title: The reaction cycle of isopenicillin N synthase observed by X-ray diffraction.
abstract: Isopenicillin N synthase (IPNS), a non-haem iron-dependent oxidase, catalyses the biosynthesis of isopenicillin N (IPN), the precursor of all penicillins and cephalosporins. The key steps in this reaction are the two iron-dioxygen-mediated ring closures of the tripeptide delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV). It has been proposed that the four-membered beta-lactam ring forms initially, associated with a highly oxidized iron(iv)-oxo (ferryl) moiety, which subsequently mediates closure of the five-membered thiazolidine ring. Here we describe observation of the IPNS reaction in crystals by X-ray crystallography. IPNS Fe2+ substrate crystals were grown anaerobically, exposed to high pressures of oxygen to promote reaction and frozen, and their structures were elucidated by X-ray diffraction. Using the natural substrate ACV, this resulted in the IPNS x Fe2+ x IPN product complex. With the substrate analogue, delta-(L-alpha-aminoadipoyl)-L-cysteinyl-L-S-methylcysteine (ACmC) in the crystal, the reaction cycle was interrupted at the monocyclic stage. These mono- and bicyclic structures support our hypothesis of a two-stage reaction sequence leading to penicillin. Furthermore, the formation of a monocyclic sulphoxide product from ACmC is most simply explained by the interception of a high-valency iron-oxo species.
authors.affiliation: The Dyson Perrins Laboratory and the Oxford Centre for Molecular Sciences, University of Oxford, UK.
authors.forename: N I
authors.initials: NI
authors.lastname: Burzlaff
authors.forename: P J
authors.initials: PJ
authors.lastname: Rutledge
authors.forename: I J
authors.initials: IJ
authors.lastname: Clifton
authors.forename: C M
authors.initials: CM
authors.lastname: Hensgens
authors.forename: M
authors.initials: M
authors.lastname: Pickford
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; D008958:Models, Molecular; D009842:Oligopeptides / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation
chemical_list: D009842:Oligopeptides; C011638:5-(2-aminoadipyl)cysteinylvaline; D010088:Oxidoreductases; C038370:isopenicillin N synthetase