title: The structural basis of cephalosporin formation in a mononuclear ferrous enzyme.
abstract: Deacetoxycephalosporin-C synthase (DAOCS) is a mononuclear ferrous enzyme that transforms penicillins into cephalosporins by inserting a carbon atom into the penicillin nucleus. In the first half-reaction, dioxygen and 2-oxoglutarate produce a reactive iron-oxygen species, succinate and CO2. The oxidizing iron species subsequently reacts with penicillin to give cephalosporin and water. Here we describe high-resolution structures for ferrous DAOCS in complex with penicillins, the cephalosporin product, the cosubstrate and the coproduct. Steady-state kinetic data, quantum-chemical calculations and the new structures indicate a reaction sequence in which a 'booby-trapped' oxidizing species is formed. This species is stabilized by the negative charge of succinate on the iron. The binding sites of succinate and penicillin overlap, and when penicillin replaces succinate, it removes the stabilizing charge, eliciting oxidative attack on itself. Requisite groups of penicillin are within 1 A of the expected position of a ferryl oxygen in the enzyme-penicillin complex.
authors.affiliation: Molecular Biophysics, Department of Cellular and Molecular Biology, Uppsala University, Box 596, S-751 24 Uppsala, Sweden.
authors.forename: Karin
authors.initials: K
authors.lastname: Valegård
authors.forename: Anke C
authors.initials: AC
authors.lastname: Terwisscha van Scheltinga
authors.forename: Alain
authors.initials: A
authors.lastname: Dubus
authors.forename: Graziella
authors.initials: G
authors.lastname: Ranghino
authors.forename: Linda M
authors.initials: LM
authors.lastname: Oster
authors.forename: Janos
authors.initials: J
authors.lastname: Hajdu
authors.forename: Inger
authors.initials: I
authors.lastname: Andersson
mesh_terms: D020134:Catalytic Domain; D002511:Cephalosporins / Q000096:biosynthesis* / Q000737:chemistry; D018360:Crystallography, X-Ray; D019751:Intramolecular Transferases / Q000737:chemistry / Q000378:metabolism*; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D010084:Oxidation-Reduction; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D055672:Static Electricity; D013302:Streptomyces / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D002511:Cephalosporins; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D011994:Recombinant Proteins; D007501:Iron; D019751:Intramolecular Transferases; C038371:deacetoxycephalosporin C synthetase