Thermostabilization of a chimeric enzyme by residue substitutions: four amino acid residues in loop regions are responsible for the thermostability of Thermus thermophilus isopropylmalate dehydrogenase.
A chimeric 3-isopropylmalate dehydrogenase, named 2T2M6T, made of parts from an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis, was found to be considerably more labile than the T. thermophilus wild-type isopropylmalate dehydrogenase. In order to identify the molecular basis of the thermal stability of the T. thermophilus isopropylmalate dehydrogenase, 11 amino acid residues in the mesophilic portion of the chimera were substituted by the corresponding residues of the T. thermophilus enzyme, and the effects of the side chain substitutions were analyzed by comparing the reaction rate of irreversible heat denaturation and catalytic parameters of the mutant chimeras with those of the original chimera, 2T2M6T. Four single-site mutants were successfully stabilized without any loss of the catalytic function. All these four sites are located in loop regions of the enzyme. Our results strongly suggest the importance of these loop structures to the extreme stability of the T. thermophilus isopropylmalate dehydrogenase.
Numata
K
K
Department of Life Science, Tokyo University of Technology, Yokohama, Japan.
Hayashi-Iwasaki
Y
Y
Kawaguchi
J
J
Sakurai
M
M
Moriyama
H
H
Tanaka
N
N
Oshima
T
T
D050539:3-Isopropylmalate Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D001120:Arginine / Q000737:chemistry; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D005998:Glycine / Q000737:chemistry; D006358:Hot Temperature; D007930:Leucine / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D055672:Static Electricity; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D001120:Arginine; D000429:Alcohol Oxidoreductases; D050539:3-Isopropylmalate Dehydrogenase; D007930:Leucine; D005998:Glycine