title: Thermostability of ancestral mutants of Caldococcus noboribetus isocitrate dehydrogenase.
abstract: 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.
authors.affiliation: Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
authors.forename: Hisako
authors.initials: H
authors.lastname: Iwabata
authors.forename: Keiko
authors.initials: K
authors.lastname: Watanabe
authors.forename: Takatoshi
authors.initials: T
authors.lastname: Ohkuri
authors.forename: Shin-Ichi
authors.initials: S
authors.lastname: Yokobori
authors.forename: Akihiko
authors.initials: A
authors.lastname: Yamagishi
mesh_terms: 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
chemical_list: D000429:Alcohol Oxidoreductases; D007521:Isocitrate Dehydrogenase; D050539:3-Isopropylmalate Dehydrogenase