Mutation design of a thermophilic Rubisco based on three-dimensional structure enhances its activity at ambient temperature.
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) plays a central role in carbon dioxide fixation on our planet. Rubisco from a hyperthermophilic archaeon Thermococcus kodakarensis (Tk-Rubisco) shows approximately twenty times the activity of spinach Rubisco at high temperature, but only one-eighth the activity at ambient temperature. We have tried to improve the activity of Tk-Rubisco at ambient temperature, and have successfully constructed several mutants which showed higher activities than the wild-type enzyme both in vitro and in vivo. Here, we designed new Tk-Rubisco mutants based on its three-dimensional structure and a sequence comparison of thermophilic and mesophilic plant Rubiscos. Four mutations were introduced to generate new mutants based on this strategy, and one of the four mutants, T289D, showed significantly improved activity compared to that of the wild-type enzyme. The crystal structure of the Tk-Rubisco T289D mutant suggested that the increase in activity was due to mechanisms distinct from those involved in the improvement in activity of Tk-Rubisco SP8, a mutant protein previously reported to show the highest activity at ambient temperature. Combining the mutations of T289D and SP8 successfully generated a mutant protein (SP8-T289D) with the highest activity to date both in vitro and in vivo. The improvement was particularly pronounced for the in vivo activity of SP8-T289D when introduced into the mesophilic, photosynthetic bacterium Rhodopseudomonas palustris, which resulted in a strain with nearly two-fold higher specific growth rates compared to that of a strain harboring the wild-type enzyme at ambient temperature. Proteins 2016; 84:1339-1346. © 2016 Wiley Periodicals, Inc.
Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-Ku, Kyoto, 606-8502, Japan.
Masahiro
M
Fujihashi
Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-Ku, Kyoto, 606-8502, Japan.
Yuichi
Y
Nishitani
Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-Ku, Kyoto, 606-8502, Japan.
Tomohiro
T
Kiriyama
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Nishikyo-Ku, Kyoto, 615-8510, Japan.
Riku
R
Aono
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Nishikyo-Ku, Kyoto, 615-8510, Japan.
Takaaki
T
Sato
Department of Biotechnology, College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga, 525-8577, Japan.
Tomoyuki
T
Takai
Department of Biotechnology, College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga, 525-8577, Japan.
Kenta
K
Tagashira
Department of Biotechnology, College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga, 525-8577, Japan.
Wakao
W
Fukuda
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Nishikyo-Ku, Kyoto, 615-8510, Japan.
Haruyuki
H
Atomi
Department of Biotechnology, College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga, 525-8577, Japan.
Tadayuki
T
Imanaka
Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-Ku, Kyoto, 606-8502, Japan. miki@kuchem.kyoto-u.ac.jp.
Kunio
K
Miki
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012241:Rhodopseudomonas / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D018724:Spinacia oleracea / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D013329:Structure-Activity Relationship; D019712:Thermococcus / Q000737:chemistry / Q000201:enzymology / Q000235:genetics
D019843:Archaeal Proteins; D001426:Bacterial Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; D012273:Ribulose-Bisphosphate Carboxylase
carbon dioxide fixation; crystal structure; protein engineering; protein-ligand complex; ribulose-1,5-bisphosphate carboxylase/oxygenase