The conversion of succinyl-coenzyme A (CoA) into methylmalonyl-CoA, catalyzed by adenosylcobalamin-dependent methylmalonyl-CoA mutase (MCM), represents an important source of building blocks for rifamycin SV biosynthesis. The structural gene for MCM from rifamycin SV-producing strain Amycolatopsis mediterranei U32 was isolated by using a heterologous gene probe encoding the MCM of Streptomyces cinnamonesis. A 7.8-kbp fragment was sequenced and four complete open reading frames (ORFs) and two incomplete ORFs were found. Two central ORFs, ORF3 and ORF4, overlap by four nucleotides and were found to encode MCM small (602 residues) and large (721 residues) subunits, respectively. Comparison showed that the MCM gene of A. mediterranei U32 was quite similar to those from other sources. The functionally unknown ORF5, immediately downstream of the mutAB gene, was quite similar to the ORFs downstream of mutAB from S. cinnamonensis and Mycobacterium tuberculosis. Such a striking cross-species conservation of gene order suggested that ORF5 could also be involved in the metabolism of methylmalonyl-CoA. MCM gene was overexpressed in Escherichia coli under T7 promoter, and MCM activity could be detected in the recombinant E. coli clone harboring MCM gene after the addition of coenzyme B12. A purification procedure based on the B12 affinity column was established to purify the MCM from E. coli. The molecular weight of purified MCM from E. coli was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, which corresponds to that calculated from the MCM protein sequence and is also the same size as that of the enzyme purified directly from A. mediterranei U32. MCM gene was overexpressed in polyketide monensin producing S. cinnamonensis, and the total monensin production was increased by 32%.