Purification and initial characterization of an enzyme with deacetoxycephalosporin C synthetase and hydroxylase activities

Abstract

Deacetoxycephalosporin C synthetase (expandase) from Cephalosporium acremonium (Acremonium chrysogenum) was purified to near homogeneity as judged by SDS/polyacrylamide-gel electrophoresis. The enzyme (Mr about 40,000) exhibited a pH optimum around 7.5. It required 2-oxoglutarate (Km 0.04 mM), Fe2+ and O2 as cofactors, and ascorbate and dithiothreitol were necessary for maximum activity. It was stable for over 4 weeks at -70 degrees C in the presence of 1 mM-dithiothreitol. Activity was inhibited by the thiol-quenching reagent N-ethylmaleimide, the metal-ion-chelating reagent bathophenanthroline, and NH4HCO3. The highly purified enzyme also showed deacetoxycephalosporin C hydroxylase (deacetylcephalosporin C synthetase) activity, indicating that both expandase and hydroxylase activities are properties of a single protein. These activities could not be separated by ion-exchange, dye-ligand, gel-filtration or hydrophobic chromatography. A beta-sulphoxide and a 3 beta-methylene hydroxy analogue of penicillin N were synthesized to test as potential intermediates in the ring-expansion reaction, Neither compound was a substrate for the enzyme. A synthetic analogue in which the 3 beta-methyl group and the 2-hydrogen atom of penicillin N were replaced by a cyclopropane ring was not a substrate but was a reversible inhibitor of the enzyme.

A spectrophotometric assay for deacetoxycephalosporin C synthase

“…Spectra were recorded on a Pye Unicam PU 8800 spectrophotometer against blanks containing Tris-HC1 buffer, pH 7.4 (50 raM). that described previously using the hole plate bioassay [3] with a broad pH optimum of 7.4-8.0. Fig.2a shows the effect of temperature on the enzyme assay, with an optimum at 35°C.…”

“…Partially purified DAOC synthase/hydroxylase (about 50°7o pure) was prepared by chromatography on Sephadex G-75 and Matrex Gel Red A (Procion Red HE3B) from Cephalosporium acremonium CO 728 essentially as described [3,8].…”

“…This was performed as described [3,7] using supersensitive E. coli ESS 21/30. In this procedure DAC as well as DAOC was determined in the assay, but the relative amount of the former was very low under the conditions used.…”

“…The cephem is then hydroxylated to deacetylcephalosporin C (DAC) [3] before further metabolism to cephalosporin C ( [1,2] reviews).…”

“…In the eukaryote Cephalosporin acremonium (Acrernonium chrysogenum) expandase and hydroxylase activities appear to be properties of a single 40 kDa monomeric protein [3][4][5] in contrast to the prokaryote Streptomyces clavuligerus, where the activities have been separated on DEAE-Trisacryl chromatography [6].…”