Integrative crystallography and molecular dynamics reveal isoform-specific ligand interaction networks in C4 and...

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 Mg²⁺, 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⁺-Mg²⁺ complexes by docking followed by MD, identifying distinct malate-Mg²⁺ 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.