Purification and Characterization of a 2-oxoglutarate-linked ATP-independent Deacetoxycephalosporin C Synthase of Streptomyces Lactamdurans

J Gen Microbiol. 1987 Nov;133(11):3165-74. doi: 10.1099/00221287-133-11-3165.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography, Gel
  • Chromatography, High Pressure Liquid
  • Chromatography, Ion Exchange
  • Hydrogen-Ion Concentration
  • Intramolecular Transferases*
  • Isomerases / isolation & purification*
  • Penicillin-Binding Proteins*
  • Streptomyces / enzymology*

Substances

  • Penicillin-Binding Proteins
  • Isomerases
  • Intramolecular Transferases
  • deacetoxycephalosporin C synthetase