Cloning and Sequencing of the Coenzyme B12-binding Domain of Isobutyryl-CoA Mutase from Streptomyces cinnamonensis, Reconstitution of Mutase Activity, and Characterization of the Recombinant Enzyme Produced inEscherichia coli

• Published in: The Journal of biological chemistry Vol. 274; no. 44; pp. 31679 - 31685
• Main Authors: Ratnatilleke, Ananda, Vrijbloed, Jan W., Robinson, John A.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 29-10-1999; American Society for Biochemistry and Molecular Biology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.44.31679

Isobutyryl-CoA mutase (ICM) catalyzes the reversible, coenzyme B12-dependent rearrangement of isobutyryl-CoA to n-butyryl-CoA, which is similar to, but distinct from, that catalyzed by methylmalonyl-CoA mutase. ICM has been detected so far in a variety of aerobic and anaerobic bacteria, where it appears to play a key role in valine and fatty acid catabolism. ICM from Streptomyces cinnamonensisis composed of a large subunit (IcmA) of 62.5 kDa and a small subunit (IcmB) of 14.3 kDa. icmB encodes a protein of 136 residues with high sequence similarity to the cobalamin-binding domains of methylmalonyl-CoA mutase, glutamate mutase, methyleneglutarate mutase, and cobalamin-dependent methionine synthase, including a conserved DXHXXG cobalamin-binding motif. Using IcmA and IcmB produced separately in Escherichia coli, we show that IcmB is necessary and sufficient with IcmA and coenzyme B12 to afford the active ICM holoenzyme. The large subunit (IcmA) forms a tightly associated homodimer, whereas IcmB alone exists as a monomer. In the absence of coenzyme B12, the association between IcmA and IcmB is weak. The ICM holoenzyme appears to comprise an α2β2-heterotetramer with up to two molecules of bound coenzyme B12. The equilibrium constant for the ICM reaction at 30 °C is 1.7 in favor of isobutyryl-CoA, and the pH optimum is near 7.4. TheKm values for isobutyryl-CoA,n-butyryl-CoA, and coenzyme B12 determined with an equimolar ratio of IcmA and IcmB are 57 ± 13, 54 ± 12, and 12 ± 2 μm, respectively. AVmax of 38 ± 3 units/mg IcmA and akcat of 39 ± 3 s−1 were determined under saturating molar ratios of IcmB to IcmA.