Presentation Information
[1ASPR-17]Structural optimization of the RubisCO large subunit (RbcL) in Nicotiana benthamiana using in silico analysis
○Misaki Kobayashi1 (1. Graduate School of Life and Earth Sciences, University of Tsukuba (Japan))
Keywords:
RubisCO,optimization,FoldX,EZSpecificity
[Purpose] To generate Nicotiana benthamiana plants with improved water use efficiency (WUE), which is the ratio of carbon gained through photosynthesis to water lost via transpiration, we aimed to optimize the structure of RubisCO to increase photosynthetic activity.
[Methode] Focusing on the activated and inactivated states of RubisCO, we analyzed the L2 structural model in which loop 6 and the 60S loop adopt open and closed conformations. Using the open and closed structures of spinach RubisCO as templates, structural models of NbRbcL in both conformational states were generated. FoldX and EZSpecificity were used to analyze and compare the open and closed states of wild-type (WT) NbRbcL and six single–amino acid substitution mutants.
[Results] FoldX analysis predicted that L74F, R312C, and P415L substantially destabilized the structure in both open and closed states compared with WT. In contrast, D72N, D397N, and A414T were predicted to either stabilize the structure or not cause significant destabilization. Prediction of enzyme–substrate specificity for the substrate RuBP using EZSpecificity showed that the difference in scores between open and closed states was greater in D72N, D397N, and A414T than in WT. Furthermore, substrate specificity toward RuBP was predicted to be higher in the closed state for D72N and A414T, and higher in the open state for D397N.
[Consideration] In A. thaliana, D72N, D397N, and A414T exhibited RubisCO content comparable to Col-0, whereas the other lines showed reduced levels. L74F, R312C, and P415L displayed growth inhibition under ambient air conditions, reduced Kcat, decreased electron transport rate (PSII), and reduced WUE expression. These results suggest that structural destabilization and impaired folding in L74F, R312C, and P415L may have promoted RubisCO degradation, leading to reduced RubisCO content and/or catalytic activity. In A. thaliana, D72N and A414T showed growth inhibition under ambient air, reduced Kcat, and decreased PSII. Since these mutants show higher RuBP affinity in the closed state, RuBP may dissociate more slowly than in WT, hindering reopening of the active site and reducing catalytic turnover. In contrast, D397N exhibited enhanced growth, increased Kcat, increased PSII, and elevated WUE expression. As this mutant was predicted to have higher affinity for RuBP in the open state, RuBP may bind more readily to the active site than in WT, resulting in enhanced catalytic efficiency.
[Conclusion] Structural stability predictions using FoldX and enzyme–substrate specificity predictions using EZSpecificity suggest that an ideal RbcL is structurally stable, with high RuBP affinity in the open state and low affinity in the closed state.
[Methode] Focusing on the activated and inactivated states of RubisCO, we analyzed the L2 structural model in which loop 6 and the 60S loop adopt open and closed conformations. Using the open and closed structures of spinach RubisCO as templates, structural models of NbRbcL in both conformational states were generated. FoldX and EZSpecificity were used to analyze and compare the open and closed states of wild-type (WT) NbRbcL and six single–amino acid substitution mutants.
[Results] FoldX analysis predicted that L74F, R312C, and P415L substantially destabilized the structure in both open and closed states compared with WT. In contrast, D72N, D397N, and A414T were predicted to either stabilize the structure or not cause significant destabilization. Prediction of enzyme–substrate specificity for the substrate RuBP using EZSpecificity showed that the difference in scores between open and closed states was greater in D72N, D397N, and A414T than in WT. Furthermore, substrate specificity toward RuBP was predicted to be higher in the closed state for D72N and A414T, and higher in the open state for D397N.
[Consideration] In A. thaliana, D72N, D397N, and A414T exhibited RubisCO content comparable to Col-0, whereas the other lines showed reduced levels. L74F, R312C, and P415L displayed growth inhibition under ambient air conditions, reduced Kcat, decreased electron transport rate (PSII), and reduced WUE expression. These results suggest that structural destabilization and impaired folding in L74F, R312C, and P415L may have promoted RubisCO degradation, leading to reduced RubisCO content and/or catalytic activity. In A. thaliana, D72N and A414T showed growth inhibition under ambient air, reduced Kcat, and decreased PSII. Since these mutants show higher RuBP affinity in the closed state, RuBP may dissociate more slowly than in WT, hindering reopening of the active site and reducing catalytic turnover. In contrast, D397N exhibited enhanced growth, increased Kcat, increased PSII, and elevated WUE expression. As this mutant was predicted to have higher affinity for RuBP in the open state, RuBP may bind more readily to the active site than in WT, resulting in enhanced catalytic efficiency.
[Conclusion] Structural stability predictions using FoldX and enzyme–substrate specificity predictions using EZSpecificity suggest that an ideal RbcL is structurally stable, with high RuBP affinity in the open state and low affinity in the closed state.
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