A 2-PROTEIN COMPONENT 7-ALPHA-CEPHEM-METHOXYLASE ENCODED BY 2 GENES OF THE CEPHAMYCIN-C CLUSTER CONVERTS CEPHALOSPORIN-C TO 7-METHOXYCEPHALOSPORIN-C

Two genes, cmcI and cmcJ, corresponding to open reading frames 7 and 8 (ORF7 and ORF8) of the cephamycin C cluster of Nocardia lactamdurans encode enzymes that convert cephalosporin C to 7-methoxycephalosporin C. Proteins P7 and P8 (the products of ORF7 and ORF8 expressed in Stre ptomyces lividans) introduce the methoxyl group at C-7 of the cephem n ucleus. Efficient hydroxylation at C-7 and transfer of the methyl grou p from S-adenosylmethionine require both proteins P7 and P8, although P7 alone shows weak C-7 hydroxylase activity and strong cephalosporin- dependent NADH oxidase activity. Both P7 and P8 appear to be synthesiz ed in a coordinated form by translational coupling of cmcI and cmcJ. P rotein P7 contains domains that correspond to conserved sequences in c holesterol 7 alpha-monooxygenases and to the active center of O-methyl transferases by comparison with the crystal structure of catechol-O-me thyltransferase. Protein P8 may act as a coupling protein for efficien t hydroxylation at C-7 in a form similar to that of the two-component system of Pseudomonas putida p-hydroxyphenylacetate-3-hydroxylase.