Structural Basis of the Stereospecificity of Bacterial B12-dependent 2-Hydroxyisobutyryl-CoA Mutase

Abstract

Background: Bacterial B 12 -dependent 2-hydroxyisobutyryl-CoA mutase specifically catalyzes the isomerization of (S)-3-hydroxybutyryl-and 2-hydroxyisobutyryl-CoA. Results: The crystal structure of 2-hydroxyisobutyryl-CoA mutase shows decisive differences in the active site when compared with the well studied methylmalonyl-CoA mutase. Conclusion: Specificity toward (S)-3-hydroxybutyryl-CoA strongly depends on the active site amino acid Asp A117 . Significance: This is the first structural characterization of a B 12 -dependent mutase with ␣ 2 ␤ 2 organization isomerizing 2-hydroxyisobutyryl-CoA.

Structural Basis for Substrate Specificity in Adenosylcobalamin-dependent Isobutyryl-CoA Mutase and Related Acyl-CoA Mutases

“…Substrate-bound structures of MCM (25,26) and HCM1 (29) as well as bioinformatic analyses (4,6,11,13) have suggested that substrate specificity in acylCoA mutases is determined by the identity of a few key amino acids. Indeed, HCM1 carrying a single active site mutation has both considerable PCM and HCM2 activity and reduced HCM1 activity (29). All other attempts to rationally alter the substrate specificity of acyl-CoA mutases by mutagenesis, however, have failed (13,34), indicating that our understanding of substrate specificity in acyl-CoA mutases is incomplete.…”

“…Of these acyl-CoA mutases, only MCM (24 -27), IcmF (28), and HCM1 (29) have been structurally characterized, and only MCM and HCM1 have been visualized with substrates bound. All three enzymes require two domains for catalytic activity as follows: a Rossmann-fold cobalamin (Cbl)-binding domain, which binds the AdoCbl cofactor in the "base-off/His-on" mode (24,27,30), and an (␣/␤) 8 triose-phosphate isomerase (TIM) barrel, which binds the substrate.…”

Thermophilic Coenzyme B 12 -Dependent Acyl Coenzyme A (CoA) Mutase from Kyrpidia tusciae DSM 2912 Preferentially Catalyzes Isomerization of ( R )-3-Hydroxybutyryl-CoA and 2-Hydroxyisobutyryl-CoA

“…Likely most interesting, Gln A208 of HCM is replaced by Ser in RCM. Thus, considering the active-site architecture of HCM (27), changes to Val and Ser at positions 117 and 208, respectively, might result in a preferred binding of (R)-3-hydroxybutyryl-CoA as observed for RCM in this study. However, a precise description of the active-site architecture of RCM cannot simply be derived from sequence alignments; models based on crystal structure analysis are required.…”

“…Accordingly, an RCM-like but less-pronounced reversion of stereospecificity has recently been obtained with an HCM variant possessing the single point mutation Asp A117 to Val A117 . Although having the same amino acid residue at position HcmA 117 as found in RCM, conversion of (R)-3-hydroxybuytryl-CoA by this enzyme, i.e., HcmA D117V reconstituted with wild-type HcmB, is not as efficient as by RCM, but only a 5-times-higher catalytic efficiency than for the (S)-enantiomer has been observed (27). This weaker reversion indicates that not only the HcmA residue at position 117 but likely also substrate interactions with other active site amino acids contribute to stereospecificity in HCM enzymes.…”

“…In the latter, residues Ile A90 and Gln A208 (numbering as in the HcmA subunit of strain A. tertiaricarbonis L108) seem to be characteristic for the HCM subfamily (7,8). Recently, by elucidating the protein structure of HCM from strain L108 (27), it could be demonstrated that Ile A90 allows orientation of Asp A117 toward the active site and thus enables formation of hydrogen bonds with the HCM main substrates (S)-3-hydroxybutyryl-and 2-hydroxyisobuytryl-CoA. Inspection of homologous residues in a couple of HCM enzymes, in RCM from K. tusciae strain DSM 2912, and in closely related mutase sequences from Bacillus thermotolerans SgZ-8 and Bacillus massiliosenegalensis JC6 revealed that residue Ile A90 is conserved in all mutases (Fig.…”