アオノ リク
青野 陸
AONO RIKU
所属 生命科学部 生物工学科
職名 助教
言語種別 英語
発行・発表の年月 2016/10
形態種別 研究論文(学術雑誌)
査読 査読あり
標題 Mutation design of a thermophilic Rubisco based on three-dimensional structure enhances its activity at ambient temperature
執筆形態 不明
掲載誌名 PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS
出版社・発行元 WILEY-BLACKWELL
巻・号・頁 84(10),1339-1346
著者・共著者 Masahiro Fujihashi,Yuichi Nishitani,Tomohiro Kiriyama,Riku Aono,Takaaki Sato,Tomoyuki Takai,Kenta Tagashira,Wakao Fukuda,Haruyuki Atomi,Tadayuki Imanaka,Kunio Miki
概要 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. (c) 2016 Wiley Periodicals, Inc.
DOI 10.1002/prot.25080
ISSNコード 08873585
PMID 27273261
PermalinkURL https://onlinelibrary.wiley.com/doi/abs/10.1002/prot.25080