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

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 (Zea mays) C₄-NADP-ME activity is much more elaborate than previously thought. Using mass spectrometry, we identified phosphorylation of the Ser419 residue of C₄-NADP-ME in protein extracts of maize leaves. The phosphorylation event increases in the light, with a peak at Zeitgeber time 2. 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 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 ZmC₄-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 CO₂ fixation rate, probably to avoid CO₂ leakiness from bundle sheath cells.

Figure 1.

TripleTOF 6600 Tandem MS Data of the Phosphopeptide acVWLVDpSK of ZmC₄-NADP-ME. The detected b (N-terminal, in red) and y (C-terminal, in blue) fragment ions are labeled in the spectrum. Ac denotes N terminus acetylation and pS denotes phosphorylated Ser. Precursor charge: +2; monoisotopic m/z: 484.7265 D (−1.60 milli-mass unit/−3.30 ppm). Confidence (ProteinPilot): 96.4% (confidence threshold for FDR ≤ 1% = 93.7%).

Figure 2.

Biochemical Characterization of Recombinant ZmC₄-NADP-ME Versions. (A) Dependence of the activity, measured using 0.5 mM NADP and 4 mM malate, on the pH of the assay. Values represent the mean ± se of at least four independent enzyme preparations, each measured in triplicate. V_o, initial velocity. (B) Kinetic parameters at pH 8.0. Kinetic data were best fitted by nonlinear regression analysis. Values represent the mean ± se of at least three independent enzyme preparations, each measured in triplicate.

Figure 3.

Exploration of the Secondary Structure Organization and Quaternary Composition of Recombinant ZmC₄-NADP-ME Versions. (A) and (B) CD spectra of ZmC₄-NADP-ME wild type (WT) and the two mutated versions obtained at pH 7.0 (A) and pH 8.0 (B). Ten accumulations each were collected from 190 to 260 nm for 0.16 mg mL⁻¹ enzyme, at 20°C. Each graph is showing the reconstructed curves obtained by applying CONTIN/LL algorithm for data evaluation as provided by the Dichroweb server. MRE, molar residue ellipticity. (C) and (D) Continuous sedimentation coefficient distribution of ZmC₄-NADP-ME wild type (WT) and the mutated versions at pH 7.0 (C) and pH 8.0 (D). Data were fitted with the ls-g*(S) model in the software package SEDFIT.

Figure 4.

Measurements of Different Parameters in Extracts of Maize Leaves during a Diurnal Cycle. (A) Quantification of total ZmC₄-NADP-ME protein by SWATH-MS. (B) Quantification of the phosphopeptide VWLVDpSK, corresponding to the phosphorylation of ZmC₄-NADP-ME at S419. (C) Total NADP-ME activity measured with 1.5 to 2.0 µg of protein extracts. (D) Malate concentration assayed by GC-MS analysis. Values represent mean ± se of four (see [A] and [B]) or three (see [C] and [D]) biological replicates. The dark period is highlighted in gray. Statistical analyses were performed in all cases (see [A] to [D]) against the first time point in the night (ZT16.5 = 16.5 h). Letters indicate that the value is statistically significant at 0.05 (a), 0.01 (b), and 0.001 (c) levels (the precise P-values are shown in Supplemental Table 3). FW, fresh weight.

Figure 5.

SbC₄-NADP-ME Crystal Structure and Amino Acids Involved in Cofactor Binding. (A) Cartoon and ribbon representation of SbC₄-NADP-ME. Monomer A (red) and B (blue) with bound cofactor NADP (colored sticks). Large parts of chains C and D are not well resolved by electron density; therefore, we depicted these chains as gray ribbons only to clarify the tetrameric assembly. (B) Monomer A from SbC₄-NADP-ME as surface representation with the bound cofactor NADP (represented by sticks). (C) Residues involved in cofactor binding are depicted as sticks with labels (distances are omitted for clarity and are listed in Supplemental Table 2).

Figure 6.

Effective Free Energies of Binding of NADP. (A) Effective binding energies computed according to the MM-PBSA approach for wild-type (WT) ZmC₄-NADP-ME, the phosphomimetic variant S419E, and pS419. The error bars indicate the se of the mean over 10 individual trajectories. Statistical significance was calculated according to Student’s t test. ΔG_eff, effective binding energy. (B) Contribution of the different energy terms, computed as variant’s (Var) energy term minus the respective energy term of the wild-type (WT) enzyme [ΔΔG_eff. = ΔG_eff(Var) − ΔG_eff(WT)]. Positive terms indicate more favorable binding to the WT enzyme, negative terms to the variant. The error bars show the se of the mean of the differences in the individual terms over the trajectories. ΔΔG_eff, relative binding free energy. (C) Molecular representation of NADP bound to the wild-type (WT) ZmC₄-NADP-ME, the phosphomimetic variant S419E, and pS419. Interactions with residue 419 are highlighted, showing hydrogen bonds for the WT enzyme with green dashed lines and charge-charge repulsion for the phosphomimetic and phosphorylated variants with red dashed lines.