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 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.
Zerbe-Burkhardt
K
K
Institute of Organic Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
Ratnatilleke
A
A
Philippon
N
N
Birch
A
A
Leiser
A
A
Vrijbloed
J W
JW
Hess
D
D
Hunziker
P
P
Robinson
J A
JA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D003038:Cobamides / Q000378:metabolism*; D015870:Gene Expression; D005798:Genes, Bacterial; D007535:Isomerases / Q000235:genetics* / Q000378:metabolism; D008765:Methylmalonyl-CoA Mutase / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*
D001426:Bacterial Proteins; D003038:Cobamides; D011994:Recombinant Proteins; D007535:Isomerases; C107545:isobutyryl-CoA mutase, Streptomyces; D008765:Methylmalonyl-CoA Mutase; C000913:cobamamide