CLONING, SEQUENCING, EXPRESSION, AND INSERTIONAL INACTIVATION OF THE GENE FOR THE LARGE SUBUNIT OF THE COENZYME B-12-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 enco des a protein of 566 residues (62,487 Da), with 43-44% sequence identi ty to the large subunit of methylmalonyl-CoA mutase (MCM) from S. cinn amonensis and Propionibacterium shermanii. Targeted disruption of the icmA gene yielded an S. cinnamonensis mutant devoid of ICM activity, T he IcmA protein is similar to 160 residues shorter than the large subu nit of the bacterial MCMs, corresponding to a loss of the entire C ter minal coenzyme B-12 binding domain. The sequence of the (beta/ alpha)( 8)-barrel comprising residues A1-A400 in P. shermanii MCM is highly co nserved in IcmA. The protein was produced in Streptomyces lividans and Escherichia coil with an N-terminal His(6) tag (His(6)-IcmA), but aft er purification His(6)-IcmA showed no ICM activity. In the presence of coenzyme B-12, protein from S. lividans and S. cinnamonensis of simil ar to 17 kDa by SDS-polyacrylamide gel electrophoresis could be select ively eluted with His(6)-IcmA from a Ni2+ affinity column, After purif ication, this small subunit showed no ICM activity but gave active enz yme when recombined with coenzyme B-12 and IcmA or His(6)-IcmA.