A Fluorescent Probe Identifies Active Site Ligands of Inositol Pentakisphosphate 2-Kinase

Inositol pentakisphosphate 2-kinase catalyzes the phosphorylation of the axial 2-OH of myo-inositol 1,3,4,5,6-pentakisphosphate for de novo synthesis of myo-inositol hexakisphosphate. Disruption of inositol pentakisphosphate 2-kinase profoundly influences cellular processes; from nuclear mRNA export and phosphate homeostasis in yeast and plants, to establishment of left-right asymmetry in zebra fish. We elaborate an active site fluorescent probe that allows high throughput screening of Arabidopsis inositol pentakisphosphate 2-kinase. We show that the probe has a binding constant comparable to the Km values of inositol phosphate substrates of this enzyme, and can be used to prospect for novel substrates and inhibitors of inositol phosphate kinases. We identify several micromolar Ki inhibitors and validate this approach by solving the crystal structure of protein in complex with purpurogallin. We additionally solve structures of protein in complexes with epimeric higher inositol phosphates. This probe may find utility in characterization of a wide family of inositol phosphate kinases

Crystal structure and enzymology of solanum tuberosum inositol tris/tetrakisphosphate kinase 1 (StITPK1)

Inositol phosphates and their pyrophosphorylated derivatives are responsive to the phosphate supply and are agents of phosphate homeostasis and other aspects of physiology. It seems likely that the enzymes that interconvert these signals work against the prevailing milieu of mixed populations of competing substrates and products. The synthesis of inositol pyrophosphates is mediated in plants by two classes of ATP-grasp fold kinase: PPIP5 kinases, known as VIH, and members of the inositol tris/tetrakisphosphate kinase (ITPK) family, specifically ITPK1/2. A molecular explanation of the contribution of ITPK1/2 to inositol pyrophosphate synthesis and turnover in plants is incomplete: the absence of nucleotide in published crystal structures limits the explanation of phosphotransfer reactions, and little is known of the affinity of potential substrates and competitors for ITPK1. Herein, we describe a complex of ADP and StITPK1 at 2.26 Å resolution and use a simple fluorescence polarization approach to compare the affinity of binding of diverse inositol phosphates, inositol pyrophosphates, and analogues. By simple HPLC, we reveal the novel catalytic capability of ITPK1 for different inositol pyrophosphates and show Ins(3,4,5,6)P4 to be a potent inhibitor of the inositol pyrophosphate-synthesizing activity of ITPK1. We further describe the exquisite specificity of ITPK1 for the myo-isomer among naturally occurring inositol hexakisphosphates

Substrate promiscuity of inositol 1,4,5-trisphosphate kinase driven by structurally-modified ligands and active site plasticity

D-myo-inositol 1,4,5-trisphosphate (InsP3) is a fundamental second messenger in cellular Ca2+ mobilization. InsP3 3-kinase, a highly specific enzyme binding InsP3 in just one mode, phosphorylates InsP3 specifically at its secondary 3-hydroxyl group to generate a tetrakisphosphate. Using a chemical biology approach with both synthetised and established ligands, combining synthesis, crystallography, computational docking, HPLC and fluorescence polarization binding assays using fluorescently-tagged InsP3, we have surveyed the limits of InsP3 3-kinase ligand specificity and uncovered surprisingly unforeseen biosynthetic capacity. Structurally-modified ligands exploit active site plasticity generating a helix-tilt. These facilitated uncovering of unexpected substrates phosphorylated at a surrogate extended primary hydroxyl at the inositol pseudo 3-position, applicable even to carbohydrate-based substrates. Crystallization experiments designed to allow reactions to proceed in situ facilitated unequivocal characterization of the atypical tetrakisphosphate products. In summary, we define features of InsP3 3-kinase plasticity and substrate tolerance that may be more widely exploitable

Effect of phytase on intestinal phytate breakdown, plasma inositol concentrations and glucose transporter type 4 abundance in muscle membranes of weanling pigs

The objective of this current study was to determine the effects of phytase dosing on growth performance, mineral digestibility, phytate breakdown and the level of glucose transporter type 4 (GLUT4) in muscle plasma membranes of weanling pigs. A total of 160 barrows were used in a randomized completely block design and assigned to four treatments for a 7-week study. Depending on the feeding phase, diets differed in dietary calcium (Ca) and phosphorus (P) levels (PC:8 to 6.8g/kg Ca; 7.3 to 6.3 g/kg P; negative control (NC):5.5 to 5.2 g/kg Ca; 5.4 to 4.7 g/kg P). NC diets were supplemented with phytase at 0 (NC); 500 (NC+500 FTU) or 2000 FTU/kg (NC+2000 FTU) phytase units/kg. Blood was collected after fasting (d 48) or feeding (d 49) for measurement of plasma inositol concentrations. On d 49, two pigs per pen were euthanized, duodenal and ileal digesta samples were collected to determine inositol phosphates (InsP6-2) concentrations. High phytase supplementation increased body weight (BW) on d 21, 35 and 49 (P <0.05). Over the entire feeding period, average daily gain (ADG), average daily feed intake (ADFI) and feed efficiency were increased by NC+2000 FTU compared to the other treatments (P <0.05). Postprandial plasma inositol concentration was increased in NC+2000 (P < 0.01), but there was only a tendency (P = 0.06) of a higher fasting plasma inositol concentration in this group. Inositol concentrations in the portal vein plasma (d 49) were not different among treatments. Duodenal digesta InsP5 and InsP6 concentrations were similar in PC and NC, but higher in these two treatments (P < 0.05) than those supplemented with phytase. Phytase supplementation decreased InsP6-4, resulting in increased InsP3-2 and myo-inositol concentrations. Similar effects were found in ileal contents. Compared to NC, phytase supplementation resulted in greater cumulative InsP6-2 disappearance (93.6% vs. 72.8% vs. 25.0%, for NC+2000 FTU, NC +500 FTU and NC, respectively, P < 0.01) till the the distal ileum. Longissimus dorsi muscle plasma membrane GLUT4 concentration was increased by NC+2000 FTU (P < 0.01) compared to NC. In summary, high phytase supplementation increased growth performance of nursery pigs. The higher myo-inositol release from phytate could contribute to the increased expression of GLUT4 in muscle plasma membranes. Further investigation is needed to determine if this is associated with enhanced cellular glucose uptake and utilization

An ATP-responsive metabolic cassette comprised of inositol tris/tetrakisphosphate kinase 1 (ITPK1) and inositol pentakisphosphate 2-kinase (IPK1) buffers diphosphosphoinositol phosphate levels

Inositol polyphosphates are ubiquitous molecular signals in metazoans, as are their pyrophosphorylated derivatives that bear a so-called 'high-energy' phosphoanhydride bond. A structural rationale is provided for the ability of Arabidopsis inositol tris/tetrakisphosphate kinase 1 to discriminate between symmetric and enantiomeric substrates in the production of diverse symmetric and asymmetric myo-inositol phosphate and diphospho-myo-inositol phosphate (inositol pyrophosphate) products. Simple tools are applied to chromatographic resolution and detection of known and novel diphosphoinositol phosphates without resort to radiolabeling approaches. It is shown that inositol tris/tetrakisphosphate kinase 1 and inositol pentakisphosphate 2-kinase comprise a reversible metabolic cassette converting Ins(3,4,5,6)P4 into 5-InsP7 and back in a nucleotide-dependent manner. Thus, inositol tris/tetrakisphosphate kinase 1 is a nexus of bioenergetics status and inositol polyphosphate/diphosphoinositol phosphate metabolism. As such, it commands a role in plants that evolution has assigned to a different class of enzyme in mammalian cells. The findings and the methods described will enable a full appraisal of the role of diphosphoinositol phosphates in plants and particularly the relative contribution of reversible inositol phosphate hydroxykinase and inositol phosphate phosphokinase activities to plant physiology

Effect of phytase supplementation on plasma and organ myo-inositol content and erythrocyte inositol phosphates in chickens

‘Woody breast’ (WB) and ‘white striping’ in broiler meat is a global problem. With unknown etiology, WB negatively impacts bird health, welfare and is a significant economic burden to the poultry industry. New evidence has shown that WB is associated with dysregulation in systemic and breast muscle-oxygen homeostasis, resulting in hypoxia and anaemia. However, it has been observed that phytase (Quantum Blue (QB) a modified, E. coli-derived 6-phytase) super dosing can reverse dysregulation of muscle-oxygen homeostasis and reduces WB severity by ~5%. The objective of this study was to assess whether levels of Ins(1,3,4,5,6)P5, the main allosteric regulator of haemoglobin, are influenced by changes in plasma myo-inositol arising from super dosing with phytase. To enable this, methods suitable for measurement of myo-inositol in tissues and inositol phosphates in blood were developed. Data were collected from independent trials, including male Ross 308 broilers fed low and adequate calcium/available phosphate (Ca/AvP) diets supplemented with QB at 1,500 phytase units (FTU)/kg, which simultaneously decreased gizzard InsP6 (P<0.001) and increased gizzard myo-inositol (P<0.001). Similarly, male Cobb 500 broiler chicks fed a negative control (NC) diet deficient in AvP, Ca and sodium or diet supplemented with the QB phytase at 500, 1000 or 2,000 FTU/kg increased plasma (P<0.001) and liver (P=0.007) myo-inositol of 18d-old birds at 2,000 FTU/kg. Finally, QB supplementation of Cobb 500 breeder flock diet at 1,250 FTU/kg increased blood myo-inositol (P<0.001) and erythrocyte Ins(1,3,4,5,6)P5 (P=0.011) of their 1d-old hatchlings. These data confirmed the ability of phytase to modulate inositol phosphate pathways by provision of metabolic precursors of important signalling molecules. The ameliorations of WB afforded by super doses of phytase may include modulation of hypoxia pathways that also involve inositol signalling molecules. Elevations of erythrocyte Ins(1,3,4,5,6)P5 by phytase supplementation may enhance systemic oxygen carrying capacity, an important factor in the amelioration of WB and WS myopathy

Regioisomeric family of novel fluorescent substrates for SHIP2

ABSTRACT: SHIP2 (SH2-domain containing inositol 5-phosphatase type 2) is a canonical 5-phosphatase which, through its catalytic action on PtdInsP3, regulates the PI3K/Akt pathway and metabolic action of insulin. It is a drug target but there is limited evidence of inhibition of SHIP2 by small molecules in the literature. With the goal to investigate inhibition, we report a homologous family of synthetic, chromophoric benzene phosphate substrates of SHIP2 that display the headgroup regiochemical hallmarks of the physiological inositide substrates that have proved difficult to crystallize with 5-phosphatases. Using time-dependent density functional theory (TD-DFT), we explore the intrinsic fluorescence of these novel substrates and show how fluorescence can be used to assay enzyme activity. The TD-DFT approach promises to inform rational design of enhanced active site probes for the broadest family of inositide-binding / metabolizing proteins, whilst maintaining the regiochemical properties of bona fide inositide substrates

Allosteric site on SHIP2 identified through fluorescent ligand screening and crystallography: a potential new target for intervention

Src Homology 2 domain-containing inositol phosphate phosphatase 2 (SHIP2) is one of ten human inositol phosphate 5-phosphatases. One of its physiological functions is dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate, PtdIns(3,4,5)P3. It is therefore a therapeutic target for pathophysiologies dependent on PtdIns(3,4,5)P3 and PtdIns(3,4)P2. Therapeutic interventions are limited by the dearth of crystallographic data describing ligand/inhibitor binding. An active site-directed fluorescent probe facilitated screening of compound libraries for SHIP2 ligands. With two additional orthogonal assays, several ligands including galloflavin were identified as low micromolar Ki inhibitors. One ligand, an oxo-linked ethylene-bridged dimer of benzene 1,2,4-trisphosphate, was shown to be an uncompetitive inhibitor that binds to a regulatory site on the catalytic domain. We posit that binding of ligands to this site restrains L4 loop motions that are key to interdomain communications that accompany high catalytic activity with phosphoinositide substrate. This site may, therefore, be a future druggable target for medicinal chemistry

Crystal structure and enzymology of Solanum tuberosum inositol tris/tetrakisphosphate kinase 1 (StITPK1)

Inositol phosphates and their pyrophosphorylated derivatives are responsive to the phosphate supply and are agents of phosphate homeostasis and other aspects of physiology. It seems likely that the enzymes that interconvert these signals work against the prevailing milieu of mixed populations of competing substrates and products. The synthesis of inositol pyrophosphates is mediated in plants by two classes of ATP-grasp fold kinase: PPIP5 kinases, known as VIH, and members of the inositol tris/tetrakisphosphate kinase (ITPK) family, specifically ITPK1/2. A molecular explanation of the contribution of ITPK1/2 to inositol pyrophosphate synthesis and turnover in plants is incomplete: the absence of nucleotide in published crystal structures limits the explanation of phosphotransfer reactions, and little is known of the affinity of potential substrates and competitors for ITPK1. Herein, we describe a complex of ADP and StITPK1 at 2.26 Å resolution and use a simple fluorescence polarization approach to compare the affinity of binding of diverse inositol phosphates, inositol pyrophosphates, and analogues. By simple HPLC, we reveal the novel catalytic capability of ITPK1 for different inositol pyrophosphates and show Ins(3,4,5,6)P4 to be a potent inhibitor of the inositol pyrophosphate-synthesizing activity of ITPK1. We further describe the exquisite specificity of ITPK1 for the myo-isomer among naturally occurring inositol hexakisphosphates

Diversification in the inositol tris/tetrakisphosphate kinase (ITPK) family: crystal structure and enzymology of the outlier AtITPK4

Myo-inositol tris/tetrakisphosphate kinases (ITPKs) catalyze diverse phosphotransfer reactions with myo-inositol phosphate and myo-inositol pyrophosphate substrates. However, the lack of structures of nucleotide-coordinated plant ITPKs thwarts a rational understanding of phosphotransfer reactions of the family. Arabidopsis possesses a family of four ITPKs of which two isoforms, ITPK1 and ITPK4, control inositol hexakisphosphate and inositol pyrophosphate levels directly or by provision of precursors. Here, we describe the specificity of Arabidopsis ITPK4 to pairs of enantiomers of diverse inositol polyphosphates and show how substrate specificity differs from Arabidopsis ITPK1. Moreover, we provide a description of the crystal structure of ATP-coordinated AtITPK4 at 2.11 Å resolution that, along with a description of the enantiospecificity of the enzyme, affords a molecular explanation for the diverse phosphotransferase activity of this enzyme. That Arabidopsis ITPK4 has a KM for ATP in the tens of micromolar range, potentially explains how, despite the large-scale abolition of InsP6, InsP7 and InsP8 synthesis in Atitpk4 mutants, Atitpk4 lacks the phosphate starvation responses of Atitpk1 mutants. We further demonstrate that Arabidopsis ITPK4 and its homologues in other plants possess an N-terminal haloacid dehalogenase-like fold not previously described. The structural and enzymological information revealed will guide elucidation of ITPK4 function in diverse physiological contexts, including InsP8-dependent aspects of plant biology