title: Purification and initial characterization of deacetoxycephalosporin C synthase from Streptomyces clavuligerus.
abstract: Deacetoxycephalosporin C synthase, the penicillin N ring expansion enzyme from Streptomyces clavuligerus, was purified to near homogeneity, as judged by sodium dodecyl sulphate - polyacrylamide gel electrophoresis. The synthase was monofunctional and could be completely separated from deacetoxycephalosporin C hydroxylase activity early in the purification sequence. Synthase specific activity was increased 97-fold over crude cell-free extracts, and the purified enzyme appeared to be a monomer with a molecular weight of 36,000 and a Km for the penicillin N substrate of 50 microM. Deacetoxycephalosporin C synthase activity required alpha-ketoglutarate, Fe2+, and oxygen and was specifically stimulated by ascorbate and dithiothreitol. The enzyme was sensitive to thiol-specific inhibitors, the most effective of which was N-ethylmaleimide.
authors.affiliation: Department of Microbiology, University of Alberta, Edmonton, Canada.
authors.forename: M J
authors.initials: MJ
authors.lastname: Rollins
authors.forename: D W
authors.initials: DW
authors.lastname: Westlake
authors.forename: S
authors.initials: S
authors.lastname: Wolfe
authors.forename: S E
authors.initials: SE
authors.lastname: Jensen
mesh_terms: D002852:Chromatography, Ion Exchange; D003067:Coenzymes / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D019751:Intramolecular Transferases; D007535:Isomerases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D046915:Penicillin-Binding Proteins; D013302:Streptomyces / Q000201:enzymology*; D013438:Sulfhydryl Compounds
chemical_list: D003067:Coenzymes; D046915:Penicillin-Binding Proteins; D013438:Sulfhydryl Compounds; D007535:Isomerases; D019751:Intramolecular Transferases; C038371:deacetoxycephalosporin C synthetase