title: Spectroscopic Evidence for the Two C-H-Cleaving Intermediates of Aspergillus nidulans Isopenicillin N Synthase.
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.
authors.forename: Esta
authors.initials: E
authors.lastname: Tamanaha
authors.forename: Bo
authors.initials: B
authors.lastname: Zhang
authors.forename: Yisong
authors.initials: Y
authors.lastname: Guo
authors.forename: Wei-Chen
authors.initials: WC
authors.lastname: Chang
authors.forename: Eric W
authors.initials: EW
authors.lastname: Barr
authors.forename: Gang
authors.initials: G
authors.lastname: Xing
authors.forename: Jennifer
authors.initials: J
authors.lastname: St Clair
authors.affiliation: Max-Planck Institute for Chemical Energy Conversion , 45470 Mülheim a. d. Ruhr, Germany.
authors.forename: Shengfa
authors.initials: S
authors.lastname: Ye
authors.affiliation: Max-Planck Institute for Chemical Energy Conversion , 45470 Mülheim a. d. Ruhr, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Neese
authors.forename: J Martin
authors.initials: JM
authors.lastname: Bollinger
authors.forename: Carsten
authors.initials: C
authors.lastname: Krebs
mesh_terms: D001233:Aspergillus nidulans / Q000201:enzymology*; D007700:Kinetics; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D011789:Quantum Theory; D013057:Spectrum Analysis
chemical_list: D010088:Oxidoreductases; C038370:isopenicillin N synthetase; D010100:Oxygen