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.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AJ246005
Using IcmA with a His6 tag attached to its N terminus, a second subunit of Isobutyryl-CoA mutase (ICM) with an apparent mass of Ϸ17 kDa as determined by SDS-polyacrylamide gel electrophoresis was isolated by affinity chromatography from S. cinnamonensis, which gave ICM activity when combined with IcmA and coenzyme B12 [5]
We describe for the first time the cloning and sequencing of the gene encoding IcmB and the first characterization of ICM reconstituted from small and large subunits produced separately in E. coli
Summary
Bacterial Strains—S. cinnamonensis A3823.5, a high-yield producer of monensin A, was a gift of Lilly. E. coli BL21(DE3) pLysS [12] was purchased from Novagen. Production of IcmA and Assay—IcmA was produced in E. coli BL21 pLysS[pET3a-icmA] as described earlier [5]. The ICM assay was as described earlier [2]. Purification of IcmB—IcmB was purified from both S. cinnamonensis and Streptomyces lividans TK64 as described earlier [5]. The N-terminal amino acid sequences of both proteins were determined by the Edman method using an automated sequencer. The N-terminal sequences obtained were as follows: S. lividans, GVAAGPIRVVVAKPGLDGHD; and S. cinnamonensis, GVAAGPIRVVVAKPGLDGHDRGAKVIARAL. Cloning icmB—General DNA manipulations were performed in E. coli [13] and Streptomyces [14] as described. Two oligonucleotides were designed based on the N-terminal protein sequence of IcmB (S ϭ G and C) (Oligo-1 and -2)
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