Adaptation of a thermophilic enzyme, 3-isopropylmalate dehydrogenase, to low temperatures.
Random mutagenesis coupled with screening of the active enzyme at a low temperature was applied to isolate cold-adapted mutants of a thermophilic enzyme. Four mutant enzymes with enhanced specific activities (up to 4.1-fold at 40 degrees C) at a moderate temperature were isolated from randomly mutated Thermus thermophilus 3-isopropylmalate dehydrogenase. Kinetic analysis revealed two types of cold-adapted mutants, i.e. k(cat)-improved and K(m)-improved types. The k(cat)-improved mutants showed less temperature-dependent catalytic properties, resulting in improvement of k(cat) (up to 7.5-fold at 40 degrees C) at lower temperatures with increased K(m) values mainly for NAD. The K(m)-improved enzyme showed higher affinities toward the substrate and the coenzyme without significant change in k(cat) at the temperatures investigated (30-70 degrees C). In k(cat)-improved mutants, replacement of a residue was found near the binding pocket for the adenine portion of NAD. Two of the mutants retained thermal stability indistinguishable from the wild-type enzyme. Extreme thermal stability of the thermophilic enzyme is not necessarily decreased to improve the catalytic function at lower temperatures. The present strategy provides a powerful tool for obtaining active mutant enzymes at lower temperatures. The results also indicate that it is possible to obtain cold-adapted mutant enzymes with high thermal stability.
Suzuki
T
T
Department of Life Science, Tokyo Institute of Technology, Nagatsuta 4259, Yokohama 226-8503, Japan.
Yasugi
M
M
Arisaka
F
F
Yamagishi
A
A
Oshima
T
T
D050539:3-Isopropylmalate Dehydrogenase; D000222:Adaptation, Physiological; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D003080:Cold Temperature; D018360:Crystallography, X-Ray; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008293:Malates / Q000378:metabolism*; D008956:Models, Chemical; D008961:Models, Structural; D016254:Mutagenesis, Insertional; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
D007527:Isoenzymes; D008024:Ligands; D008293:Malates; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; D050539:3-Isopropylmalate Dehydrogenase