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

Abstract

Purification of the coenzyme B12-dependent isobutyryl-CoA mutase (ICM) from Streptomyces cinnamonensis gave a protein of ~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 ~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 (β/α)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 ~17 kDa by SDS-polyacrylamide gel electrophoresis could be selectively eluted with His6IcmA 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.