Search

In plant mitochondria, nicotinamide adenine dinucleotide-malic enzyme (NAD-ME) has a housekeeping function in malate respiration. In different plant lineages, NAD-ME was independently co-opted in C4 photosynthesis. In the C4 Cleome species, Gynandropsis gynandra and Cleome angustifolia, all NAD-ME genes (NAD-MEα, NAD-MEβ1, and NAD-MEβ2) were affected by C4 evolution and are expressed at higher levels than their orthologs in the C3 species Tarenaya hassleriana. In T. hassleriana, the NAD-ME housekeeping function is performed by two heteromers, NAD-MEα/β1 and NAD-MEα/β2, with similar biochemical properties. In both C4 species, this role is restricted to NAD-MEα/β2. In the C4 species, NAD-MEα/β1 is exclusively present in the leaves, where it accounts for most of the enzymatic activity. Gynandropsis gynandra NAD-MEα/β1 (GgNAD-MEα/β1) exhibits high catalytic efficiency and is differentially activated by the C4 intermediate aspartate, confirming its role as the C4-decarboxylase. During C4 evolution, NAD-MEβ1 lost its catalytic activity; its contribution to the enzymatic activity results from a stabilizing effect on the associated α-subunit and the acquisition of regulatory properties. We conclude that in bundle sheath cell mitochondria of C4 species, the functions of NAD-ME as C4 photosynthetic decarboxylase and as a housekeeping enzyme coexist and are performed by isoforms that combine the same α-subunit with differentially adapted β-subunits., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Evolution of the C 4 photosynthetic pathway involved in some cases recruitment of housekeeping proteins through gene duplication and their further neofunctionalization. NADP-malic enzyme (ME), the most widespread C 4 decarboxylase, has increased its catalytic efficiency and acquired regulatory properties that allowed it to participate in the C 4 pathway. Here, we show that regulation of maize ( Zea mays ) C 4 -NADP-ME activity is much more elaborate than previously thought. Using mass spectrometry, we identified phosphorylation of the Ser419 residue of C 4 -NADP-ME in protein extracts of maize leaves. The phosphorylation event increases in the light, with a peak at Zeitgeber time 2. Phosphorylation of ZmC 4 -NADP-ME drastically decreases its activity as shown by the low residual activity of the recombinant phosphomimetic mutant. Analysis of the crystal structure of C 4 -NADP-ME indicated that Ser419 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 ZmC 4 -NADP-ME at Ser419 during the first hours in the light is a cellular mechanism that fine tunes the enzymatic activity to coordinate the carbon concentration mechanism with the CO 2 fixation rate, probably to avoid CO 2 leakiness from bundle sheath cells., (© 2019 American Society of Plant Biologists. All rights reserved.)