A two-protein component 7 alpha-cephem-methoxylase encoded by two genes of the cephamycin C cluster converts cephalosporin C to 7-methoxycephalosporin C

Abstract

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 Streptomyces lividans) introduce the methoxyl group at C-7 of the cephem nucleus. Efficient hydroxylation at C-7 and transfer of the methyl group 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 synthesized in a coordinated form by translational coupling of cmcI and cmcJ. Protein P7 contains domains that correspond to conserved sequences in cholesterol 7␣-monooxygenases and to the active center of Omethyltransferases by comparison with the crystal structure of catechol-O-methyltransferase. Protein P8 may act as a coupling protein for efficient hydroxylation at C-7 in a form similar to that of the two-component system of Pseudomonas putida p-hydroxyphenylacetate-3-hydroxylase.Cephamycin C is synthesized in Nocardia lactamdurans and Streptomyces clavuligerus from precursor amino acids L-␣-aminoadipic acid, L-cysteine, L-valine, and L-methionine. The first three amino acids are linked to form the corresponding tripeptide by the multienzyme ␣-aminoadipyl-cysteinyl-valine synthetase (2, 5), which is encoded by the pcbAB gene (10). The ␣-aminoadipyl-cysteinyl-valine tripeptide is cyclized to form isopenicillin N, and this intermediate is epimerized to form penicillin N, which is later converted to deacetoxycephalosporin C by deacetoxycephalosporin C synthase (expandase). The genes encoding these three enzymatic steps, pcbC, cefD, and cefE, in N. lactamdurans are known to be clustered with lat (which encodes lysine-6-aminotransferase) and pcbAB (9, 11). Further reactions are involved in the synthesis of the C-7 methoxyl group and in the attachment of the carbamoyl group at C-3Ј. Little information is available about the so-called late genes, which convert deacetoxycephalosporin C into cephamycin C. Deacetoxycephalosporin C is known to be hydroxylated to deacetylcephalosporin C in S. clavuligerus by an ␣-ketoglutarate-requiring 3Ј-methylcephem hydroxylase (4, 16). Deacetylcephalosporin C is converted into O-carbamoyldeacetylcephalosporin C by an O-carbamoyltransferase that transfers a carbamoyl group from carbamoylphosphate to the 3Ј-hydroxyl group of deacetylcephalosporin C (6). The methoxyl group at C-7 in the cephamycins derives from molecular oxygen (15) and methionine (18) by the apparent action of an oxygenase and a methyltransferase.At least three types of oxygenases are involved in hydroxylations of microbial metabolites. The first class are ␣-ketoglutarate dependent and require Fe 2ϩ ions to introduce one of the oxygen atoms from O 2 into the substrate (1). A second class of flavin monooxygenases (e.g., p-hydroxybenzoate hydroxylase, salicylate hydroxylase, phenol hydroxylase, melilolate hydro...

A regulatory gene (ccaR) required for cephamycin and clavulanic acid production in Streptomyces clavuligerus: amplification results in overproduction of both beta-lactam compounds

“…Cephamycin and clavulanic acid levels were routinely determined by bioassay as described previously (36), and these assays were confirmed by high-pressure liquid chromatography (6).…”

Allophane increases the protein levels of several cephamycin biosynthetic enzymes in Nocardia lactamdurans

“…Four other genes are involved in the so-called ' late ' steps of the cephamycin biosynthesis in N. lactamdurans, namely c$F (3-methylcephem hydroxylase) (Coque e t al., 1996a), cmcH (3'-hydroxymethylcephem O-carbamoyltransferase) (Coque e t al. , 1995a), and cmcl-cmcJ which encode the twocomponent (proteins P7 and P8) 7a-cephem methoxylase (Coque et al, 1995b). It is of great interest to know how phosphate and other regulatory effectors influencing cephamycin biosynthesis affect the expression of these genes.…”

Association of newly discovered IS elements with the dichloromethane utilization genes of methylotrophic bacteria

“…A modest level of 27 O/O amino acid identity was also noted between Orf353 and protein P8 of Nocardia lactamdurans. Protein P8 is one of the proteins implicated in converting cephalosporin C to 7-methoxycephalosporin C (Coque et al, 1995), but it is unclear if this reaction relates in any way to dihalomethane metabolism.…”