Mutation design of a thermophilic Rubisco based on three-dimensional structure enhances its activity at ambient temperature

Masahiro Fujihashi, Yuichi Nishitani, Tomohiro Kiriyama, Riku Aono, Takaaki Sato, Tomoyuki Takai, Kenta Tagashira, Wakao Fukuda, Haruyuki Atomi, Tadayuki Imanaka, Kunio Miki*

*この研究の対応する著者

研究成果: Article査読

6 被引用数 (Scopus)

抄録

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.

本文言語English
ページ(範囲)1339-1346
ページ数8
ジャーナルProteins: Structure, Function and Bioinformatics
84
10
DOI
出版ステータスPublished - 2016 10月 1
外部発表はい

ASJC Scopus subject areas

  • 構造生物学
  • 生化学
  • 分子生物学

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