In streptomycetes, the conversion of succinyl-coenzyme A (CoA) into methylmalonyl-CoA, catalyzed by methylmalonyl-CoA mutase, most likely represents an important source of building blocks for polyketide antibiotic biosynthesis. In this work, the structural gene for methylmalonyl-CoA mutase from Streptomyces cinnamonensis was cloned by using a heterologous gene probe encoding the mutase from Propionibacterium shermanii. A 5,732-bp fragment was sequenced, within which four open reading frames were identified on one DNA strand. The two largest (mutA and mutB) overlap by 1 nucleotide and encode proteins of 616 and 733 residues showing high amino acid sequence similarities to each other and to methylmalonyl-CoA mutases from P. shermanii and mammalian sources. The transcriptional start of the mutA-mutB message, determined by S1 mapping, coincides with the first nucleotide of the translational start codon. Evidence that these two open reading frames encode a functional mutase in S. cinnamonensis was obtained by subcloning and expression in Streptomyces lividans TK64. The mut4 and mutB gene products were detected in Western blots (immunoblots) with mutase-specific antibodies and by direct detection of mutase activity with a newly developed assay method.The methylmalonyl-CoA mutase was unable to catalyze the conversion of isobutyryl-CoA into n-butyryl-CoA, another closely related adenosylcobalamin-dependent rearrangement known to occur in S. cinnamonensis.

Purification of the coenzyme B12-dependent isobutyryl-CoA mutase (ICM) from Streptomyces cinnamonensis gave a protein of approximately 65 kDa by SDS-polyacrylamide gel electrophoresis, whose gene icmA was cloned using sequences derived from tryptic peptide fragments. The gene encodes a protein of 566 residues (62, 487 Da), with 43-44% sequence identity to the large subunit of methylmalonyl-CoA mutase (MCM) from S. cinnamonensis and Propionibacterium shermanii. Targeted disruption of the icmA gene yielded an S. cinnamonensis mutant devoid of ICM activity. The IcmA protein is approximately 160 residues shorter than the large subunit of the bacterial MCMs, corresponding to a loss of the entire C-terminal coenzyme B12 binding domain. The sequence of the (beta/alpha)8-barrel comprising residues A1-A400 in P. shermanii MCM is highly conserved in IcmA. The protein was produced in Streptomyces lividans and Escherichia coli with an N-terminal His6 tag (His6-IcmA), but after purification His6-IcmA showed no ICM activity. In the presence of coenzyme B12, protein from S. lividans and S. cinnamonensis of approximately 17 kDa by SDS-polyacrylamide gel electrophoresis could be selectively eluted with His6-IcmA from a Ni2+ affinity column. After purification, this small subunit showed no ICM activity but gave active enzyme when recombined with coenzyme B12 and IcmA or His6-IcmA.

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Genetic instability in Streptomyces glaucescens characteristically involves the occurrence of gross genomic rearrangements including high-level sequence amplification and extensive deletion. We investigated the relationship of the unstable melC and strS loci and a 100 kb region of the chromosome which frequently gives rise to intense heterogeneous DNA amplification. Standard chromosome walking using a cosmid bank in conjunction with a "reverse-blot" procedure enabled us to construct a contiguous genomic BamHI map of the unstable region exceeding 900 kb. The unstable genes and the amplifiable region (AUD locus) are physically linked within a 600 kb segment of the chromosome. The previously characterized deletions which affect these loci are merely components of much larger deletions ranging from 270 to over 800 kb which are polar in nature, effecting the sequential loss of the strS and melC loci. The more extensive deletions terminate either adjacent to, or in the vicinity of DNA reiterations at the AUD locus. Additionally, a deletion junction fragment and the corresponding deletion ends were cloned and analysed at the sequence level.

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