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 Šresolution 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.

Insights to the Structural Basis for the Stereospecificity of the Escherichia coli Phytase, AppA.

AppA, the Escherichia coli periplasmic phytase of clade 2 of the histidine phosphatase (HP2) family, has been well-characterized and successfully engineered for use as an animal feed supplement. AppA is a 1D-6-phytase and highly stereospecific but transiently accumulates 1D-myo-Ins(2,3,4,5)P4 and other lower phosphorylated intermediates. If this bottleneck in liberation of orthophosphate is to be obviated through protein engineering, an explanation of its rather rigid preference for the initial site and subsequent cleavage of phytic acid is required. To help explain this behaviour, the role of the catalytic proton donor residue in determining AppA stereospecificity was investigated. Four variants were generated by site-directed mutagenesis of the active site HDT amino acid sequence motif containing the catalytic proton donor, D304. The identity and position of the prospective proton donor residue was found to strongly influence stereospecificity. While the wild-type enzyme has a strong preference for 1D-6-phytase activity, a marked reduction in stereospecificity was observed for a D304E variant, while a proton donor-less mutant (D304A) displayed exclusive 1D-1/3-phytase activity. High-resolution X-ray crystal structures of complexes of the mutants with a non-hydrolysable substrate analogue inhibitor point to a crucial role played by D304 in stereospecificity by influencing the size and polarity of specificity pockets A and B. Taken together, these results provide the first evidence for the involvement of the proton donor residue in determining the stereospecificity of HP2 phytases and prepares the ground for structure-informed engineering studies targeting the production of animal feed enzymes capable of the efficient and complete dephosphorylation of dietary phytic acid.

Snapshots during the catalytic cycle of a histidine acid phytase reveal an induced-fit structural mechanism.

Highly engineered phytases, which sequentially hydrolyze the hexakisphosphate ester of inositol known as phytic acid, are routinely added to the feeds of monogastric animals to improve phosphate bioavailability. New phytases are sought as starting points to further optimize the rate and extent of dephosphorylation of phytate in the animal digestive tract. Multiple inositol polyphosphate phosphatases (MINPPs) are clade 2 histidine phosphatases (HP2P) able to carry out the stepwise hydrolysis of phytate. MINPPs are not restricted by a strong positional specificity making them attractive targets for development as feed enzymes. Here, we describe the characterization of a MINPP from the Gram-positive bacterium Bifidobacterium longum (BlMINPP). BlMINPP has a typical HP2P-fold but, unusually, possesses a large α-domain polypeptide insertion relative to other MINPPs. This insertion, termed the U-loop, spans the active site and contributes to substrate specificity pockets underpopulated in other HP2Ps. Mutagenesis of U-loop residues reveals its contribution to enzyme kinetics and thermostability. Moreover, four crystal structures of the protein along the catalytic cycle capture, for the first time in an HP2P, a large ligand-driven α-domain motion essential to allow substrate access to the active site. This motion recruits residues both downstream of a molecular hinge and on the U-loop to participate in specificity subsites, and mutagenesis identified a mobile lysine residue as a key determinant of positional specificity of the enzyme. Taken together, these data provide important new insights to the factors determining stability, substrate recognition, and the structural mechanism of hydrolysis in this industrially important group of enzymes.

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.

Arabidopsis inositol phosphate kinases IPK1 and ITPK1 constitute a metabolic pathway in maintaining phosphate homeostasis.

Emerging studies have suggested that there is a close link between inositol phosphate (InsP) metabolism and cellular phosphate (Pi ) homeostasis in eukaryotes; however, whether a common InsP species is deployed as an evolutionarily conserved metabolic messenger to mediate Pi signaling remains unknown. Here, using genetics and InsP profiling combined with Pi -starvation response (PSR) analysis in Arabidopsis thaliana, we showed that the kinase activity of inositol pentakisphosphate 2-kinase (IPK1), an enzyme required for phytate (inositol hexakisphosphate; InsP6 ) synthesis, is indispensable for maintaining Pi homeostasis under Pi -replete conditions, and inositol 1,3,4-trisphosphate 5/6-kinase 1 (ITPK1) plays an equivalent role. Although both ipk1-1 and itpk1 mutants exhibited decreased levels of InsP6 and diphosphoinositol pentakisphosphate (PP-InsP5 ; InsP7 ), disruption of another ITPK family enzyme, ITPK4, which correspondingly caused depletion of InsP6 and InsP7 , did not display similar Pi -related phenotypes, which precludes these InsP species from being effectors. Notably, the level of d/l-Ins(3,4,5,6)P4 was concurrently elevated in both ipk1-1 and itpk1 mutants, which showed a specific correlation with the misregulated Pi phenotypes. However, the level of d/l-Ins(3,4,5,6)P4 is not responsive to Pi starvation that instead manifests a shoot-specific increase in the InsP7 level. This study demonstrates a more nuanced picture of the intersection of InsP metabolism and Pi homeostasis and PSRs than has previously been elaborated, and additionally establishes intermediate steps to phytate biosynthesis in plant vegetative tissues.

Lab-scale preparation and QC of phytase assay substrate from rice bran.

Phytases are involved in the phosphate acquisition and remobilization in plants, microbes and animals. They have become important technical enzymes in the feed industry and are used to make phosphate, present in animal feed as phytate, available for monogastric animal nutrition. Phytases may also be beneficial to human nutrition because phytate is known to interfere with the uptake of important micronutrients. Accordingly, phytases attract considerable research attention and phytate substrate lacking contaminants that interfere with commonly used phosphate-release assays is essential for this field of science. A procedure to prepare suitable sodium phytate from rice bran is presented. Extracted phytate is precipitated with barium hydroxide and re-dissolved in methanol after washing steps and sulphuric acid treatment. Remaining impurities are precipitated before the dissolved phytate is recovered as the sodium salt by addition of sodium hydroxide. In order to make the substrate widely available for research communities, the procedure relies solely on basic laboratory equipment and materials. Methods for quality control and monitoring of the purified sodium phytate or commercial alternatives are also presented.

The crystal structure of mammalian inositol 1,3,4,5,6-pentakisphosphate 2-kinase reveals a new zinc-binding site and key features for protein function.

Inositol 1,3,4,5,6-pentakisphosphate 2-kinases (IP5 2-Ks) are part of a family of enzymes in charge of synthesizing inositol hexakisphosphate (IP6) in eukaryotic cells. This protein and its product IP6 present many roles in cells, participating in mRNA export, embryonic development, and apoptosis. We reported previously that the full-length IP5 2-K from Arabidopsis thaliana is a zinc metallo-enzyme, including two separated lobes (the N- and C-lobes). We have also shown conformational changes in IP5 2-K and have identified the residues involved in substrate recognition and catalysis. However, the specific features of mammalian IP5 2-Ks remain unknown. To this end, we report here the first structure for a murine IP5 2-K in complex with ATP/IP5 or IP6 Our structural findings indicated that the general folding in N- and C-lobes is conserved with A. thaliana IP5 2-K. A helical scaffold in the C-lobe constitutes the inositol phosphate-binding site, which, along with the participation of the N-lobe, endows high specificity to this protein. However, we also noted large structural differences between the orthologues from these two eukaryotic kingdoms. These differences include a novel zinc-binding site and regions unique to the mammalian IP5 2-K, as an unexpected basic patch on the protein surface. In conclusion, our findings have uncovered distinct features of a mammalian IP5 2-K and set the stage for investigations into protein-protein or protein-RNA interactions important for IP5 2-K function and activity.

Land-use influences phosphatase gene microdiversity in soils.

Phosphorus cycling exerts significant influence upon soil fertility and productivity - processes largely controlled by microbial activity. We adopted phenotypic and metagenomic approaches to investigate phosphatase genes within soils. Microbial communities in bare fallowed soil showed a marked capacity to utilise phytate for growth compared with arable or grassland soil communities. Bare fallowed soil contained lowest concentrations of orthophosphate. Analysis of metagenomes indicated phoA, phoD and phoX, and histidine acid and cysteine phytase genes were most abundant in grassland soil which contained the greatest amount of NaOH-EDTA extractable orthophosphate. Beta-propeller phytase genes were most abundant in bare fallowed soil. Phylogenetic analysis of metagenome sequences indicated the phenotypic shift observed in the capacity to mineralise phytate in bare fallow soil was accompanied by an increase in phoD, phoX and beta-propeller phytase genes coding for exoenzymes. However, there was a remarkable degree of genetic similarity across the soils despite the differences in land-use. Predicted extracellular ecotypes were distributed across a greater range of soil structure than predicted intracellular ecotypes, suggesting that microbial communities subject to the dual stresses of low nutrient availability and reduced access to organic material in bare fallowed soils rely upon the action of exoenzymes.

Cephalosporinases associated with outer membrane vesicles released by Bacteroides spp. protect gut pathogens and commensals against ß-lactam antibiotics.

OBJECTIVES: To identify ß-lactamase genes in gut commensal Bacteroides species and to assess the impact of these enzymes, when carried by outer membrane vesicles (OMVs), in protecting enteric pathogens and commensals. METHODS: A deletion mutant of the putative class A ß-lactamase gene (locus tag BT_4507) found in the genome of the human commensal Bacteroides thetaiotaomicron was constructed and a phenotypic analysis performed. A phylogenetic tree was built from an alignment of nine Bacteroides cephalosporinase protein sequences, using the maximum likelihood method. The rate of cefotaxime degradation after incubation with OMVs produced by different Bacteroides species was quantified using a disc susceptibility test. The resistance of Salmonella Typhimurium and Bifidobacterium breve to cefotaxime in liquid culture in the presence of B. thetaiotaomicron OMVs was evaluated by measuring bacterial growth. RESULTS: The B. thetaiotaomicron BT_4507 gene encodes a ß-lactamase related to the CepA cephalosporinase of Bacteroides fragilis. OMVs produced by B. thetaiotaomicron and several other Bacteroides species, except Bacteroides ovatus, carried surface-associated ß-lactamases that could degrade cefotaxime. ß-Lactamase-harbouring OMVs from B. thetaiotaomicron protected Salmonella Typhimurium and B. breve from an otherwise lethal dose of cefotaxime. CONCLUSIONS: The production of membrane vesicles carrying surface-associated ß-lactamases by Bacteroides species, which constitute a major part of the human colonic microbiota, may protect commensal bacteria and enteric pathogens, such as Salmonella Typhimurium, against ß-lactam antibiotics.

Evidence for a SAL1-PAP chloroplast retrograde pathway that functions in drought and high light signaling in Arabidopsis.

Compartmentation of the eukaryotic cell requires a complex set of subcellular messages, including multiple retrograde signals from the chloroplast and mitochondria to the nucleus, to regulate gene expression. Here, we propose that one such signal is a phosphonucleotide (3'-phosphoadenosine 5'-phosphate [PAP]), which accumulates in Arabidopsis thaliana in response to drought and high light (HL) stress and that the enzyme SAL1 regulates its levels by dephosphorylating PAP to AMP. SAL1 accumulates in chloroplasts and mitochondria but not in the cytosol. sal1 mutants accumulate 20-fold more PAP without a marked change in inositol phosphate levels, demonstrating that PAP is a primary in vivo substrate. Significantly, transgenic targeting of SAL1 to either the nucleus or chloroplast of sal1 mutants lowers the total PAP levels and expression of the HL-inducible ASCORBATE PEROXIDASE2 gene. This indicates that PAP must be able to move between cellular compartments. The mode of action for PAP could be inhibition of 5' to 3' exoribonucleases (XRNs), as SAL1 and the nuclear XRNs modulate the expression of a similar subset of HL and drought-inducible genes, sal1 mutants accumulate XRN substrates, and PAP can inhibit yeast (Saccharomyces cerevisiae) XRNs. We propose a SAL1-PAP retrograde pathway that can alter nuclear gene expression during HL and drought stress.

Optimized barley phytase gene expression by focused FIND-IT screening for mutations in cis-acting regulatory elements.

Introduction: Induced modification of plant gene expression is of both fundamental and applied importance. Cis-acting regulatory elements (CREs) are major determinants of the spatiotemporal strength of gene expression. Yet, there are few examples where induced genetic variation in predetermined CREs has been exploited to improve or investigate crop plants. Methods: The digital PCR based FIND-IT technology was applied to discover barley mutants with CRE variants in the promoter of the nutritional important barley grain phytase (PAPhy_a) gene. Results and discussion: Mutants with higher or lower gene expression and ultimately higher or lower mature grain phytase activity (MGPA), respectively, were discovered. Field trials and inositol phosphate profiling during germination showed that PAPhy_a does not influence agronomic performance under the trial conditions but it does shorten the lag time of phosphate mobilization during germination. Higher endogenous MGPA is an improvement of grain quality for feed use as it improves the phosphate bioavailability for monogastric animals. Moreover, as the targeted CRE motifs of the PAPhy_a promoter are shared with a range of seed expressed genes like key cereal and legume storage genes, the current results demonstrates a concept for modulating individual gene expression levels of a range of seed genes.

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.

Conformational changes in inositol 1,3,4,5,6-pentakisphosphate 2-kinase upon substrate binding: role of N-terminal lobe and enantiomeric substrate preference.

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP(5) 2-K) catalyzes the synthesis of inositol 1,2,3,4,5,6-hexakisphosphate from ATP and IP(5). Inositol 1,2,3,4,5,6-hexakisphosphate is implicated in crucial processes such as mRNA export, DNA editing, and phosphorus storage in plants. We previously solved the first structure of an IP(5) 2-K, which shed light on aspects of substrate recognition. However, failure of IP(5) 2-K to crystallize in the absence of inositide prompted us to study putative conformational changes upon substrate binding. We have made mutations to residues on a region of the protein that produces a clasp over the active site. A W129A mutant allowed us to capture IP(5) 2-K in its different conformations by crystallography. Thus, the IP(5) 2-K apo-form structure displays an open conformation, whereas the nucleotide-bound form shows a half-closed conformation, in contrast to the inositide-bound form obtained previously in a closed conformation. Both nucleotide and inositide binding produce large conformational changes that can be understood as two rigid domain movements, although local changes were also observed. Changes in intrinsic fluorescence upon nucleotide and inositide binding are in agreement with the crystallographic findings. Our work suggests that the clasp might be involved in enzyme kinetics, with the N-terminal lobe being essential for inositide binding and subsequent conformational changes. We also show how IP(5) 2-K discriminates between inositol 1,3,4,5-tetrakisphosphate and 3,4,5,6-tetrakisphosphate enantiomers and that substrate preference can be manipulated by Arg(130) mutation. Altogether, these results provide a framework for rational design of specific inhibitors with potential applications as biological tools for in vivo studies, which could assist in the identification of novel roles for IP(5) 2-K in mammals.

Jasmonate and phytochrome A signaling in Arabidopsis wound and shade responses are integrated through JAZ1 stability.

Jasmonate (JA) activates plant defense, promotes pollen maturation, and suppresses plant growth. An emerging theme in JA biology is its involvement in light responses; here, we examine the interdependence of the JA- and light-signaling pathways in Arabidopsis thaliana. We demonstrate that mutants deficient in JA biosynthesis and signaling are deficient in a subset of high irradiance responses in far-red (FR) light. These mutants display exaggerated shade responses to low, but not high, R/FR ratio light, suggesting a role for JA in phytochrome A (phyA) signaling. Additionally, we demonstrate that the FR light-induced expression of transcription factor genes is dependent on CORONATINE INSENSITIVE1 (COI1), a central component of JA signaling, and is suppressed by JA. phyA mutants had reduced JA-regulated growth inhibition and VSP expression and increased content of cis-(+)-12-oxophytodienoic acid, an intermediate in JA biosynthesis. Significantly, COI1-mediated degradation of JASMONATE ZIM DOMAIN1-beta-glucuronidase (JAZ1-GUS) in response to mechanical wounding and JA treatment required phyA, and ectopic expression of JAZ1-GUS resulted in exaggerated shade responses. Together, these results indicate that JA and phyA signaling are integrated through degradation of the JAZ1 protein, and both are required for plant responses to light and stress.

Synthesis of inositol phosphate ligands of plant hormone-receptor complexes: pathways of inositol hexakisphosphate turnover.

Reduction of phytate is a major goal of plant breeding programs to improve the nutritional quality of crops. Remarkably, except for the storage organs of crops such as barley, maize and soybean, we know little of the stereoisomeric composition of inositol phosphates in plant tissues. To investigate the metabolic origins of higher inositol phosphates in photosynthetic tissues, we have radiolabelled leaf tissue of Solanum tuberosum with myo-[2-3H]inositol, undertaken a detailed analysis of inositol phosphate stereoisomerism and permeabilized mesophyll protoplasts in media containing inositol phosphates. We describe the inositol phosphate composition of leaf tissue and identify pathways of inositol phosphate metabolism that we reveal to be common to other kingdoms. Our results identify the metabolic origins of a number of higher inositol phosphates including ones that are precursors of cofactors, or cofactors of plant hormone-receptor complexes. The present study affords alternative explanations of the effects of disruption of inositol phosphate metabolism reported in other species, and identifies different inositol phosphates from that described in photosynthetic tissue of the monocot Spirodela polyrhiza. We define the pathways of inositol hexakisphosphate turnover and shed light on the occurrence of a number of inositol phosphates identified in animals, for which metabolic origins have not been defined.

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase is a distant IPK member with a singular inositide binding site for axial 2-OH recognition.

Inositol phosphates (InsPs) are signaling molecules with multiple roles in cells. In particular (InsP(6)) is involved in mRNA export and editing or chromatin remodeling among other events. InsP(6) accumulates as mixed salts (phytate) in storage tissues of plants and plays a key role in their physiology. Human diets that are exclusively grain-based provide an excess of InsP(6) that, through chelation of metal ions, may have a detrimental effect on human health. Ins(1,3,4,5,6)P(5) 2-kinase (InsP(5) 2-kinase or Ipk1) catalyses the synthesis of InsP(6) from InsP(5) and ATP, and is the only enzyme that transfers a phosphate group to the axial 2-OH of the myo-inositide. We present the first structure for an InsP(5) 2-kinase in complex with both substrates and products. This enzyme presents a singular structural region for inositide binding that encompasses almost half of the protein. The key residues in substrate binding are identified, with Asp368 being responsible for recognition of the axial 2-OH. This study sheds light on the unique molecular mechanism for the synthesis of the precursor of inositol pyrophosphates.

Accentuating the positive and eliminating the negative: Efficacy of TiO2 as digestibility index marker for poultry nutrition studies.

Inert digestibility index markers such as titanium dioxide are universally accepted to provide simple measurement of digestive tract retention and relative digestibility in poultry feeding trials. Their use underpins industry practice: specifically dosing regimens for adjunct enzymes added to animal feed. Among these, phytases, enzymes that degrade dietary phytate, inositol hexakisphosphate, represent a billion-dollar sector in an industry that raises ca. 70 billion chickens/annum. Unbeknown to the feed enzyme sector, is the growth in cell biology of use of titanium dioxide for enrichment of inositol phosphates from extracts of cells and tissues. The adoption of titanium dioxide in cell biology arises from its affinity under acid conditions for phosphates, suggesting that in feeding trial contexts that target phytate degradation this marker may not be as inert as assumed. We show that feed grade titanium dioxide enriches a mixed population of higher and lower inositol phosphates from acid solutions. Additionally, we compared the extractable inositol phosphates in gizzard and ileal digesta of 21day old male Ross 308 broilers fed three phytase doses (0, 500 and 6000 FTU/kg feed) and one inositol dose (2g/kg feed). This experiment was performed with or without titanium dioxide added as a digestibility index marker at a level of 0.5%, with all diets fed for 21 days. Analysis yielded no significant difference in effect of phytase inclusion in the presence or absence of titanium dioxide. Thus, despite the utility of titanium dioxide for recovery of inositol phosphates from biological samples, it seems that its use as an inert marker in digestibility trials is justified-as its inclusion in mash diets does not interfere with the recovery of inositol phosphates from digesta samples.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of the apo form of InsP5 2-K from Arabidopsis thaliana.

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP(5) 2-K) is a key enzyme that catalyzes the synthesis of phytic acid (IP(6)) from inositol 1,3,4,5,6-pentakisphosphate (IP(5)) and ATP. The first structure of IP(5) 2-K, that from Arabidopsis thaliana, has been solved previously; it only crystallized in the presence of inositol, either the substrate IP(5) or the product IP(6), and failed to crystallize in its free state (without inositol). Based on structural analysis, a point mutation of IP(5) 2-K (W129A) has been produced in order to overcome this limitation and obtain information about protein conformational changes upon substrate binding. Here, the production and crystallization of W129A IP(5) 2-K in its free state and with bound nucleotide is described. These crystals differed from the native crystals and belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 66.00, b = 68.23, c = 105.80 Å and a = 63.06, b = 71.80, c = 100.23 Å, respectively. The crystals diffracted to resolutions of 2.22 Å (apo) and 2.05 Å (nucleotide bound) using synchrotron radiation and contained one molecule per asymmetric unit. The structures have been determined using the molecular-replacement method and refinement is being undertaken.

Phytase dose-dependent response of kidney inositol phosphate levels in poultry.

Phytases, enzymes that degrade phytate present in feedstuffs, are widely added to the diets of monogastric animals. Many studies have correlated phytase addition with improved animal productivity and a subset of these have sought to correlate animal performance with phytase-mediated generation of inositol phosphates in different parts of the gastro-intestinal tract or with release of inositol or of phosphate, the absorbable products of phytate degradation. Remarkably, the effect of dietary phytase on tissue inositol phosphates has not been studied. The objective of this study was to determine effect of phytase supplementation on liver and kidney myo-inositol and myo-inositol phosphates in broiler chickens. For this, methods were developed to measure inositol phosphates in chicken tissues. The study comprised wheat/soy-based diets containing one of three levels of phytase (0, 500 and 6,000 FTU/kg of modified E. coli 6-phytase). Diets were provided to broilers for 21 D and on day 21 digesta were collected from the gizzard and ileum. Liver and kidney tissue were harvested. Myo-inositol and inositol phosphates were measured in diet, digesta, liver and kidney. Gizzard and ileal content inositol was increased progressively, and total inositol phosphates reduced progressively, by phytase supplementation. The predominant higher inositol phosphates detected in tissues, D-and/or L-Ins(3,4,5,6)P4 and Ins(1,3,4,5,6)P5, differed from those (D-and/or L-Ins(1,2,3,4)P4, D-and/or L-Ins(1,2,5,6)P4, Ins(1,2,3,4,6)P5, D-and/or L-Ins(1,2,3,4,5)P5 and D-and/or L-Ins(1,2,4,5,6)P5) generated from phytate (InsP6) degradation by E. coli 6-phytase or endogenous feed phytase, suggesting tissue inositol phosphates are not the result of direct absorption. Kidney inositol phosphates were reduced progressively by phytase supplementation. These data suggest that tissue inositol phosphate concentrations can be influenced by dietary phytase inclusion rate and that such effects are tissue specific, though the consequences for physiology of such changes have yet to be elucidated.