The oxygenation of the 3-methyl group of 7beta-(5-D-aminoadipamido)-3-methylceph-3-em-4-carboxylic acid (desacetoxycephalosporin C) by extracts of Acremonium chrysogenum [proceedings

Abstract

The final stages in the biosynthesis of cephalosporin C are thought to involve the oxidation of desacetoxycephalosporin C to desacetylcephalosporin C (Fujisawa et al., 1975). Licrsch et al. (1976) showed that, in the presence of acetyl-CoA, cell-free extracts of Acremonium chrysogenum convert desacetoxycephalosporin C into cephalosporin C. They presented indirect evidence for an intermediate oxidation of the 3-methyl group on the cephalosporin nucleus by a mono-oxygenase dependent on NADH and MnZ+. Using an assay which measures the conversion of [3-Me-3H]desacetoxycephalosporin C into [3H]desacetylcephalosporin C, we now show that this oxidation is in fact catalysed by a 2-oxoglutarate-linked dioxygenase similar to the enzymes described by Abbott & Udenfrieiid (1 974). Desacetoxycephalosporin C was prepared by hydrogenolysis of cephalosporin C by using a large excess of palladium oxide on keiselguhr (Stedman et al., 1934). [3-Me-3H]Desacetoxycephalosporin C was prepared at The Radiochemical Centre (Amersham, Bucks., U.K.) by a similar procedure but with 3H. Chromatography on DEAE-cellulose eluted with acetic acid followed by preparative t.1.c. on cellulose (Eastman Kodak, type 13254) eluted with propan-1-ol/water/acetic acid (5: 2: 1, by vol.) yielded a product with a specific radioactivity of about 2Ci/mmol. A mutant of A. chrysogenum yielding high titres of cephalosporin C was grown on slopes containing 4% (w/v) maltose, 2.4% Oxoid malt extract, 1 % Oxoid peptone and 2% agar for 7 days at 28°C. Mycelium from half the slope was transferred to a 250ml flaskcontaining40ml of2.5 %corn steepliquor,0.55 %ammonium acetate, 2.5 %sucrose