioxygen analogue, NO and ACV bound to the active-site iron supports this hypothesis.

Transition States in a Protein Environment − ONIOM QM:MM Modeling of Isopenicillin N Synthesis

“…There are fully synthetic routes to generate IPN, 25 Isopenicillin N synthase is a suitable target for QM/MM investigations because X-ray structures have been collected at different stages of the reaction, including the IPN product and an analogue of the β-lactam intermediate. 20,27 Independent deuterium kinetic isotope effects for both the cysteine β-carbon and the valine β-carbon suggests that both C-H bond activation steps are at least partially rate-limiting, 28 which gives two steps where the calculated barrier can be compared to experiment.…”

Terminally Truncated Isopenicillin N Synthase Generates a Dithioester Product: Evidence for a Thioaldehyde Intermediate during Catalysis and a New Mode of Reaction for Non‐Heme Iron Oxidases

“…Oxygen binds end‐on in the site opposite Asp216,6 avoiding the bridged binding mode seen with other non‐heme iron enzymes and thus enabling oxidase activity (the primary reaction pathway for IPNS) in preference to oxygenase activity. Oxygen sits adjacent to the cysteinyl β‐carbon of ACV and can then selectively abstract the pro‐ S hydrogen from this position to form a thioaldehyde/Fe‐peroxide intermediate 24 .…”

“…It has been observed previously that, in the complex of wildtype IPNS with ACV, these seven residues “adopt a conformation that extends the final helix (α‐10) relative to the Mn:IPNS structure and encloses the substrate in the active site.”6 Thus, the C‐terminal residues shield the active site and protect the reactive species present in intermediates 23 – 27 (Figure 7 a) from alternative reaction paths. It follows that, in a mutant enzyme lacking these residues, the active site is more exposed and open.…”

“…An initial structure was generated by rigid body refinement of protein atoms from the IPNS:Fe II :ACV model (PDB 1BK0)6 against the new data. Further refinement was carried out using REFMAC67 and Coot for model building 68.…”

“…The mechanism of IPNS has been studied using a wide range of ACV analogues in solution,12 in crystallisation studies,5, 6, 13, 14 and by turnover within the crystalline protein (with reaction initiated by exposing anaerobic crystals to pressurised oxygen gas) 7, 15, 16, 17, 18. IPNS catalyses an array of different oxidation reactions both in solution and in crystals; many of the ACV analogues studied, particularly those altered in the third residue (valine), are oxidised to alternative cyclic and acyclic products.…”