Journal of Biological Chemistry 1999-10-29

Cloning and sequencing of the coenzyme B(12)-binding domain of isobutyryl-CoA mutase from Streptomyces cinnamonensis, reconstitution of mutase activity, and characterization of the recombinant enzyme produced in Escherichia coli.

A Ratnatilleke, J W Vrijbloed, J A Robinson

Index: J. Biol. Chem. 274 , 31679-31685, (1999)

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Abstract

Isobutyryl-CoA mutase (ICM) catalyzes the reversible, coenzyme B(12)-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 cinnamonensis is 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 B(12) 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 B(12), the association between IcmA and IcmB is weak. The ICM holoenzyme appears to comprise an alpha(2)beta(2)-heterotetramer with up to two molecules of bound coenzyme B(12). The equilibrium constant for the ICM reaction at 30 degrees C is 1.7 in favor of isobutyryl-CoA, and the pH optimum is near 7.4. The K(m) values for isobutyryl-CoA, n-butyryl-CoA, and coenzyme B(12) determined with an equimolar ratio of IcmA and IcmB are 57 +/- 13, 54 +/- 12, and 12 +/- 2 microM, respectively. A V(max) of 38 +/- 3 units/mg IcmA and a k(cat) of 39 +/- 3 s(-1) were determined under saturating molar ratios of IcmB to IcmA.