Phosphatase activity of PMM1 monitored by ³¹P-NMR spectroscopy.

<p>PMM1 (20 μg) was incubated with Glc-1,6-P₂ 0.55 mM at 27°C, with and without IMP 0.17 mM. Panel A) Spectrum of reagents, Glc-1,6-P₂ and IMP, prior to PMM1 addition. Panel B) Spectrum of products and IMP after PMM1 addition. Panel C) Spectrum of reagents, Glc-1,6-P₂, prior to PMM1 addition. Panel D) Spectrum of products after PMM1 addition. Creatine phosphate (1 mM) was added as an internal standard and all the spectra were referred to it (0 ppm). Resonance assignment was obtained by comparison with pure compounds: <b>1</b> and <b>2</b>, Glc-1,6-P₂; <b>3</b> Glc-1-P+P_i; <b>4</b>, Glc-6-P; <b>5</b>, IMP; *, inorganic phosphate.</p

Ligand binding interactions.

<p>Panel A) A zoom-in of the interaction between IMP and PMM1 as it is seen in the low energy structure 52 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189629#pone.0189629.g007" target="_blank">Fig 7</a>, panel A): the cap domain is shadowed in light magenta, the core domain is shadowed in grey. Panel B) Refined binding modes: details from energetic minima structures from 3 different trajectories (asterisk -traj5, section mark -traj13, circle -traj14 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189629#pone.0189629.g009" target="_blank">Fig 9</a>, panel B).</p

Effects of bisphosphonates on the phosphatase and glucomutase activities of PMM1, QDK-PMM1 and PMM2.

<p>Panel A) Phosphoglucomutase activity of PMM1 and PMM2 was measured in the presence of Glc-1-P 40 μM and Glc-1,6-P₂ 27 μM. Panel B) Glc-1,6-P₂-phosphatase activity of PMM1, QDK-PMM1 and PMM2 was measured in the presence of Glc-1,6-P₂ 0.145 mM. In both cases the activities were also measured in the presence of clodronate (2.8 mM) or neridronate (1.5 mM).</p

Phosphomannomutase activity of PMM1 and PMM2 on Man-6-P monitored by ³¹P-NMR spectroscopy.

<p>PMM1 or PMM2 (54 μg) were incubated with Man-6-P 1mM and Glc-1,6-P₂ 0.1 mM at 27°C. Panel A) Spectrum of reagents prior to PMM1 addition. Panel B) Spectrum of products after PMM1 addition. Panel C) Spectrum of reagents prior to PMM2 addition. Panel D) Spectrum of products after PMM2 addition. The spectra were accumulated over 40 min. Creatine phosphate (1 mM) was added as an internal standard and all the spectra were referred to it (0 ppm). Resonance assignment was obtained by comparison with pure compounds: <b>1</b>, Glc-6-P; <b>2</b>, Man-6-P; <b>3</b> and <b>5</b>, Man-1,6-P₂; <b>4</b>, Man-1-P; <b>6</b> and <b>7</b>, Glc-1,6-P₂; *, inorganic phosphate.</p

In silico docking of IMP and PMM1.

<p>Panel A) Unconstrained simulation. Only 5 trajectories are shown. Structure 52, used for the next refinement simulation, is identified by an asterisk. Panel B) Refinement simulation for structure 52 from traj1 in panel A. The circle, the section mark and the asterisk represent the structure further analyzed (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189629#pone.0189629.g010" target="_blank">Fig 10</a> panel B).</p

Thermal stability of phosphomannomutases.

<p>Panel A) Melting temperatures of PMM1 and QDK-PMM1 (0.5 mg/ml) were measured in the presence of different ligands: Glc-1-P 0.5 mM, Glc-1-P 0.5 mM + vanadate 0.5 mM, vanadate 0.5 mM, Glc-1,6-P₂ 0.5 mM, IMP 0.17 mM. Panel B). Melting temperatures of PMM1 and PMM2 (0.5 mg/ml) were measured in the presence of different concentrations of Glc-1,6-P₂ (ranging from 0 to 1 mM). The experiments were conducted at pH 7.5 in the presence of 2.4x SyproOrange by Thermal Shift Assay and the temperature increase was 1°/min from 20 to 90°C.</p

Competitive inhibition of PMM1 mutase activity by IMP.

<p>Phosphoglucomutase activity of PMM1 (Glc-1,6-P2 1 μM and Glc-1-P ranging from 0 to 60 μM) was measured at 0, 2, 4, 6 and 10 μM IMP. Data are shown as Lineweaver-Burk plots.</p

PMM1 and PMM2 sequence alignment.

<p>Active site residues are highlighted in grey, residues that are conserved in PMM1 orthologous proteins, but not in PMM2 orthologous proteins are in bold.</p

2–6 dithiopurine stabilizes human lysosomal alpha-galactosidase in thermal shift assay.

<p>Panel A. Fabrazyme^® (in Na-Hepes 20 mM, NaCl 150 mM, pH 7.4) was equilibrated in the presence of ligands dissolved in DMSO 20%: DGJ 1 microM (empty triangle) or 40 microM (empty circle); DTP 6 mM (empty diamond); DTP 6 mM plus DGJ 1 microM (filled circle); DTP 6 mM plus DGJ 40 microM (filled triangle). A control (with only DMSO) was also shown (x). Panel B. Fabrazyme^® (in Na-Hepes 20 mM, NaCl 150 mM, pH 7.4) was incubated in the presence of DTP 6 mM for 1 hour at 4°C then DTP was eliminated by dialysis. A control experiment was conducted by incubating the enzyme only with DMSO. Both the aliquots of Fabrazyme^® were analysed by thermal shift assay in the presence of DTP 6 mM or in the presence of DMSO. Filled squares: DTP/DTP; open squares: DMSO/DTP; filled circles: DTP/DMSO; open circles: DMSO/DMSO where the first word of the label corresponds to the pretreatment and the second part corresponds to the presence of the compound during the thermal scan. The protein samples were heated from 20 to 90° at 1°C/min in the presence of Sypro Orange 2.5x. Data were shown as normalized curves.</p

2–6 dithiopurine has no effect on mutations affecting the allosteric site.

<p>COS-7 cells were cultured in conventional medium and transfected with plasmid containing A37T, P40S, M42T, M42V mutants. Cells were treated with DTP 6 mM dissolved in DMSO and an appropriate control was realized. After 48 h incubation, the cells were scraped and lysed then water-soluble extracts were analysed by western blotting (A and B) and enzyme assay (C). Standard deviations are indicated by bars.</p