Novel B(12)-dependent acyl-CoA mutases and their biotechnological potential

Abstract

The recent spate of discoveries of novel acyl-CoA mutases has engendered a growing appreciation for the diversity of 5'-deoxyadenosylcobalamin-dependent rearrangement reactions. The prototype of the reaction catalyzed by these enzymes is the 1,2 interchange of a hydrogen atom with a thioester group leading to a change in the degree of carbon skeleton branching. These enzymes are predicted to share common architectural elements: a Rossman fold and a triose phosphate isomerase (TIM)-barrel domain for binding cofactor and substrate, respectively. Within this family, methylmalonyl-CoA mutase (MCM) is the best studied and is the only member found in organisms ranging from bacteria to man. MCM interconverts (2R)-methylmalonyl-CoA and succinyl-CoA. The more recently discovered family members include isobutyryl-CoA mutase (ICM), which interconverts isobutyryl-CoA and n-butyryl-CoA; ethylmalonyl-CoA mutase, which interconverts (2R)-ethylmalonyl-CoA and (2S)-methylsuccinyl-CoA; and 2-hydroxyisobutyryl-CoA mutase, which interconverts 2-hydroxyisobutyryl-CoA and (3S)-hydroxybutyryl-CoA. A variant in which the two subunits of ICM are fused to a G-protein chaperone, IcmF, has been described recently. In addition to its ICM activity, IcmF also catalyzes the interconversion of isovaleryl-CoA and pivalyl-CoA. This review focuses on the involvement of acyl-CoA mutases in central carbon and secondary bacterial metabolism and on their biotechnological potential for applications ranging from bioremediation to stereospecific synthesis of C2-C5 carboxylic acids and alcohols, and for production of potential commodity and specialty chemicals.

Figure 1

Reactions catalyzed by acyl-CoA mutases. A generalized 1,2-rearrangement reaction catalyzed by AdoCbl-dependent mutases is shown in the top row followed by reactions catalyzed by MCM, ICM, IcmF, HCM and ECM.

Figure 2

Domain organization of acyl-CoA mutases. The domains/subunits are color-coded as follows: AdoCbl-binding domain (red), substrate-binding domain (yellow), methylmalonyl-CoA epimerase (navy) and G-protein chaperone (cyan). The oligomeric state of the proteins is shown on the right. The following proteins were used as examples: MCMs (Propionibacterium shermanii (αβ)(P11653, P11652), E. coli (α2)(AAA69084), Metallosphaera sedula (α2β2)(Msed_0638, Msed_2055) and Bacillus tusciae (α2β2)(YP_003589181)); ICM (Streptomyces cinnamonensis (α2β2)(AAC08713, CAB59633)), IcmF (Geobacillus kaustophilus (α2)(YP_149244)), HCM (Rhodobacter sphaeroides (α2β2)(Rsph17029_3657, Rsph17029_3654)), and ECM (Rhodobacter sphaeroides (α2)(ACJ71670)).

Figure 3

Active site of MCM and corresponding amino acids substitutions in acyl-CoA mutases. (A) MCM from P. shermanii is a αβ heterodimer (PDB file: 4REQ) and (B) close-up of active site of MCM, where two key active site residues (green) important for catalysis, are shown in stick representation.

Figure 4

Pathways in which MCM and ICM/IcmF participate. Reactions shown in red are found in both bacteria and higher organisms while those in blue have been found only in bacteria.

Figure 5

Pathways involving ECM, MCM and HCM. Reactions shown in red represent the ethylmalonyl-CoA pathway for acetyl-CoA assimilation while those in black represent reactions within this pathway that lead to glyoxylate regeneration that supports the serine cycle. The pathway for degradation of MBTE is shown in blue.

Figure 6

An alternative pathway for isolvaleryl-CoA synthesis and a proposed pivalyl-CoA pathway. The pathway for biosynthesis of isovaleryl-CoA as shown was characterized in M. xanthus (47).