Fig 2: Structure of the hMESH1 (D66K)-NADPH complex.a, The architecture of the hMESH1 active site. MESH1 is shown in the ribbon diagram, NADPH in the stick model, and the Zn2+ (grey) and Na+ (pink) ions are shown as spheres. Secondary structures are labeled, with MESH1-NADPH-interacting motifs annotated in magenta. b, Coordination of the active site Zn2+ ion in a distorted octahedral geometry. NADPH and sidechains of the Zn2+-binding residues are shown in the stick model. The zinc ion (grey) and its coordinating water molecule (red) are shown as spheres. The signature ‘HD’ motif is annotated in blue. c, The schematic illustration of the NADPH recognition by MESH1. Polar interactions are denoted with dashed lines, and vdW contacts are shown with curved lines. The locations of the metal ions and NADPH-interacting water molecules are denoted as circles. d, Molecular recognition of the 2’-phospho-adenosine diphosphate moiety. e, Molecular recognition of the nicotinamide riboside moiety.

Fig 3: MESH1 contributes to the NADPH phosphatase activity in cells.NADPH phosphatase activity of HEK-293T was estimated by incubating NADPH with cell lysates with different MESH1 status; phosphate release over time was measured by the malachite green assay and normalized by the total lysate protein (μmol/min/mg total lysate protein). a, Significantly decreased cellular NADPH phosphatase activity by silencing MESH1 in HEK-293T cells. b, Increased cellular NADPH phosphatase activity with overexpression of WT MESH1, but not with overexpression of enzymatically deficient MESH1 (MESH1-E65A) in HEK-293T cells. Black dots represent siNT or empty vector samples, blue dots represent siMESH1 samples, red dots represent MESH1-WT samples, and grey dots represent MESH1-E65A samples. Error bars represent s.d. (n=3 biologically independent samples per condition). Statistical analysis: one way ANOVA with Tukey HSD post-hoc, **P<0.01, N.S., not significant.

Fig 4: MESH1-depletion increased the ratio of reduced glutathione over oxidized glutathione.a-d, The level of indicated metabolites in RCC4 transfected with non-targeting (NT) siRNA or MESH1-targeting siRNA were quantified by mass spectrometry and normalized by DNA content, a, cysteine; b, oxidized glutathione (GSSG); c, reduced glutathione (GSH) (effect size 1.746, p=0.0491); and d, normalized intensity ratio of GSH/GSSG. n=5 biologically independent samples per condition. Each box plot was defined by a lower line (at the 25th percentile), a central line (at the centre), and a higher line (at the 75th percentile). The whisker boundaries marked the minima and the maxima of the data. Statistical analysis: two-tail student’s t-test, *P<0.05.

Fig 5: MESH1 is a mammalian NADPH phosphatase.a, Chemical similarity between NADPH and ppGpp, and the proposed chemical reaction of MESH1. b, Detection of the hMESH1-catalyzed phosphate release from NADPH by color change (yellow to green) in the malachite green assay. c, Linear product accumulation of the NADPH dephosphorylation reaction catalyzed by hMESH1 (n=3 biologically independent samples, error bars indicate s.d.). d, Validation of the formation of NADH by mass spectrometry analysis. This experiment was repeated for three independent times with similar results. e, Enzymatic characterization of hMESH1 toward NADPH and NADP+ (mean ± s.d., n=4 independent experiments). f, Effects of Zn2+ substitution and active site mutation on the specific activity of purified recombinant hMESH1 (mean ± s.d., n=3 independent experiments). g. hMESH1 is a significant contributor to the NADPH phosphatase activity in RCC4 lysates. Black dots represent siNT samples, and blue dots represent siMESH1 samples. n=4 biologically independent samples for each condition; Samples were analyzed by Western blot to confirm knock-down efficiency. h, Cell fractionation separating cytosol from nucleus and mitochondria, showing that MESH1 is specifically enriched in the cytosolic compartment. This representative western blot was repeated independently for 3 times on RCC4 cells and HEK-293T cells with similar results. Error bars indicate s.d. One-way ANOVA with Tukey HSD post-hoc test, *P<0.05, **P<0.01.