Unexpected Oxidation of a Depsipeptide Substrate Analogue in Crystalline Isopenicillin N Synthase

Abstract

Isopenicillin N synthase (IPNS) is a non-heme iron(ii)-dependent oxidase that is central to penicillin biosynthesis. Herein, we report mechanistic studies of the IPNS reaction in the crystalline state, using the substrate analogue delta-(L-alpha-aminoadipoyl)-(3R)-methyl-L-cysteine D-alpha-hydroxyisovaleryl ester (AmCOV) to probe the early stages of the catalytic cycle. The X-ray crystal structure of the anaerobic IPNS:Fe(II):AmCOV complex was solved to 1.40 A resolution, and it reveals several subtle differences in the active site relative to the complex of the enzyme with its natural substrate. The crystalline IPNS:Fe(II):AmCOV complex was then exposed to oxygen gas at high pressure; this brought about reaction to give what appears to be a hydroxymethyl/ene-thiol product. A mechanism for this reaction is proposed. These results offer further insight into the delicate interplay of steric and electronic effects in the IPNS active site and the mechanistic intricacies of this remarkable enzyme.

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

“…It has long been thought that such a species is involved in this reaction; however, directly characterising it has proved difficult 15, 16, 17, 18, 24. The dithioester product 7 / 8 reported here is most plausibly formed through interception of a thioaldehyde at the IPNS active site by a second ACV thiol/thiolate (Figure 6 b).…”

“…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.…”

The crystal structure of an isopenicillin N synthase complex with an ethereal substrate analogue reveals water in the oxygen binding site

“…In the complex of IPNS with its natural substrate ACV (Fig. 2a) [10] and complexes with substrate analogues containing third residues of a similar size [22,24,48], only one water ligand is present at iron, opposite His214 (#713 in Fig. 2a).…”

“…X‐ray crystal structures of apo‐IPNS [9] and the enzyme:substrate complex [10] paint a detailed structural picture of the enzyme active site. Time‐resolved studies using high‐pressure oxygenation to initiate reaction within crystalline IPNS have enabled step‐by‐step elucidation of the reaction mechanism on a structural level [11,22–25], while structures of the enzyme with ACV analogues reveal a range of binding modes and the interplay between steric and electronic effects at the IPNS active site [26–32]. Seeking.…”

Membrane‐Surface Anchoring of Charged Diacylglycerol‐Lactones Correlates with Biological Activities

“…The mechanism of IPNS catalysis has been studied extensively by spectroscopy,3–6 by solution‐phase turnover studies with substrate analogues,7 and more recently by protein crystallography 8–18. IPNS was the first member of the non‐heme iron oxidase (NHIO) family of enzymes to be characterised crystallographically8 and subsequent studies have led to crystal structures for enzyme–substrate complexes,9 the enzyme in complex with a monocyclic β‐lactam intermediate (intercepted chemically in the active site),10 and the enzyme–product complex,10 plus a range of enzyme–analogue complexes and intercepted reaction products 11–17…”