Probing the penicillin sidechain selectivity of recombinant deacetoxycephalosporin C synthase

Deacetoxycephalosporin C synthase from Streptomyces clavuligerus catalyses the conversion of the five-membered penicillin ring to the unsaturated six- membered cephem ring of deacetoxycephalosporin C. The effects on enzyme act ivity of the penicillin substrate sidechain and various cofactors were inve stigated using a continuous spectrophotometric assay. The conversion of pen icillin G to phenylacetyl-7-aminodeacetoxycephalo sporanic acid (G-7-ADCA) was confirmed, and further details of the reaction were elucidated. The con version of ampicillin to cephalexin was faster than that of acetyl-6-APA to acetyl-7-ADCA k(cat) = 0.120 +/- 0.001 s(-1) versus 0.035 +/- 0.001 s(-1), but they had similar K-m values: 4.86 +/- 0.12 and 3.28 +/- 0.26 mM, respe ctively. Amoxycillin and penicillin V were also converted at low levels. Co nversion was not detected for penicillanate, 6-aminopenicillanate, carbenic illin, temocillin, ticarcillin or benzylpenicilloic acid, suggesting that t he enzyme has a relatively strict selectivity for the sidechain of the peni cillin substrate.