title: Purification and characterization of a 2-oxoglutarate-linked ATP-independent deacetoxycephalosporin C synthase of Streptomyces lactamdurans.
abstract: The deacetoxycephalosporin C (DAOC) synthase (expandase) of Streptomyces lactamdurans was highly purified, as shown by SDS-PAGE and isoelectric focusing. The enzyme catalysed the oxidative ring expansion that converts penicillin N into DAOC. The enzyme was very unstable but could be partially stabilized in 25 mM-Tris/HCl, pH 9.0, in the presence of DTT (0.1 mM). The enzyme required 2-oxoglutarate, oxygen and Fe2+, but did not need ATP, ascorbic acid, Mg2+ or K+. The optimum temperature was between 25 and 30 degrees C. The DAOC synthase showed a high specificity for the penicillin substrate. Only penicillin N but not isopenicillin N, penicillin G or 6-aminopenicillanic acid served as substrates. 2-Oxoglutarate analogues were not used as substrates although 2-oxobutyrate and 3-oxoadipate inhibited the enzyme by 100% and 56% respectively. The enzyme was strongly inhibited by Cu2+, Co2+ and Zn2+. The apparent Km values for penicillin N, 2-oxoglutarate and Fe2+ were 52 microM, 3 microM and 71 microM respectively. The enzyme was a monomer with a molecular mass of 27,000 Da +/- 1,000.
authors.affiliation: Departamento de Microbiología, Facultad de Biología, Universidad de León, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Cortés
authors.forename: J F
authors.initials: JF
authors.lastname: Martín
authors.forename: J M
authors.initials: JM
authors.lastname: Castro
authors.forename: L
authors.initials: L
authors.lastname: Láiz
authors.forename: P
authors.initials: P
authors.lastname: Liras
mesh_terms: D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006863:Hydrogen-Ion Concentration; D019751:Intramolecular Transferases; D007535:Isomerases / Q000302:isolation & purification*; D046915:Penicillin-Binding Proteins; D013302:Streptomyces / Q000201:enzymology*
chemical_list: D046915:Penicillin-Binding Proteins; D007535:Isomerases; D019751:Intramolecular Transferases; C038371:deacetoxycephalosporin C synthetase