title: Alternative oxidation by isopenicillin N synthase observed by X-ray diffraction.
abstract: BACKGROUND
  Isopenicillin N synthase (IPNS) catalyses formation of bicyclic isopenicillin N, precursor to all penicillin and cephalosporin antibiotics, from the linear tripeptide delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine. IPNS is a non-haem iron(II)-dependent enzyme which utilises the full oxidising potential of molecular oxygen in catalysing the bicyclisation reaction. The reaction mechanism is believed to involve initial formation of the beta-lactam ring (via a thioaldehyde intermediate) to give an iron(IV)-oxo species, which then mediates closure of the 5-membered thiazolidine ring.
  
  
  RESULTS
  Here we report experiments employing time-resolved crystallography to observe turnover of an isosteric substrate analogue designed to intercept the catalytic pathway at an early stage. Reaction in the crystalline enzyme-substrate complex was initiated by the application of high-pressure oxygen, and subsequent flash freezing allowed an oxygenated product to be trapped, bound at the iron centre. A mechanism for formation of the observed thiocarboxylate product is proposed.
  
  
  CONCLUSIONS
  In the absence of its natural reaction partner (the N-H proton of the L-cysteinyl-D-valine amide bond), the proposed hydroperoxide intermediate appears to attack the putative thioaldehyde species directly. These results shed light on the events preceding beta-lactam closure in the IPNS reaction cycle, and enhance our understanding of the mechanism for reaction of the enzyme with its natural substrate.
authors.affiliation: The Dyson Perrins Laboratory, University of Oxford, UK.
authors.forename: J M
authors.initials: JM
authors.lastname: Ogle
authors.forename: I J
authors.initials: IJ
authors.lastname: Clifton
authors.forename: P J
authors.initials: PJ
authors.lastname: Rutledge
authors.forename: J M
authors.initials: JM
authors.lastname: Elkins
authors.forename: N I
authors.initials: NI
authors.lastname: Burzlaff
authors.forename: R M
authors.initials: RM
authors.lastname: Adlington
authors.forename: P L
authors.initials: PL
authors.lastname: Roach
authors.forename: J E
authors.initials: JE
authors.lastname: Baldwin
mesh_terms: D018360:Crystallography, X-Ray; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D010088:Oxidoreductases; C038370:isopenicillin N synthetase