Integrative crystallography and molecular dynamics reveal isoform‐specific ligand interaction networks in C4 and nonC4 plant NADP ‐malic enzyme

SUMMARY NADP‐dependent malic enzyme (NADP‐ME) has been repeatedly co‐opted into distinct metabolic roles across plants, most prominently as the decarboxylase of NADP‐ME‐type C4 photosynthesis. In maize, the plastidic C4‐ and nonC4‐NADP‐ME isoforms are closely related in sequence yet display strikingly different catalytic properties, suggesting that small changes in ligand recognition can re‐tune reaction chemistry. However, mechanistic interpretation has been hampered by the scarcity of plant NADP‐ME structures captured in catalytically informative, ligand‐bound states. Here, we integrate X‐ray crystallography with structure‐guided docking and atomistic molecular dynamics (MD) to resolve ligand‐site interaction networks across reaction states. We determined a 2.55 Å structure of maize plastidic nonC4‐NADP‐ME bound to NADP + , pyruvate, and Mg 2+ , revealing a conserved NADP‐ME fold with localized active‐site flexibility. Comparison with maize C4‐NADP‐ME uncovers isoform‐specific rewiring of NADP + and pyruvate contacts, with the nonC4 enzyme forming a denser product–cofactor interaction network. To access substrate‐bound states, we reconstructed malate‐NADP + ‐Mg 2+ complexes by docking followed by MD, identifying distinct malate‐Mg 2+ coordination geometries and alternative NADP + positioning between isoforms. Together, these structures and simulations provide a network‐level framework for plastidic NADP‐ME functional diversification and generate testable hypotheses for how ligand coordination drives isoform‐specific catalysis.