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Abstract The evolution of C4 photosynthesis required extensive modification of ancestral enzymes enabling the development of an efficient carbon concentrating mechanism. A key example is NADP-malic enzyme (NADP-ME), which, in maize and sorghum—members of the same C4 lineage—underwent gene duplication and neofunctionalization, resulting in 2 plastidic isoforms with distinct oligomeric states: a tetrameric C4-specific isoform and a dimeric housekeeping (nonC4) isoform. In this study, we resolve the structural basis of this oligomeric divergence using X-ray crystallography, cryo-electron microscopy, and molecular modeling combined with targeted biochemical analysis. Our findings demonstrate that the N-terminal region of nonC4-NADP-ME is involved in its oligomeric organization, whereas a suite of adaptive substitutions at the dimer interface drives the transition to the stable tetramer characteristic of the C4 isoform. Moreover, the C-terminal region stabilizes the oligomeric states of C4- and nonC4-NADP-ME through specific interactions with adaptive residues. We propose that tetramerization mitigates aggregation at the high expression levels demanded by the C4 cycle and likely creates a scaffold for the emergence of regulatory properties. Collectively, the data show that remodeling of terminal domains and inter-subunit interfaces rewires the quaternary architecture of the enzymes, illustrating how subtle structural changes can drive the evolution of complex innovations such as C4 photosynthesis.

The C4 carbon concentrating mechanism relies on specialized enzymes that have evolved unique expression patterns and biochemical properties distinct to their ancestral housekeeping forms. In maize and sorghum, the evolution of C4-NADP-malic enzyme (C4-NADP-ME) involved gene duplication and neofunctionalization, leading to the emergence of two plastidic isoforms: C4-NADP-ME and nonC4-NADP-ME, each with distinct kinetic and structural features. While C4-NADP-ME functions primarily as a tetramer, nonC4-NADP-ME exists in an equilibrium between dimeric and tetrameric forms, favoring the dimer in solution. This study shows which evolutionary changes in amino acid sequences influence the structure and function of these isoforms. By integrating X-ray crystallography, cryo-electron microscopy, computational molecular modeling and targeted biochemical analysis of mutant and truncated protein variants, we identify crucial roles for the N- and C-terminal regions and specific amino acid residues in governing isoform oligomerization. Our results reveal that the N-terminal region is essential for stabilizing the dimeric form of nonC4-NADP-ME, whereas specific adaptive substitutions and interactions with the C-terminal region enhance the stability of the tetrameric state characteristic of the C4-adapted isoform. We propose that differences in the N-terminal domain between the C4 and nonC4 isoforms reflect distinct selective pressures, which have driven their evolutionary divergence to fulfill specialized cellular functions.Competing Interest StatementThe authors have declared no competing interest.