title: Studies on deacetoxycephalosporin C synthase support a consensus mechanism for 2-oxoglutarate dependent oxygenases.
abstract: Deacetoxycephalosporin C synthase (DAOCS) catalyzes the oxidative ring expansion of penicillin N (penN) to give deacetoxycephalosporin C (DAOC), which is the committed step in the biosynthesis of the clinically important cephalosporin antibiotics. DAOCS belongs to the family of non-heme iron(II) and 2-oxoglutarate (2OG) dependent oxygenases, which have substantially conserved active sites and are proposed to employ a consensus mechanism proceeding via formation of an enzyme·Fe(II)·2OG·substrate ternary complex. Previously reported kinetic and crystallographic studies led to the proposal of an unusual "ping-pong" mechanism for DAOCS, which was significantly different from other members of the 2OG oxygenase superfamily. Here we report pre-steady-state kinetics and binding studies employing mass spectrometry and NMR on the DAOCS-catalyzed penN ring expansion that demonstrate the viability of ternary complex formation in DAOCS catalysis, arguing for the generality of the proposed consensus mechanism for 2OG oxygenases.
authors.affiliation: Chemistry Research Laboratory, University of Oxford , 12 Mansfield Road, Oxford, OX1 3TA, United Kingdom.
authors.forename: Hanna
authors.initials: H
authors.lastname: Tarhonskaya
authors.forename: Andrea
authors.initials: A
authors.lastname: Szöllössi
authors.forename: Ivanhoe K H
authors.initials: IK
authors.lastname: Leung
authors.forename: Jacob T
authors.initials: JT
authors.lastname: Bush
authors.forename: Luc
authors.initials: L
authors.lastname: Henry
authors.forename: Rasheduzzaman
authors.initials: R
authors.lastname: Chowdhury
authors.forename: Aman
authors.initials: A
authors.lastname: Iqbal
authors.forename: Timothy D W
authors.initials: TD
authors.lastname: Claridge
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Schofield
authors.forename: Emily
authors.initials: E
authors.lastname: Flashman
mesh_terms: D002384:Catalysis; D018360:Crystallography, X-Ray; D019751:Intramolecular Transferases / Q000737:chemistry*; D007656:Ketoglutaric Acids / Q000737:chemistry*; D007700:Kinetics; D013058:Mass Spectrometry; D019906:Nuclear Magnetic Resonance, Biomolecular; D010105:Oxygenases / Q000737:chemistry*; D046915:Penicillin-Binding Proteins / Q000737:chemistry*
chemical_list: D007656:Ketoglutaric Acids; D046915:Penicillin-Binding Proteins; D010105:Oxygenases; D019751:Intramolecular Transferases; C038371:deacetoxycephalosporin C synthetase