Cloning, sequencing, expression, and insertional inactivation of the gene for the large subunit of the coenzyme B12-dependent isobutyryl-CoA mutase from Streptomyces cinnamonensis

Purification of the coenzyme B-12-dependent isobutaryl-CoA mutase (ICM) from Streptomyces cinnamonensis gave a protein of similar to 65 kDa by SDS-polyacrylamide gel electrophoresis, whose gene icmA was cloned using se quences 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 similar to 160 residues shorter than the large subunit of the bacterial MCMs, corresponding to a loss of the entire C terminal coenzyme B-12 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 His(6) tag (His(6)-IcmA), but after purification His(6)-IcmA showed no ICM activity. In the presence of coenzyme B-12, protein from S. lividans and S. cinnamonensis of similar to 17 kDa by SDS-polyacrylamide gel electrophoresis could be selectively eluted with His(6)-IcmA from a Ni2+ affinity column, After purification, this small subunit showed no ICM activity but gave active enzyme when recombined with coenzyme B-12 and IcmA or His(6)-IcmA.