title: Studies on the ring-cyclization and ring-expansion enzymes of beta-lactam biosynthesis in Cephalosporium acremonium.
abstract: Micrococcus luteus was found to be very sensitive to isopenicillin N and was used as assay organism for purification of the enzyme isopenicillin N synthetase, which cyclizes delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N. Purification of the enzyme from the crude extract obtained by sonication of mycelia of Cephalosporium acremonium CW-19 was carried out by ammonium sulfate precipitation, desalting with Sephadex G-25, gel filtration on LKB ultrogel AcA44 or ion-exchange chromatography on DEAE-Sepharose. The cyclization enzyme was separated from the ring-expansion enzyme and was purified considerably more than 50-fold by this procedure. Using the purified enzyme, we found that the disulfide bis-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine required reduction to delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine in order to behave as a substrate. The enzyme activity was stimulated by FeSO4 and ascorbate, but other cofactors, including alpha-ketoglutarate, were inactive. In addition to delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine, the enzyme converted adipyl-L-cysteinyl-D-valine, N-acetyl-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine, and glycyl-delta-(L-alpha-aminoadipyl)L-cysteinyl-D-valine to penicillins. All of these latter peptides were competitive inhibitors of the cyclization reaction. The Km of the cyclization enzyme is 10 times higher than that of the ring-expansion enzyme, deacetoxycephalosporin C synthetase. The pH and temperature optima of the two enzymes were rather similar. Phosphate inhibited ring expansion, but not cyclization. Both enzymes appear to be soluble enzymes of about 31 000 molecular weight.
mesh_terms: D000164:Acremonium / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D004798:Enzymes / Q000302:isolation & purification / Q000378:metabolism; D019751:Intramolecular Transferases; D007535:Isomerases / Q000302:isolation & purification / Q000378:metabolism; D010088:Oxidoreductases; D046915:Penicillin-Binding Proteins; D047090:beta-Lactams
chemical_list: D000900:Anti-Bacterial Agents; D004798:Enzymes; D046915:Penicillin-Binding Proteins; D047090:beta-Lactams; D010088:Oxidoreductases; C038370:isopenicillin N synthetase; D007535:Isomerases; D019751:Intramolecular Transferases; C038371:deacetoxycephalosporin C synthetase