A spectrophotometric assay for deacetoxycephalosporin C synthase.
A continuous direct spectrophotometric assay for deacetoxycephalosporin C synthase was developed, based on the absorption at 260 nm characteristic of the dihydrothiazine moiety of cephalosporins. Km values of 0.18 mM for penicillin N and 0.16 mM for alpha-ketoglutarate were determined. A coupled assay using succinate thiokinase, pyruvate kinase and lactate dehydrogenase showed that succinate was a product of both deacetoxycephalosporin C synthase and hydroxylase reactions. The expandase reaction exhibited a 1:1.06 stoichiometry for deacetoxycephalosporin C and succinate.
D000164:Acremonium / Q000201:enzymology; D019751:Intramolecular Transferases; D007535:Isomerases / Q000032:analysis* / Q000378:metabolism; D007656:Ketoglutaric Acids; D007700:Kinetics; D007770:L-Lactate Dehydrogenase; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D011770:Pyruvate Kinase; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D013386:Succinates; D019802:Succinic Acid
D007656:Ketoglutaric Acids; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D013386:Succinates; D019802:Succinic Acid; D007770:L-Lactate Dehydrogenase; D011770:Pyruvate Kinase; D007535:Isomerases; D019751:Intramolecular Transferases; C038371:deacetoxycephalosporin C synthetase; C006414:penicillin N