Skip to main content
. 2019 Aug 22;8:e43582. doi: 10.7554/eLife.43582

Figure 5. The AtVIH2 kinase domain has 1-kinase activity and produces 1PP-InsP5 and InsP8, the phosphatase domain is a 1 - and 5 - pyrophosphatase.

(A, B) 2D 1H-13C-HMBC spectra of the products produced by plant AtVIH2-KD (blue trace) and human HsPPIP5K2-KD (red trace) in the presence of InsP6 (A) or 5PP-InsP5 (B). Substrate standards are colored in yellow. (C) Decay of the InsP6 or 5-PP-InsP5 substrate during the NMR-time course experiment shown in (A) and (B), respectively. A fit of the initial decay indicates a turnover number of ~0.4/min with InsP6 as a substrate and ~1/min using 5-PP-InsP5 as a substrate. (D) Qualitative native PAGE phosphatase activity assay. Reactions containing recombinant phosphatase domain of AtVIH2 (AtVIH2-PD,~5 µg) or ScVip1 (ScVip1-PD,~27 µg) were incubated with 175 μM of either InsP6 (black), 1PP-InsP5 (blue), 5PP-InsP5 (gray) or a non-hydrolyzable 5PCP-InsP5 analog (orange) for 16 hr at 37°C. 40 μl of the reaction were separated in a 35% acrylamide gel. The bands corresponding to InsP6 and 1 or 1/5PP-InsP5 are indicated by an arrowhead. (E) Malachite green-based phosphatase activity assay. Reactions containing recombinant AtVIH2-PD (~5 µg) or ScVip1-PD (~27 µg) were incubated with 175 μM InsP6, 1PP-InsP5, 5PP-InsP5 or 5PCP-InsP5 for 16 hr at 37°C (left). 1 mM Mg2+ or 5 mM EDTA were supplemented as indicated (right; 5PP-InsP5 only). Recombinant HsPPIP5K2-KD (~17 µg) was used as a negative control and tested only with 5PP-InsP5 (right). Reactions were performed in quadruplicates and released orthophosphate was quantified using a malachite green assay (Baykov et al., 1988). (F) NMR time course experiment comparing the phosphatase activities of 2 µM ScVip1-PD and ScVip1-PDRH/AA using 40 µM [13C6]5PP-InsP5 as substrate. Samples were measured in a pseudo-2D spin-echo difference experiment and the relative intensities of the C2 peaks of InsP6 and 5PP-InsP5 were quantified.

Figure 5.

Figure 5—figure supplement 1. The purification of recombinant proteins.

Figure 5—figure supplement 1.

(A) Size-exclusion chromatography traces of purified recombinant HsPPIP5K2 kinase domain (HsPPIP5K2-KD, residues 41–366), AtVIH2 kinase domain (AtVIH2-KD, residues 11–338), AtVIH2 kinase domain mutant (AtVIH2-KDKD/AA, residues 11–338), and CrVip1 kinase domain from Chlamydomonas (CrVip1-KD, residues 20–350). Fractions isolated for enzymatic assays are indicated below. (B) Size-exclusion chromatography traces of purified recombinant AtVIH2 phosphatase domain (AtVIH2-PD, Gly359-Arg1002), AtVIH2 phosphatase domain mutant (AtVIH2-PDRH/AA, residues 359–1002), ScVip1 phosphatase domain (ScVip1-PD, residues 515–1088), and ScVip1 phosphatase domain mutant (ScVip1-PDRH/AA, residues 515–1088). For enzyme activity assays, the indicated fractions containing the purified recombinant protein were pooled (fractions A12-A15 for AtVIH2-PD; A12-B13 for AtVIH2-PDRH/AA; and A15-B13 for both ScVip1-PD and ScVip1-PDRH/AA). (C) Coomassie-stained SDS-PAGE gel showing the recombinant proteins used in this study. For each lane, around 3 μg of protein from the respective concentrated fractions were loaded.
Figure 5—figure supplement 2. 1D 31P and 2D 1H-31P-HMBC spectra of the products produced by AtVIH2-KD, and 2D 1H-13C-HMBC spectra of the products produced by plant AtVIH2-KDKD/AA.

Figure 5—figure supplement 2.

(A,B) 1D 31P spectra of the products produced by the plant AtVIH2-KD in the presence of InsP6 (A) and 5PP-InsP5 (B). Inset panels shows the 2D 1H-31P-HMBC spectra. 1,5(PP)-InsP4 refers to InsP8. (C, D) Decay of the InsP6 (C) or 5-PP-InsP5 (D) substrate during the NMR-time course experiment. (E, F) 2D 1H-13C-HMBC spectra of the products produced by plant AtVIH2-KDKD/AA (blue trace) in the presence of InsP6 (E) or 5PP-InsP5 (F).
Figure 5—figure supplement 3. The ScVip1-PDRH/AA and AtVIH2-PDRH/AA recombinant enzymes show reduced phosphatase activity.

Figure 5—figure supplement 3.

Qualitative native PAGE phosphatase activity assay. Reactions containing 2 µM ScVip1-PD (black), ScVip1-PDRH/AA(gray), AtVIH2-PD (cyan) and AtVIH2-PDRH/AA (magenta) were incubated with 80 μM 5PP-InsP5 for 16 hr at 37°C. The reactions were then separated in a 35% acrylamide gel. The bands corresponding to InsP6 and 1 or 5PP-InsP5 are indicated by an arrow head.