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Spectroscopic Evidence for the Two C–H-Cleaving Intermediates of Aspergillus nidulans Isopenicillin N Synthase
- Source :
- Journal of the American Chemical Society. 138:8862-8874
- Publication Year :
- 2016
- Publisher :
- American Chemical Society (ACS), 2016.
-
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 co-substrate 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.
- Subjects :
- Stereochemistry
Thiazolidine
Isopenicillin N synthase
Tripeptide
010402 general chemistry
Cleavage (embryo)
Ring (chemistry)
01 natural sciences
Biochemistry
Aspergillus nidulans
Article
Catalysis
Cofactor
chemistry.chemical_compound
Colloid and Surface Chemistry
Cleave
biology
010405 organic chemistry
Spectrum Analysis
Substrate (chemistry)
General Chemistry
0104 chemical sciences
Oxygen
Kinetics
chemistry
biology.protein
Quantum Theory
Oxidoreductases
Subjects
Details
- ISSN :
- 15205126 and 00027863
- Volume :
- 138
- Database :
- OpenAIRE
- Journal :
- Journal of the American Chemical Society
- Accession number :
- edsair.doi.dedup.....efdf13e5bb1f935fd966d415d02b00a8