Abstract
5'-Deoxyadenosylcobalamin (AdoCbl)-dependent isomerases catalyze carbon skeleton rearrangements using radical chemistry. We have recently characterized a fusion protein that comprises the two subunits of the AdoCbl-dependent isobutyryl-CoA mutase flanking a G-protein chaperone and named it isobutyryl-CoA mutase fused (IcmF). IcmF catalyzes the interconversion of isobutyryl-CoA and n-butyryl-CoA, whereas GTPase activity is associated with its G-protein domain. In this study, we report a novel activity associated with IcmF, i.e. the interconversion of isovaleryl-CoA and pivalyl-CoA. Kinetic characterization of IcmF yielded the following values: a K(m) for isovaleryl-CoA of 62 ± 8 μM and V(max) of 0.021 ± 0.004 μmol min(-1) mg(-1) at 37 °C. Biochemical experiments show that an IcmF in which the base specificity loop motif NKXD is modified to NKXE catalyzes the hydrolysis of both GTP and ATP. IcmF is susceptible to rapid inactivation during turnover, and GTP conferred modest protection during utilization of isovaleryl-CoA as substrate. Interestingly, there was no protection from inactivation when either isobutyryl-CoA or n-butyryl-CoA was used as substrate. Detailed kinetic analysis indicated that inactivation is associated with loss of the 5'-deoxyadenosine moiety from the active site, precluding reformation of AdoCbl at the end of the turnover cycle. Under aerobic conditions, oxidation of the cob(II)alamin radical in the inactive enzyme results in accumulation of aquacobalamin. Because pivalic acid found in sludge can be used as a carbon source by some bacteria and isovaleryl-CoA is an intermediate in leucine catabolism, our discovery of a new isomerase activity associated with IcmF expands its metabolic potential.
Highlights
isobutyryl-CoA mutase fused (IcmF) is a fusion between a coenzyme B12-dependent isobutyryl-CoA/n-butyryl-CoA isomerase and a G-protein chaperone
Gene Neighborhood Analysis for icmF—In several bacteria, the icmF gene is located in the same operon with or in close proximity to genes encoding enzymes involved in fatty acid metabolism [3]
Our discovery of isobutyryl-CoA mutase (ICM) activity in the IcmF fusion protein, which is widely distributed in bacteria, suggests its involvement in metabolic processes beyond polyketide synthesis [3]
Summary
IcmF is a fusion between a coenzyme B12-dependent isobutyryl-CoA/n-butyryl-CoA isomerase and a G-protein chaperone. 5-Deoxyadenosylcobalamin (AdoCbl)-dependent isomerases catalyze carbon skeleton rearrangements using radical chemistry. 2 The abbreviations used are: MCM, methylmalonyl-CoA mutase; AdoCbl, 5Ј-deoxyadenosylcobalamin; IcmF, isobutyryl-CoA mutase fused; ICM, min (AdoCbl)-dependent isomerases that catalyze 1,2 rearrangements of methylmalonyl-CoA to succinyl-CoA and isobutyryl-CoA to n-butyryl-CoA, respectively (Fig. 1) [1, 2]. A family of enzymes that are similar in their primary sequence to MCM catalyzes AdoCbl-dependent carbon skeleton rearrangements and include, in addition to ICM, 2-hydroxyisobutyryl-CoA mutase (HCM) [5] and ethylmalonylCoA mutase (ECM) [6] (Fig. 1). We report a novel AdoCbl-dependent 1,2 rearrangement reaction catalyzed by IcmF and demonstrate that in the presence of GTP the isovaleryl-CoA mutase activity of IcmF is partially protected from the inactivation
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