Epistasis effects of multiple ancestral-consensus amino acid substitutions on the thermal stability of glycerol kinase from Cellulomonas sp. NT3060.
Thermostable variants of the Cellulomonas sp. NT3060 glycerol kinase have been constructed by through the introduction of ancestral-consensus mutations. We produced seven mutants, each having an ancestral-consensus amino acid residue that might be present in the common ancestors of both bacteria and of archaea, and that appeared most frequently at the position of 17 glycerol kinase sequences in the multiple sequence alignment. The thermal stabilities of the resulting mutants were assessed by determining their melting temperatures (Tm), which was defined as the temperature at which 50% of the initial catalytic activity is lost after 15 min of incubation, as well as when the half-life of the catalytic activity occurs at a temperature of 60°C (t1/2). Three mutants showed increased stabilities compared to the wild-type protein. We then produced five more mutants with multiple amino acid substitutions. Some of the resulting mutants showed thermal stabilities much greater than those expected given the stabilities of the respective mutants with single mutations. Therefore, the effects of mutations are not always simply additive and some amino acid substitutions, which do not affect or only slightly improve stability when individually introduced into the protein, show substantial stabilizing effects in combination with other mutations.
Graduate School of Environmental and Life Science, Okayama University, 1-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan.
Yasuhisa
Y
Fukuda
Graduate School of Environmental and Life Science, Okayama University, 1-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan.
Asuka
A
Abe
Graduate School of Environmental and Life Science, Okayama University, 1-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan.
Takashi
T
Tamura
Biochemical Department, Toyobo Co. Ltd., 2-2-8 Dojima Hama, Kita-ku, Osaka 530-8230, Japan.
Takahide
T
Kishimoto
Biochemical Department, Toyobo Co. Ltd., 2-2-8 Dojima Hama, Kita-ku, Osaka 530-8230, Japan.
Atsushi
A
Sogabe
Faculty of Human Sciences, Waseda University, 2-579-15 Mikajima, Tokorozawa, Saitama 359-1192, Japan.
Satoshi
S
Akanuma
Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Shin-Ichi
S
Yokobori
Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Akihiko
A
Yamagishi
Department of Macromolecular Science, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka 560-0043, Japan.
Katsumi
K
Imada
Graduate School of Environmental and Life Science, Okayama University, 1-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan. Electronic address: kinagaki@okayama-u.ac.jp.
Kenji
K
Inagaki
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D055162:Biocatalysis; D040143:Cellulomonas / Q000201:enzymology* / Q000235:genetics; D016384:Consensus Sequence / Q000235:genetics*; D004795:Enzyme Stability / Q000235:genetics; D004843:Epistasis, Genetic; D005991:Glycerol Kinase / Q000737:chemistry* / Q000235:genetics*; D006207:Half-Life; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D016415:Sequence Alignment; D013696:Temperature
D005991:Glycerol Kinase
Ancestral mutation; Cellulomonas; Diagnostic enzyme; Epistasis effect; Glycerol kinase; Thermostabilization