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J Biol Chem, Vol. 274, Issue 44, 31679-31685, October 29, 1999

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 in Escherichia coli

From the Institute of Organic Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland

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 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 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 ₂₂-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. The K_m 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. A V_max of 38 ± 3 units/mg IcmA and a k_cat of 39 ± 3 s¹ were determined under saturating molar ratios of IcmB to IcmA.

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