IcmF Is a Fusion between the Radical B12 Enzyme Isobutyryl-CoA Mutase and Its G-protein Chaperone

Abstract

Coenzyme B 12 is used by two highly similar radical enzymes, which catalyze carbon skeleton rearrangements, methylmalonyl-CoA mutase and isobutyryl-CoA mutase (ICM). ICM catalyzes the reversible interconversion of isobutyryl-CoA and n-butyryl-CoA and exists as a heterotetramer. In this study, we have identified >70 bacterial proteins, which represent fusions between the subunits of ICM and a P-loop GTPase and are currently misannotated as methylmalonyl-CoA mutases. We designate this fusion protein as IcmF (isobutyryl-CoA mutase fused). All IcmFs are composed of the following three domains: the N-terminal 5-deoxyadenosylcobalamin binding region that is homologous to the small subunit of ICM (IcmB), a middle P-loop GTPase domain, and a C-terminal part that is homologous to the large subunit of ICM (IcmA). The P-loop GTPase domain has very high sequence similarity to the Methylobacterium extorquens MeaB, which is a chaperone for methylmalonyl-CoA mutase. We have demonstrated that IcmF is an active ICM by cloning, expressing, and purifying the IcmFs from Geobacillus kaustophilus, Nocardia farcinica, and Burkholderia xenovorans. This finding expands the known distribution of ICM activity well beyond the genus Streptomyces, where it is involved in polyketides biosynthesis, and suggests a role for this enzyme in novel bacterial pathways for amino acid degradation, myxalamid biosynthesis, and acetyl-CoA assimilation.

Visualization of a radical B 12 enzyme with its G-protein chaperone

“…2B), whereas Arg263 points in the opposite direction and directly contacts the Cbl-domain via a bifurcated interaction with Gln75 and Glu76. Gln75, Glu76, Leu259, Asp262, Arg263, and Arg265 are all fully conserved in IcmF sequences (20).…”

“…metallidurans icmF was amplified from genomic DNA and ligated into a pET28a vector (Novagen) at the NdeI and BamHI sites. C. metallidurans IcmF was purified as described previously (20,21). IcmF-SeMet was expressed using the methionine biosynthesis inhibition method.…”

“…Termed IcmF (20), the fusion protein exhibits both GTPase activity and AdoCbl-dependent carbon skeleton isomerase activity, interconverting isobutyryl-CoA and n-butyryl-CoA, as well as pivalyl-CoA and isovaleryl-CoA ( Fig. S1 B and C) (20,21). The latter activity is newly discovered and may have relevance to the biodegradation of branched compounds (22).…”

“…An ortholog of MeaB, MeaI, is found fused to isobutyryl-CoA mutase. Termed IcmF (20), the fusion protein exhibits both GTPase activity and AdoCbl-dependent carbon skeleton isomerase activity, interconverting isobutyryl-CoA and n-butyryl-CoA, as well as pivalyl-CoA and isovaleryl-CoA ( Fig. S1 B and C) (20,21).…”

Retro-biosynthetic screening of a modular pathway design achieves selective route for microbial synthesis of 4-methyl-pentanol

“…For example, an isobutyryl-CoA mutase or branched a-keto-acid decarboxylase route could be used to generate the isobutyryl-CoA precursor in place of modules 1 and 2 (ref. 65). Similarly, a FAS route could be substituted for module 3 to generate the longer saturated acid substrate for module 4 (ref.…”