Identification of a novel trifunctional homoisocitrate dehydrogenase and modulation of the broad substrate specificity through site-directed mutagenesis

Biochem Biophys Res Commun. 2005 Oct 21;336(2):596-602. doi: 10.1016/j.bbrc.2005.08.139.


A gene encoding homoisocitrate dehydrogenase (HICDH) of Deinococcus radiodurans was cloned, sequenced, and overexpressed in Escherichia coli. The amino acid sequence was 63% identical to HICDH from Thermus thermophilus (Tth-HICDH). Similar to Tth-HICDH, purified, recombinant Dra-HICDH was a tetramer, required K+ and Mn2+ for activity, and used NAD+ as a coenzyme. However, unlike Tth-HICDH, which has a 20-fold preference for isocitrate over homoisocitrate, Dra-HICDH preferred homoisocitrate to isocitrate by 1.5-fold. Moreover, it catalyzed the oxidation of 3-isopropylmalate, albeit at approximately 0.1% the rate seen with homoisocitrate and isocitrate. Saturation mutagenesis of Dra-HICDH Arg87 was next performed because an orthologous Arg85 to valine mutation in Tth-HICDH results in loss of activity toward isocitrate, but in retention of activity toward homoisocitrate. Unexpectedly, the Arg85Val variant became able to catalyze the oxidation of 3-isopropylmalate. Screening of the saturation mutagenesis library identified two variants, Arg87Val and Arg87Thr, that were able to catalyze the oxidation of homoisocitrate, but not isocitrate or 3-isopropylmalate. Deletion of Dra-HICDH Ala80, a residue missing from Tth-HICDH and predicted to reside at the entrance of alpha-helix Arg87, resulted in alterations in substrate specificity that rendered Dra-HICDH similar to Tth-HICDH; i.e., a 4-fold preference for isocitrate over homoisocitrate and inability to catalyze the oxidation of 3-isopropylmalate. Seemingly minor changes in primary sequence result in changes in substrate specificity of beta-decarboxylating dehydrogenases.

MeSH terms

  • Alcohol Oxidoreductases / chemistry*
  • Alcohol Oxidoreductases / genetics
  • Alcohol Oxidoreductases / metabolism*
  • Amino Acid Sequence
  • Amino Acid Substitution
  • Deinococcus / enzymology*
  • Enzyme Activation
  • Molecular Sequence Data
  • Molecular Weight
  • Mutagenesis, Site-Directed
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Structure-Activity Relationship
  • Substrate Specificity


  • Recombinant Proteins
  • Alcohol Oxidoreductases
  • homoisocitrate dehydrogenase