Role of PDXP in hippocampal PLP homeostasis.

(a) Age-dependent expression of PDXK and PDXP in murine hippocampi. Left panels, representative Western blots of three hippocampi for each genotype. The same blots were reprobed with α-actin antibodies as a loading control. The age of the investigated mice is indicated above the blots. Right panel, densitometric quantification of hippocampal PDXP and PDXK Western blot signals, corrected by the corresponding actin signals. Young mice were 18-42 days old, older mice were 252-351 days old; n=7 hippocampi were analyzed per group. Data are mean values ± S.D. Statistical analysis was performed with unpaired, two-sided t-tests; p-values are indicated. (b) Age-dependent, total PLP-concentrations in isolated hippocampi of PDXP-WT and PDXP-KO mice. PLP was derivatized with semicarbazide and analyzed by HPLC. Each symbol represents the result of the PLP determination in an individual hippocampus. Data were fitted by Gaussian least-squares analyses. (c) Determination of protein-bound and protein-depleted PLP in PDXP-WT and PDXP-KO hippocampal lysates of young (18-42 days old) and older mice (252-352 days old). The number of analyzed hippocampi is indicated in the bars. Data are mean values ± S.D. Statistical analysis was performed with two-way ANOVA and Tukey’s multiple comparisons test. Significant differences (adjusted P-values) in protein-depleted PLP levels are indicated. The exact age of analyzed mice is listed in Figure 1 – supplementary figure 1. Source data are available for this Figure.

Characterization of the 7,8-DHF/PDXP interaction.

(a) Determination of half-maximal inhibitory constants (IC50) of 7,8-DHF (2D-structure shown on top) for purified murine or human PDXP, using pyridoxal 5’-phosphate (PLP) as a substrate. Phosphatase activities in the presence of 7,8-DHF were normalized to the respective enzyme activities measured in the presence of the DMSO solvent control. Data are mean values ± S.D. of n=3 (human PDXP) and n=4 (murine PDXP) independent experiments. (b) IC50 values of different flavones for purified murine PDXP with PLP as a substrate. Phosphatase activities in the presence of flavones were normalized to the respective enzyme activities in the presence of the DMSO solvent control. All data are mean values ± S.D. The inhibition of PDXP by 3,7,8-trihydroxyflavone-4’-hydroxyphenyl (2D-structure shown on top) was assessed in n=6 independent experiments. All other data are from n=3 biologically independent experiments. Apparently missing error bars are hidden by the symbols. (c) Biolayer interferometry (BLI) measurements of the interaction of 7,8-DHF with purified murine PDXP. Left panel, example sensorgram overlayed with the global 1:1 binding model (red) and the negative control (gray). The dashed line indicates the start of the dissociation phase. Right panel, steady-state dose-response analysis for 7,8-DHF based on n=4 measurements. (d) Sensitivity of the indicated HAD phosphatases to 7,8-DHF. Phosphatase activities in the presence of 7,8-DHF were normalized to the respective enzyme activities measured in the presence of the DMSO solvent control. Data are mean values ± S.D. of n=4 (PGP) or n=3 independent experiments (all other phosphatases). Phosphatase substrates and cap types are indicated in the legend. PGP, phosphoglycolate phosphatase; LHPP, phospholysine phosphohistidine inorganic pyrophosphate phosphatase; PHOP2, phosphatase orphan 2; PSPH, phosphoserine phosphatase; MDP1, magnesium-dependent phosphatase-1. Source data are available for this Figure.

Kinetic constants of PDXP-catalyzed PLP hydrolysis in the presence of 7,8-DHF.

X-ray crystal structure of murine PDXP in complex with 7,8-DHF.

(a) The model was refined to a resolution of 2.0 Å (PDB code 8QFW). Protomer A of full-length, homodimeric PDXP is shown in pink and protomer B in green. 7,8-DHF is shown in sphere representation with its C-atoms in pink and the catalytically essential Mg2+ ions are shown as cyan spheres. (b) Structural overlay of apo-PDXP (PDB code 4BX3, chain A, in brown) and the 7,8-DHF-bound PDXP protomer A (in pink). 7,8-DHF is shown in stick representation with its C-atoms in pink. The catalytic Asp25 residues are shown in red or purple sticks, respectively, and the Mg2+ ion is depicted as a cyan sphere. The substrate specificity loops are highlighted in yellow. (c) 2Fo-Fc electron density map contoured at an RMSD of 1 in blue, overlaid with the refined model. The Fo-Fc map of 7,8-DHF contoured at an RMSD of 3 is shown with positive densities in green mesh. (d) Arg62 blocks 7,8-DHF binding in the B-protomer. A salt bridge between Arg62 (C-atoms shown as green spheres) in the B-protomer (in green) and Asp14 (C-atoms shown as pink spheres) of a symmetry-related A-protomer (in pink) blocks the 7,8-DHF binding site. 7,8-DHF (in stick representation with gray C-atoms) is modeled based on the A-protomer. (e) Comparison of the 7,8-DHF and PLP binding sites. The active site of 7,8-DHF-bound PDXP is in gray. 7,8-DHF is shown in stick representation (pink C-atoms), and the Mg2+ ion as a cyan sphere. PLP (in stick representation with blue C-atoms) was modelled based on a superposition of the human PDXP-PLP complex (PDB code 2CFT). (f) Structural details of bound 7,8-DHF and adjacent residues of the active site. (g) Verification of 7,8-DHF - PDXP interactions. Left panel, phosphatase activity of purified PDXP or the indicated PDXP variants. Data are mean values ± S.D. of n=3 independent experiments. Right panel, determination of the IC50 values of 7,8-DHF for purified PDXP or the indicated PDXP variants. Data are mean values ± S.D. of n=3 independent experiments. Apparently missing error bars are hidden by the symbols. Source data are available for this Figure.

Data Collection and Refinement Statistics.

Effect of 7,8-DHF on the PLP/PL ratio in cultured hippocampal neurons from WT or PDXP-KO mice.

(a) Effect of long-term PDXP deficiency on total PLP levels in hippocampal neurons. Data are mean values ± S.E. of n=4 independent experiments. Statistical significance was assessed with a two-tailed, unpaired t-test. A representative image of primary hippocampal neurons stained for the neuronal marker protein MAP2 is shown in the insert (pixel intensities were color-inverted for better visualization). Scale bar, 100 µm. (b) Western blot analysis of PDXP and PDXK expression in hippocampal neuron samples shown in (a). The same blots were reprobed with α-actin antibodies as a loading control. The densitometric quantification of PDXK signals is shown on the right; data are mean values ± S.E. of n=4 independent experiments. (c) Effect of 7,8-DHF (20 µM, 45 min) or the DMSO solvent control (0.02% v/v, 45 min) on the PLP/PL ratio in hippocampal neurons of PDXP-WT or PDXP-KO mice. Source data are available for this Figure.

Analysis of total hippocampal PLP levels in PDXP-WT and PDXP-KO mice.

Each value represents the result of the PLP determination in an individual hippocampus. Analysis for a statistically significant difference between PLP levels in PDXP-WT and PDXP-KO hippocampi (all ages combined; two-tailed, unpaired t-test) p<0.0001. Bold table entries indicate those hippocampal extracts that were further separated for an analysis of protein-depleted and protein-bound PLP (see Fig. 1c).

Identification of PDXP inhibitors.

A primary screen was conducted using 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as an artificial substrate. Out of 41,182 screened compounds, 256 compounds were discarded that showed very high autofluorescence (as recognized by elevated fluorescence at the start of the kinetic curve); 26 compounds showed statistically significant PDXP activation, and 255 compounds showed PDXP inhibition (as recognized by an elevated or decreased slope of the kinetic curve, respectively). The average Z’-factor of the screen was 0.75 ± 0.112. These 281 compounds were selected for DiFMUP-based concentration-dependent validation, and the 46 most potent compounds were selected. A counter-screening was conducted in parallel, also in a concentration-dependent fashion, against the PDXP paralog and closest relative phosphoglycolate phosphatase (PGP). The 14 compounds that were inactive against PGP were validated in a secondary assay, using the PDXP substrate pyridoxal 5’-phosphate (PLP). Two PDXP inhibitor hits blocked PLP dephosphorylation by ≥50%. Source data are available for this Figure.

PDXP inhibitor hits.

Determination of half-maximal inhibitory constants (IC50) of 14 PDXP inhibitory compounds (see InChI Key for chemical substance identification) using purified murine PDXP and pyridoxal 5’-phosphate (PLP) as a substrate. Data marked with an asterisk (*) are results of n=3 independent experiments. Because of the limited quantity of most compounds available for these assays, all other data are results of n=1 determinations. 7,8-DHF, 7,8-dihydroxyflavone; ∼, approximate IC50 value. FMP-1, FMP-2 and FMP-3 are compounds obtained from academic donors whose structure is undisclosed for intellectual property reasons.

BLI measurements of the interaction of 7,8-DHF with purified murine PDXP.

Sensorgrams of three additional experiments overlayed with the global 1:1 binding model (red) and the negative control (gray). The dashed line indicates the start of the dissociation phase.

Unit cell environment of the 7,8-DHF · PDXP crystal.

The 7,8-DHF-bound protomer A is depicted in pink, the inhibitor-free protomer B is colored in light green. 7,8-DHF is shown in stick representation (C-atoms in pink), and its position is marked with a white asterisk. Arg62 and Glu148 at the active site entrances of protomers A and B are highlighted in blue. Surrounding symmetry mates within 4 Å of the centrally located 7,8-DHF-PDXP/apo-PDXP dimer are shown in surface representation in magenta or dark green. Whereas the active site in 7,8-DHF-bound protomer A is accessible, the corresponding site in protomer B is shielded by two crystallographic neighbors (white arrow). The marked area is enlarged in the bottom panel. Source data are available for this Figure.

Alignment of human and murine PDXP.

Protein sequences of human PDXP (UniProtKB Q96GD0) and murine PDXP (UniProtKB P60487) were aligned with the EMBL-EBI multiple sequence alignment tool Clustal Omega version 1.2.4. PDXP residues found to engage in 7,8-DHF interactions (highlighted in red color) are identical in human and murine PDXP.

Purity of the employed PDXP and PDXP variants.

A Coomassie Blue-stained gel is shown.