Copurification and Characterization of Deacetoxycephalosporin C synthetase/hydroxylase From Cephalosporium Acremonium

J Bacteriol. 1987 Apr;169(4):1611-8. doi: 10.1128/jb.169.4.1611-1618.1987.

Abstract

Deacetoxycephalosporin C synthetase (expandase), which catalyzes ring expansion of penicillin N to deacetoxycephalosporin C (DAOC), has been stabilized in vitro and purified to near homogeneity from the industrially important fungus Cephalosporium acremonium. Throughout the purification, the expandase activity remained physically associated with and in a constant ratio of 7:1 to DAOC hydroxylase activity. The latter activity mediates hydroxylation of DAOC to deacetylcephalosporin C (DAC). The copurified expandase/hydroxylase appeared to be monomeric, with a molecular weight of 41,000 +/- 2,000 and an isoelectric point of 6.3 +/- 0.3. Both catalytic activities required alpha-ketoglutarate, Fe2+, and O2 and were stimulated by ascorbate, dithiothreitol, and ATP. The Fe2+ requirement was specific, and sulfhydryl groups in the purified protein were apparently essential for both ring expansion and hydroxylation. The kinetics and stoichiometry of DAOC/DAC formation from the expandase/hydroxylase-catalyzed reactions suggested that ring expansion of penicillin N preceded hydroxylation of DAOC.

MeSH terms

  • Acremonium / enzymology*
  • Amino Acids / analysis
  • Cephalosporins / biosynthesis
  • Chelating Agents / pharmacology
  • Hydrogen-Ion Concentration
  • Intramolecular Transferases*
  • Isoelectric Point
  • Isomerases / analysis
  • Isomerases / isolation & purification*
  • Isomerases / metabolism
  • Metals / pharmacology
  • Molecular Weight
  • Oxygenases / analysis
  • Oxygenases / isolation & purification*
  • Oxygenases / metabolism
  • Penicillin-Binding Proteins*
  • Penicillins / metabolism
  • Substrate Specificity
  • Sulfhydryl Reagents / pharmacology
  • Temperature

Substances

  • Amino Acids
  • Cephalosporins
  • Chelating Agents
  • Metals
  • Penicillin-Binding Proteins
  • Penicillins
  • Sulfhydryl Reagents
  • deacetoxycephalosporin C
  • Oxygenases
  • deacetoxycephalosporin C hydroxylase
  • Isomerases
  • Intramolecular Transferases
  • deacetoxycephalosporin C synthetase
  • penicillin N