Over the past five years, the biochemistry laboratory classes at Kenyon College have used bioinformatic software to identify sixteen mutant 3-hydroxyisobutyrate dehydrogenase proteins from the model organism Arabidopsis thaliana (AtHIBDH1). In response, the Rouhier lab repeated and standardized the expression, purification, and enzyme assay protocols for this enzyme. AtHIBDH1 plays a key role in valine and propionyl-CoA metabolism where it catalyzes the oxidation of 3-hydroxyisobutyrate or 3-hydroxypropionate to (methyl)malonate semialdehyde, respectively. While the active site is critical for substrate binding and activity, we identified additional amino acid sites that are also critical. Single-point mutations were generated via PCR using the plasmid for the wild-type gene. Positive colonies were selected and sequenced to ensure complete generation of the mutation. E. coli containing each mutant were then grown, lysed, and the target protein purified and concentrated using affinity chromatography. After initial screening for any enzyme activity, active enzymes were tested with varying concentrations of (S)-3-hydroxyisobutyrate (S-HIBA) and the co-factor, NAD+. Previous research showed that S-HIBA binds to the active-site lysine at position 219. Our sixteen mutations were scattered throughout the binding sites for NAD+, S-HIBA, and near the surface of the protein. We used an absorbance-based kinetic assay to compare Vmax, Km, and Kcat, for wild-type and mutant proteins. Of these tests, most proteins with mutations at or near the catalytic lysine were found to deactivate the enzyme’s activity. Mutations a bit further out from the active site on nearby well-conserved alpha helices showed slightly altered activity, while mutations far from the active site had little effect.

Functional characterization of sixteen 3-hydroxyisobutyrate dehydrogenase mutants from Arabidopsis thaliana

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