Evolutionary optimization of enzymes for their operation in the C4 photosynthetic pathway: the case of NADP-malic enzyme
Plant Biochemistry and Biophysics
Final Report Abstract
We focused our research on the structural evolution of C4-NADP-ME, specifically addressing Tasks 1, 2, and 4 and the regulatory effect of malate, addressing Task 3. Using advanced structural biology techniques - including X-ray crystallography, cryo-electron microscopy (Cryo-EM), and computational molecular modeling - alongside detailed biochemical analyses of targeted mutants, we identified critical roles for the N-terminal region and specific amino acid residues in regulating isoform oligomerization. Our findings reveal that the N-terminal region is essential for stabilizing the dimeric form of nonC4-NADP-ME, whereas specific adaptive mutations enhance the tetrameric stability unique to C4-NADP-ME. This differential evolution of the N-terminal domains suggests distinct selective pressures on C4 and non-C4 isoforms, likely reflecting their specialized tissue-specific roles. Recently, we obtained high-resolution structural insights into nonC4-NADP-ME using X-ray crystallography and Cryo-EM. We are currently refining these structural data. Additionally, we completed Task 3, investigating the molecular mechanisms behind malate inhibition. Our results demonstrate that proline residue 433 does not play a role in malate inhibition. Through tryptophan fluorescence quenching assays, thermal shift analyses, and molecular dynamics simulations, we found that distinct interactions of malate at the active site, modulated by its binding at the putative allosteric site, likely account for the observed pH-dependent regulatory behaviors in C4- and nonC4-NADP-ME isoforms. Furthermore, the dimeric C4 mutant G200R does not exhibit malate inhibition, leading us to hypothesize that tetramerization of C4-NADP-ME may be linked to the acquisition of this regulatory trait. Future studies will involve detailed structural and functional analyses of C4-NADP-ME mutants and exploration of additional residues potentially involved in regulation. Our project findings have been presented at various congresses and meetings, leading to invitations to contribute a review on the adaptive diversity in structure and function of C4 photosynthetic components.
Publications
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Adaptive diversity in structure and function of C4 photosynthetic components. Biochemical Society Transactions, 51(3), 1157-1168.
Alvarez, Clarisa E. & Maurino, Veronica G.
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Crystal structure of the G200R mutant from the maize chloroplastic photosynthetic NADP(+)-dependent malic enzyme. Worldwide Protein Data Bank. Worldwide Protein Data Bank.
Klinke, S.; Schneberger, N.; Boehm, J.M.; Willms, S.; Hagelueken, G.; Geyer, M.; Maurino, V. & Alvarez, C.E.
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Lighting the way: Compelling open questions in photosynthesis research. The Plant Cell, 36(10), 3914-3943.
Eckardt, Nancy A.; Allahverdiyeva, Yagut; Alvarez, Clarisa E.; Büchel, Claudia; Burlacot, Adrien; Cardona, Tanai; Chaloner, Emma; Engel, Benjamin D.; Grossman, Arthur R.; Harris, Dvir; Herrmann, Nicolas; Hodges, Michael; Kern, Jan; Kim, Tom Dongmin; Maurino, Veronica G.; Mullineaux, Conrad W.; Mustila, Henna; Nikkanen, Lauri; Schlau-Cohen, Gabriela ... & Yano, Junko
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From dimer to tetramer: the evolutionary trajectory of C4 photosynthetic-NADP-ME oligomeric state in Poaceae. openRxiv.
Böhm, Jonas M.; Willms, Simone; Ferrao, Oja; Buitrago-Arango, Martin; Hüdig, Meike; Poschmann, Gereon; Fazelnia, Nazanin; Nagel-Steger, Luitgard; Klinke, Sebastián; Drakonaki, Athina; Gatsogiannis, Christos; Tronconi, Marcos A.; Alvarez, Clarisa E. & Maurino, Veronica G.
