Isopenicillin N Synthase: Crystallographic Studies

Abstract

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 IPNSÀ FeÀ 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.

The biosynthetic logic and enzymatic machinery of approved fungi-derived pharmaceuticals and agricultural biopesticides

“…IPNS belongs to the intermolecular dioxygenases, and its functional activity requires the presence of Fe 2+ , molecular oxygen and ascorbate. 135,136 IPNS is an exceptional enzyme due its multiple functions, and the IPNScatalyzed reactions are of particular interest as the proteinsubstrate complexes, including enzyme-substrate, enzyme- Natural Product Reports Review product and enzyme-trapped-intermediates, have brought a wealth of new insights into the mechanistic activities of the non-heme iron oxidase IPNS. 134,137,138 However, some details of IPNS catalysis are not yet fully understood.…”

“…134,137,138 However, some details of IPNS catalysis are not yet fully understood. 136 In the next steps of penicillin production, the rst committed intermediate 86 can be further converted to the nal hydrophobic penicillins [penicillin G (87) or phenoxymethylpenicillin (88)] by the multifunctional enzyme acyl-CoA:IPN acyltransferase (IAT) encoded by penDE when phenylacetic acid or phenoxyacetic acid was added to the culture medium in Penicillium or Aspergillus, respectively. [139][140][141] Specically, IAT catalyzes six different reactions, including cleavage of the a-aminoadipic side-chain in 86 by releasing 6-aminopenicillanic acid (6-APA, 89), and introduces the formation of an amide bond between 89 and the activated aromatic acids, which takes place either directly or in two steps (Fig.…”

Which is the real oxidant in competitive ligand self-hydroxylation and substrate oxidation—a biomimetic iron(ii)-hydroperoxo species or an oxo-iron(iv)-hydroxy one?

“…In other words, 1 might either directly carry out the oxidation reactions or undergo O-O bond heterolysis to yield an oxo-iron (IV)-hydroxo intermediate (2 in Scheme 1), which works in the following oxidation reactions. However, no isotope labelling 18 O incorporation from water molecules into the final oxidation product during the labelling experiments with either H 2 18 O/ 16 O 2 or H 2 16 O/ 18 O 2 could only confirm that the environmental water molecule either should not be involved in the formation of reactive species or did not exchange its oxygen with the reactive species before the oxidation reactions took place. 3,36 This is different from the previous findings about some iron(III)-hydroperoxo species in which the water oxygen can incorporate into the final oxidation product, [44][45][46][47][48] which means that either the O-O bond cleavage of the iron(III)-hydroperoxo complex needs the assistance of the environmental water molecule or the oxygen exchange between the reactive species and the environmental water should be faster than the following oxidations.…”

“…17 However, in 2-ODDs (e.g., isopenicillin N synthase), an oxo-iron(IV)-hydroxo intermediate is postulated to be responsible for the cyclisation reaction. 18 However, direct experimental evidence for such reactive intermediates in the catalytic cycle of 2-ODDs is still absent. To understand the mechanistic details of enzymatic catalysis, a number of nonheme iron complexes have been developed to mimic the catalytic processes of enzymes.…”

Electronic Structure and Reactivity of Dioxygen-Derived Aliphatic Thiolate-Ligated Fe-Peroxo and Fe(IV) Oxo Compounds

“…Cysteinate-ligated nonheme iron enzymes play a key role in the biosynthesis of β-lactam antibiotics by isopenicillin N synthase (IPNS), − as well as the regulation of cysteine by cysteine dioxygenase (CDO), − toxic levels of which can lead to neurological disorders or metastases of cancerous tumors. − Synthetic routes to β-lactam antibiotics lack the efficiency of the IPNS catalyzed route; therefore, IPNS provides one of few weapons currently available for fighting bacterial infections . The mechanism of IPNS involves the oxidative bicyclization of the cysteine-containing tripeptide ACV to afford β-lactam and thiazolidine rings . The ACV substrate binds to Fe­(II) via the cysteinate sulfur (Figure ), which has been theoretically predicted to lower the activation barrier to O 2 binding .…”