Identification of a novel trifunctional homoisocitrate dehydrogenase and modulation of the broad substrate specificity through site-directed mutagenesis.
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.
Miyazaki
Kentaro
K
Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan. miyazaki-kentaro@aist.go.jp
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D034301:Deinococcus / Q000201:enzymology*; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C021368:homoisocitrate dehydrogenase