Characterization of the cmcH genes of Nocardia lactamdurans and Streptomyces clavuligerus encoding a functional 3'-hydroxymethylcephem O-carbamoyltransferase for cephamycin biosynthesis.

Sequencing of ORF10 (gene cmcH) of the Nocardia lactamdurans cephamycin gene cluster proved that it encodes a protein with a deduced molecular mass of 57,149 Da. This protein showed significant similarity to the putative O-carbamoyltransferases (O-Cases) encoded by the nodU genes of Rhizobium fredii and Bradyrhizobium japonicum, involved in the synthesis of nodulation factors. The carbamoyl-phosphate (CP)-binding amino-acid sequence of human OTCase is conserved in the cmcH product. A similar cmcH (80% identify in a 160-nt fragment) in the cephamycin (CmC) cluster of cmc genes of Streptomyces clavuligerus was partially sequenced. The cmcH gene is closely linked to and in the same orientation as cefF in both organisms. Both cmcH were subcloned in pIJ702 and expressed in Streptomyces lividans. Extracts of transformants could carbamoylate decarbamoylcefuroxime. A similar cmcH was found by Southern hybridization in Streptomyces cattleya, but not in Streptomyces griseus or Streptomyces lipmanii which produce non-carbamoylated CmC.