Posttranslational Modification of the NADP-Malic Enzyme Involved in C₄ Photosynthesis Modulates the Enzymatic Activity during the Day

Abstract

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.

Developmental Effects on Relative Use of PEPCK and NADP-ME Pathways of C4 Photosynthesis in Maize

“…Less consideration has been given to the plasticity of the C 4 pathways within a species. NADP-ME is strongly light regulated by light quality (Casati et al, 1998) and quantity (Hatch and Kagawa, 1976;Bellasio and Griffiths, 2014b), and by time of day, through phosphorylation, with its activity peaking at two hours after dawn and decreasing slowly throughout the day (Bovdilova et al, 2019). Leaves positioned lower in the canopy may not receive the quality and quantity of light to activate NADP-ME to the levels observed at the top of the canopy (Figure 4, Figure 6).…”

A Partial C4 Photosynthetic Biochemical Pathway in Rice

“…Both enzymes are regulated in the same manner even when expressed within C 3 leaves ( Fukayama et al, 2001 ; Taniguchi et al, 2008 ). A recent study has shown that C 4 NADP-ME is also regulated in the light by reversible phosphorylation at Ser419 which is involved in the binding of NADP at the active site ( Bovdilova et al, 2019 ). In contrast, PEPC is regulated by both metabolite effectors and reversible phosphorylation, but the mechanisms of regulation in C 3 and C 4 leaves are different ( Vidal and Chollet, 1997 ).…”

Investigating LGALS3BP/90 K glycoprotein in the cerebrospinal fluid of patients with neurological diseases

“…Trapping was performed at 2 µl/min on a Nano cHiPLC Trap column (Sciex 200 µm × 0.5 mm, ChromXP C18-CL, 3 µm, 120 Å) with 100% A for 10 min. The separation was performed at 300 nl/min, on a Nano cHiPLC column (Sciex 75 µm × 15 cm, ChromXP C18-CL, 3 µm, 120 Å) 40 . The gradient was as follows: 0-1 min, 5% B (0.1% formic acid in acetonitrile, Fisher Chemicals, Geel, Belgium); 1-46 min, 5-35% B; 46-48 min, 35-80% B; 48-54 min, 80% B; 54-57 min, 80-5% B; 57-75 min, 5% B.…”

“…A UniProt reviewed database (20,413 entries, accessed on 08/01/2019) containing the sequences of the proteins from Human was used. The following search parameters were set: Iodoacetamide, as Cys alkylation; Trypsin, as digestion; TripleTOF 6600, as the Instrument; gel-based ID as Special factors; Biological modifications as ID focus; Search effort, as thorough; and an FDR analysis 40 . Only the proteins with Unused Protein Score above 1.3 and 95% confidence were considered.…”