Copurification and characterization of deacetoxycephalosporin C synthetase/hydroxylase from Cephalosporium acremonium

Abstract

Deacetoxycephalosporin C synthetase (expandase), which catalyzes ring expansion of penicillin N to deacetoxycephalosporin C (DAOC), has been stabilized in vitro and purified to near homogeneity from the industrially important fungus Cephalosporium acremonium. Throughout the purification, the expandase activity remained physically associated with and in a constant ratio of 7:1 to DAOC hydroxylase activity. The latter activity mediates hydroxylation of DAOC to deacetylcephalosporin C (DAC). The copurified expandase/hydroxylase appeared to be monomeric, with a molecular weight of 41,000 +/- 2,000 and an isoelectric point of 6.3 +/- 0.3. Both catalytic activities required alpha-ketoglutarate, Fe2+, and O2 and were stimulated by ascorbate, dithiothreitol, and ATP. The Fe2+ requirement was specific, and sulfhydryl groups in the purified protein were apparently essential for both ring expansion and hydroxylation. The kinetics and stoichiometry of DAOC/DAC formation from the expandase/hydroxylase-catalyzed reactions suggested that ring expansion of penicillin N preceded hydroxylation of DAOC.

A spectrophotometric assay for deacetoxycephalosporin C synthase

“…Fig.2c shows double reciprocal plots for Pen N and o~-ketoglutarate, with Km values of 0.18 mM and 0.16 raM, respectively. These values are slightly higher than those quoted for o~-ketoglutarate using the hole plate bioassay (0.04 mM [3]) and Pen N and a~-ketoglutarate using HPLC assays (0.029 mM and 0.022 mM [4]). …”

“…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].…”

Cloning and sequencing of the beta-lactam hydroxylase gene (cefF) from Streptomyces clavuligerus: gene duplication may have led to separate hydroxylase and expandase activities in the actinomycetes

“…The next step in the pathway is the hydroxylation of deacetoxycephalosporin C to deacetylcephalosporin C catalyzed by deacetylcephalosporin C synthetase (hydroxylase). In Cephalosporium acremonium, these two steps are carried out by a single enzyme (2,14,15). However, in Streptomyces clavuligerus, these reactions have been shown to be catalyzed by separate enzymes (4).…”

“…The plasmid, pOW399, was used for the high-level expression of hydroxylase in E. coli JM109. Cultures of E. coli JM109(pOW399) were induced for protein production and cell extracts were assayed for the presence of hydroxylase and expandase activity (2,7,20). E. coli JM109 (pOW399) cell extracts contained hydroxylase activity, whereas E. coli JM109 has no hydroxylase activity.…”

Purification and characterization of the isopenicillin N epimerase from Nocardia lactamdurans

“…The availability of purified IPN synthases (Wamos et al, 1985;Jensen et al, 1986;Castro el al., 1988) and deacetoxycephalosporin C synthases (Kupka et al, 1983;Dotzlaf & Yeh, 1987;Cortks et al, 1987) from different species of fungi and Streptomyces has created a great deal of interest in the utilization of these enzymes to obtain new antibiotics from modified substrates (Jensen , 1986) or in the use of immobilized biosynthetic enzymes . The possibility of utilizing the epimerase coupled to other biosynthetic enzymes in enzyme reactors illustrates the need to characterize this enzyme from different cephamycin-producing actinomycetes in order to find one with better stability.…”