Thermostability of ancestral mutants of Caldococcus noboribetus isocitrate dehydrogenase.
We constructed mutant genes of Caldococcus noboribetus isocitrate dehydrogenase containing ancestral amino acid residues that were inferred using the maximal likelihood method and a composite phylogenetic tree of isocitrate dehydrogenase and 3-isopropylmalate dehydrogenase. The mutant genes were expressed in Escherichia coli and the protein products purified. Thermostabilities, reported as the half-inactivation temperatures, for the purified enzymes were determined and compared with that of the wild-type enzyme. Four of the five mutant enzymes have greater thermal stabilities than wild-type isocitrate dehydrogenase. The results are compatible with the hyperthermophilic universal ancestor (commonote) hypothesis. Incorporation of ancestral residues into a modern-day protein sequence can be used to improve protein thermostability.
Iwabata
Hisako
H
Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Watanabe
Keiko
K
Ohkuri
Takatoshi
T
Yokobori
Shin-Ichi
S
Yamagishi
Akihiko
A
D050539:3-Isopropylmalate Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000235:genetics; D000595:Amino Acid Sequence; D041521:Desulfurococcales / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D019143:Evolution, Molecular; D006358:Hot Temperature; D007521:Isocitrate Dehydrogenase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010802:Phylogeny
D000429:Alcohol Oxidoreductases; D007521:Isocitrate Dehydrogenase; D050539:3-Isopropylmalate Dehydrogenase