Abstract
Bacterial coenzyme B12-dependent 2-hydroxyisobutyryl-CoA mutase (HCM) is a radical enzyme catalyzing the stereospecific interconversion of (S)-3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA. It consists of two subunits, HcmA and HcmB. To characterize the determinants of substrate specificity, we have analyzed the crystal structure of HCM from Aquincola tertiaricarbonis in complex with coenzyme B12 and the substrates (S)-3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA in alternative binding. When compared with the well studied structure of bacterial and mitochondrial B12-dependent methylmalonyl-CoA mutase (MCM), HCM has a highly conserved domain architecture. However, inspection of the substrate binding site identified amino acid residues not present in MCM, namely HcmA Ile(A90) and Asp(A117). Asp(A117) determines the orientation of the hydroxyl group of the acyl-CoA esters by H-bond formation, thus determining stereospecificity of catalysis. Accordingly, HcmA D117A and D117V mutations resulted in significantly increased activity toward (R)-3-hydroxybutyryl-CoA. Besides interconversion of hydroxylated acyl-CoA esters, wild-type HCM as well as HcmA I90V and I90A mutant enzymes could also isomerize pivalyl- and isovaleryl-CoA, albeit at >10 times lower rates than the favorite substrate (S)-3-hydroxybutyryl-CoA. The nonconservative mutation HcmA D117V, however, resulted in an enzyme showing high activity toward pivalyl-CoA. Structural requirements for binding and isomerization of highly branched acyl-CoA substrates such as 2-hydroxyisobutyryl- and pivalyl-CoA, possessing tertiary and quaternary carbon atoms, respectively, are discussed.
Highlights
Bacterial B12-dependent 2-hydroxyisobutyryl-CoA mutase catalyzes the isomerization of (S)-3hydroxybutyryl- and 2-hydroxyisobutyryl-CoA
Determinants of hydroxyisobutyryl-CoA mutase (HCM) Substrate Specificity—The most significant structural difference between methylmalonyl-CoA mutase (MCM), isobutyryl-CoA mutase (ICM), and HCM substrates is the type of the substituents attached to the thioester-linked carbon atom (Fig. 1)
The structure of HCM reveals that the orientation of the hydroxyl group of HCM substrates corresponds to the hydrogen attached to the thioester-linked carbon of MCM substrates (Fig. 5C)
Summary
Bacterial B12-dependent 2-hydroxyisobutyryl-CoA mutase catalyzes the isomerization of (S)-3hydroxybutyryl- and 2-hydroxyisobutyryl-CoA. Specific substrate-enzyme interaction might be controlled by different interactions than those mediated by Tyr and Arg207 in PfMCM, e.g. the two methyl groups of the ICM substrate isobutyryl-CoA cannot form H-bonds with the corresponding active site amino acids PheA80 and GlnA198 or any other residue as the free substrate carboxyl group does in MCM. In contrast to both PfMCM and HsMCM, in bacterial ICM and HCM as well as in recently described archaeal MCMs [19, 20], acyl-CoA and coenzyme B12 binding sites are distributed on two subunits of significantly different size (i.e. large and small subunits A and B, respectively). In comparison with the structure of MCM, decisive differences in the active site could be identified, enabling the specific interconversion of (S)-3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA
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