The oxygenation of [3-methyl-3H]desacetoxycephalosporin C [7β-(5-d-aminadipamido)-3-methylceph-3-em-4-carboxylic acid] to [3-hydroxymethyl-3H]desacetylcephalosporin C by 2-oxoglutarate-linked dioxygenases from Acremonium chrysogenum and Streptomyces clavuligerus

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Abstract
Cell-free extracts of A. chrysogenum and S. clavuligerus oxidize the 3-methyl group of desacetoxycephalosporin C to a 3-hydroxymethyl group. The enzyme responsible for this reaction in these organisms was purified 20- and 30-fold, respectively, by chromatography on DEAE-cellulose. The enzymes, which were assayed with [3-methyl-3H]desacetoxycephalosporin C as substrate, have the properties expected of 2-oxoglutarate-linked dioxygenases. They require 2-oxoglutarate, Fe2+ cations and a mixture of reducing agents (dithiothreitol and ascorbate) for full activity. The enzyme from A. chrysogenum, but not that from S. clavuligerus, is activated about 10-fold when it is preincubated with a reaction mixture from which either desacetoxycephalosporin C or 2-oxoglutarate is omitted. Fe2+ cations seem to play a key role in this activation. Both enzymes seem highly specific for cephalosporins with the natural 7.beta.-(5-D-aminoadipamido) side chain and are likely to be responsible for the oxidation of the 3-methylcephem nucleus in vivo.