Copurification and characterization of deacetoxycephalosporin C synthetase/hydroxylase from Cephalosporium acremonium.
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.
D000164:Acremonium / Q000201:enzymology*; D000596:Amino Acids / Q000032:analysis; D002511:Cephalosporins / Q000096:biosynthesis; D002614:Chelating Agents / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D019751:Intramolecular Transferases; D007526:Isoelectric Point; D007535:Isomerases / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D010105:Oxygenases / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000378:metabolism; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013696:Temperature
D000596:Amino Acids; D002511:Cephalosporins; D002614:Chelating Agents; D008670:Metals; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D013439:Sulfhydryl Reagents; C025578:deacetoxycephalosporin C; D010105:Oxygenases; C045288:deacetoxycephalosporin C hydroxylase; D007535:Isomerases; D019751:Intramolecular Transferases; C038371:deacetoxycephalosporin C synthetase; C006414:penicillin N