Screening of stable proteins in an extreme thermophile, Thermus thermophilus.
The leuB gene codes for 3-isopropylmalate dehydrogenase of the leucine biosynthetic pathway in an extreme thermophile, Thermus thermophilus. The leuB gene of the thermophile was replaced with a temperature-sensitive chimeric leuB gene. The resultant transformant was adapted to high temperature, a thermostable mutant strain being obtained. A single base substitution that replaces isoleucine at 93 with leucine was found in the chimeric leuB gene of the thermostable mutant. The resultant amino acid residue coincided with the corresponding residue of the T. thermophilus enzyme. It was confirmed that the mutant enzyme is more stable than the original chimeric enzyme. This system can be used to produce stabilized mutants of other enzymes without structural knowledge of them.
D050539:3-Isopropylmalate Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D004795:Enzyme Stability; D004926:Escherichia coli; D005798:Genes, Bacterial; D006358:Hot Temperature; D007700:Kinetics; D007930:Leucine / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; D050539:3-Isopropylmalate Dehydrogenase; D007930:Leucine