Structure, interactions and action of Mycobacterium tuberculosis 3-hydroxyisobutyric acid dehydrogenase

Abstract

Biochemical and crystallographic studies on 3-hydroxyisobutyric acid dehydrogenase (HIBADH), a member of the 3-hydroxyacid dehydrogenase superfamily, have been carried out. Gel filtration and blue native PAGE of HIBADH show that the enzyme is a dimer. The enzyme preferentially uses NAD as the cofactor and is specific to -hydroxyisobutyric acid (HIBA). It can also use-HIBA, l-serine and 3-hydroxypropanoic acid (3-HP) as substrates, but with much less efficiency. The pH optimum for activity is ∼11. Structures of the native enzyme, the holoenzyme, binary complexes with NAD, -HIBA,-HIBA, l-serine and 3-HP and ternary complexes involving the substrates and NAD have been determined. None of the already known structures of HIBADH contain a substrate molecule at the binding site. The structures reported here provide for the first time, among other things, a clear indication of the location and interactions of the substrates at the active site. They also define the entrance of the substrates to the active site region. The structures provide information on the role of specific residues at the active site and the entrance. The results obtained from crystal structures are consistent with solution studies including mutational analysis. They lead to the proposal of a plausible mechanism of the action of the enzyme.

Role of a cysteine residue in substrate entry and catalysis in MtHIBADH: Analysis by chemical modifications and site‐directed mutagenesis

“…We have carried out detailed biochemical and structural studies of Mtb 3-hydroxyisoutyrate dehydrogenase (MtHIBADH) enzyme that converts 3-HIBA to its aldehydic form and thus catalyzes the sixth step in valine catabolism pathway. 18 The final step is catalyzed by methylmalonate semialdehyde dehydrogenase that converts methyl malonate semialdehyde to propionyl Co-A. 19 HIBADH enzyme belongs to β-hydroxyacid dehydrogenase family that catalyzes the oxidation of specific β-hydroxy-carboxylic acid substrates.…”

“…MtHIBADH is dimer in solution, selects NAD over NADP cofactor, and shows a preference for S-enantiomer of HIBA substrate. 18 The closest structural homologs of MtHIBADH are the enzymes from Pseudomonas aeruginosa and human. The active site for substrate binding in MtHIBADH requires three sets of interactions; Lys-168 and Asn-172 residues tether hydroxyl group, 118-VSGG-121 motif residues stabilize carboxylate group, and hydrophobic interaction involving Phe-236 stabilizes the methyl group of the substrate S-HIBA.…”

“…The active site for substrate binding in MtHIBADH requires three sets of interactions; Lys-168 and Asn-172 residues tether hydroxyl group, 118-VSGG-121 motif residues stabilize carboxylate group, and hydrophobic interaction involving Phe-236 stabilizes the methyl group of the substrate S-HIBA. 18,21 The active site is highly susceptible to any change in residues either required for the entry of the substrate or its binding at the reaction center. 18,21,22 Although the substrate interacting residues are conserved in HIBADH enzymes, the enzymes from diverse sources show a different preference for substrates, viz.…”

“…18,21 The active site is highly susceptible to any change in residues either required for the entry of the substrate or its binding at the reaction center. 18,21,22 Although the substrate interacting residues are conserved in HIBADH enzymes, the enzymes from diverse sources show a different preference for substrates, viz. L-serine in HIBADH from P. aeruginosa, 21 2-methyl-DL-serine in HIBADH from P. putida.…”

“…L-serine in HIBADH from P. aeruginosa, 21 2-methyl-DL-serine in HIBADH from P. putida. 23 HIBADH enzyme of T. thermophilus can use serine or HIBA substrates irrespective of their chirality, 22 while the enzyme from pig kidney, human liver, Bacillus cereus, and Mtb prefers S-HIBA substrate 18,24,25 and enzymes from Candida rugosa and rabbit liver utilize both enantiomers of HIBA. 26 Chemical modification of HIBADH from mammalian origin showed the enzyme to be sensitive to the reagents that modify their cysteine and tyrosine residues.…”