Spectroscopic Evidence for the Two C-H-Cleaving Intermediates of Aspergillus nidulans Isopenicillin N Synthase

Esta Tamanaha, Bo Zhang, Yisong Guo, Wei Chen Chang, Eric W. Barr, Gang Xing, Jennifer St Clair, Shengfa Ye, Frank Neese, J. Martin Bollinger, Carsten Krebs

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Abstract

The enzyme isopenicillin N synthase (IPNS) installs the β-lactam and thiazolidine rings of the penicillin core into the linear tripeptide l-δ-aminoadipoyl-l-Cys-d-Val (ACV) on the pathways to a number of important antibacterial drugs. A classic set of enzymological and crystallographic studies by Baldwin and co-workers established that this overall four-electron oxidation occurs by a sequence of two oxidative cyclizations, with the β-lactam ring being installed first and the thiazolidine ring second. Each phase requires cleavage of an aliphatic C-H bond of the substrate: the pro-S-CCys,β-H bond for closure of the β-lactam ring, and the CVal,β-H bond for installation of the thiazolidine ring. IPNS uses a mononuclear non-heme-iron(II) cofactor and dioxygen as cosubstrate to cleave these C-H bonds and direct the ring closures. Despite the intense scrutiny to which the enzyme has been subjected, the identities of the oxidized iron intermediates that cleave the C-H bonds have been addressed only computationally; no experimental insight into their geometric or electronic structures has been reported. In this work, we have employed a combination of transient-state-kinetic and spectroscopic methods, together with the specifically deuterium-labeled substrates, A[d2-C]V and AC[d8-V], to identify both C-H-cleaving intermediates. The results show that they are high-spin Fe(III)-superoxo and high-spin Fe(IV)-oxo complexes, respectively, in agreement with published mechanistic proposals derived computationally from Baldwin's founding work.

Original languageEnglish (US)
Pages (from-to)8862-8874
Number of pages13
JournalJournal of the American Chemical Society
Volume138
Issue number28
DOIs
StatePublished - Jul 20 2016

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All Science Journal Classification (ASJC) codes

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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