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

Abstract

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.

An Insight of RuBisCO Evolution through a Multilevel Approach

“…Therefore, an increase in the loop flexibility between αB and βC, Lys322 or residues in the vicinity of the catalytic center is important to increase the catalytic activity of RuBisCO from T. kodakarensis at room temperature. Finally, our MD simulation results corroborate very well the work of Satagopan et al [ 63 ] and Fujihashi et al [ 12 ]. Likewise, the regions of greater flexibility and active sites exhibit highly correlated or anticorrelated movements between the different isoforms ( Figure 8 and Figure 9 ), building a dynamic correlation network where information signal are transmitted [ 48 ].…”

“…Thermococcus kodakarensis is a hyperthermophilic archaea. Its optimal growth is at 85 °C, and it can exceed the activity of RuBisCO spinach by 20 times [ 12 , 83 ], but at room temperature its activity is only one-eighth [ 12 ]. For this reason, it is necessary to develop new T. kodakarensis strains with good photosynthetic performance at room temperatures.…”

“…For this reason, it is necessary to develop new T. kodakarensis strains with good photosynthetic performance at room temperatures. In this sense, 3WQP T289D [ 12 ] and 3KDO SP6 [ 10 ] mutants were developed. Their results show an increase in the carboxylase activity of 24% and 31%, respectively.…”

“…Isoform III is found in archaeas and consists of a toroid-shaped pentagonal decamer composed of L subunits [ 9 ]. In addition, the enzyme shows extreme thermostability with high carboxylase activity at high temperatures [ 10 , 11 ] and exceeds the RuBisCO activity of spinach by 20 times, but it is not efficient at room temperature [ 12 ]. Moreover, it is not affected by the presence of oxygen [ 9 , 13 , 14 ].…”

Selection of Cyanobacterial ( Synechococcus sp. Strain PCC 6301) RubisCO Variants with Improved Functional Properties That Confer Enhanced CO 2 -Dependent Growth of Rhodobacter capsulatus, a Photosynthetic Bacterium

“…capsulatus, R. palustris, and R. eutropha have been exploited for selection studies with heterologous RubisCOs (23–25, 39, 47, 48). The absolute dependence on RubisCO for CO 2 -dependent growth, coupled with the ability to grow under heterotrophic growth conditions (i.e., RubisCO and the Calvin-Benson-Bassham [CBB] cycle are dispensable), allows for a convenient means to select suppressor mutations in RubisCO genes that overcome an initial negative-growth phenotype.…”