Purification and Characterization of a 2-Oxoglutarate-linked ATP-independent Deacetoxycephalosporin C Synthase of Streptomyces lactamdurans

Abstract

SUMMARY: The deacetoxycephalosporin C (DAOC) synthase (expandase) of Streptomyces lactamduranswas 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 Fe^{2 +}, but did not need ATP, ascorbic acid, Mg^{2 +} or K⁺. The optimum temperature was between 25 and 30 °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 Cu^{2 +}, Co^{2 +}and Zn^{2 +}. The apparent K_m values for penicillin N, 2-oxoglutarate and Fe^{2 +}were 52 μm, 3 μmand 71 μmrespectively. The enzyme was a monomer with a molecular mass of 27000 Da ± 1000.