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Abstract

Evolution of the C4 photosynthetic pathway involved in some cases recruitment of housekeeping proteins through gene duplication and their further neofunctionalization. NADP-malic enzyme (ME), the most widespread C4 decarboxylase, has increased its catalytic efficiency and acquired regulatory properties that allowed it to participate in the C4 pathway. Here, we show that regulation of maize C4-NADP-ME activity is much more elaborated than until now indicated. Using mass spectrometry, we identified phosphorylation of the serine 419 (S419) of C4-NADP-ME in protein extracts of maize leaves. The phosphorylation event increases after the light turns on, with a peak at ZT2. Phosphorylation of ZmC4-NADP-ME drastically decreases its activity as shown by the low residual activity of the recombinant phosphomimetic mutant. Analysis of the crystal structure of C4-NADP-ME indicated that S419 is involved in the binding of NADP at the active site. Molecular dynamics simulations and effective binding energy computations indicate a less favorable binding of the cofactor NADP in the phosphomimetic and the phosphorylated variants. We propose that phosphorylation of ZmC4-NADP-ME at S419 during the first hours in the light is a cellular mechanism to fine-tune the enzymatic activity to coordinate the carbon concentration mechanism with the CO2 fixation rate, most probably to avoid CO2 leakiness from bundle sheath cells.