Molecular adaptations of NADP-malic enzyme for its function in C₄ photosynthesis in grasses

In C₄ grasses of agronomical interest, malate shuttled into the bundle sheath cells is decarboxylated mainly by nicotinamide adenine dinucleotide phosphate (NADP)-malic enzyme (C₄-NADP-ME). The activity of C₄-NADP-ME was optimized by natural selection to efficiently deliver CO₂ to Rubisco. During its evolution from a plastidic non-photosynthetic NADP-ME, C₄-NADP-ME acquired increased catalytic efficiency, tetrameric structure and pH-dependent inhibition by its substrate malate. Here, we identified specific amino acids important for these C₄ adaptions based on strict differential conservation of amino acids, combined with solving the crystal structures of maize and sorghum C₄-NADP-ME. Site-directed mutagenesis and structural analyses show that Q503, L544 and E339 are involved in catalytic efficiency; E339 confers pH-dependent regulation by malate, F140 is critical for the stabilization of the oligomeric structure and the N-terminal region is involved in tetramerization. Together, the identified molecular adaptations form the basis for the efficient catalysis and regulation of one of the central biochemical steps in C₄ metabolism.