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.

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.

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.

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.

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.

Characterisation of a soil MINPP phytase with remarkable long-term stability and activity from Acinetobacter sp.

Phylogenetic analysis, homology modelling and biochemical methods have been employed to characterize a phytase from a Gram-negative soil bacterium. Acinetobacter sp. AC1-2 phytase belongs to clade 2 of the histidine (acid) phytases, to the Multiple Inositol Polyphosphate Phosphatase (MINPP) subclass. The enzyme was extraordinarily stable in solution both at room temperature and 4°C, retaining near 100% activity over 755 days. It showed a broad pH activity profile from 2-8.5 with maxima at 3, 4.5-5 and 6. The enzyme showed Michaelis-Menten kinetics and substrate inhibition (Vmax, Km, and Ki, 228 U/mg, 0.65 mM and 2.23 mM, respectively). Homology modelling using the crystal structure of a homologous MINPP from a human gut commensal bacterium indicated the presence of a potentially stabilising polypeptide loop (a U-loop) straddling the active site. By employ of the enantiospecificity of Arabidopsis inositol tris/tetrakisphosphate kinase 1 for inositol pentakisphosphates, we show AC1-2 MINPP to possess D6-phytase activity, which allowed modelling of active site specificity pockets for InsP6 substrate. While phytase gene transcription was unaltered in rich media, it was repressed in minimal media with phytic acid and orthophosphate as phosphate sources. The results of this study reveal AC1-2 MINPP to possess desirable attributes relevant to biotechnological use.

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.

Structure of a cereal purple acid phytase provides new insights to phytate degradation in plants.

Grain phytate, a mixed metal ion salt of inositol hexakisphosphate, accounts for 60%-80% of stored phosphorus in plants and is a potent antinutrient of non-ruminant animals including humans. Through neofunctionalization of purple acid phytases (PAPhy), some cereals such as wheat and rye have acquired particularly high mature grain phytase activity. As PAPhy activity supplies phosphate, liberates metal ions necessary for seedling emergence, and obviates antinutrient effects of phytate, its manipulation and control are targeted crop traits. Here we show the X-ray crystal structure of the b2 isoform of wheat PAPhy induced during germination. This high-resolution crystal structure suggests a model for phytate recognition that, validated by molecular dynamics simulations, implicates elements of two sequence inserts (termed PAPhy motifs) relative to a canonical metallophosphoesterase (MPE) domain in forming phytate-specific substrate specificity pockets. These motifs are well conserved in PAPhys from monocot cereals, enzymes which are characterized by high specificity for phytate. Tested by mutagenesis, residues His229 in PAPhy motif 4 and Lys410 in the MPE domain, both conserved in PAPhys, are found to strongly influence phytase activity. These results explain the observed phytase activity of cereal PAPhys and open the way to the rational engineering of phytase activity in planta.

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 the 10 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.

Improved sensitivity, accuracy and prediction provided by a high-performance liquid chromatography screen for the isolation of phytase-harbouring organisms from environmental samples.

HPLC methods are shown to be of predictive value for classification of phytase activity of aggregate microbial communities and pure cultures. Applied in initial screens, they obviate the problems of 'false-positive' detection arising from impurity of substrate and imprecision of methodologies that rely on phytate-specific media. In doing so, they simplify selection of candidates for biotechnological applications. Combined with 16S sequencing and simple bioinformatics, they reveal diversity of the histidine phosphatase class of phytases most commonly exploited for biotechnological use. They reveal contribution of multiple inositol-polyphosphate phosphatase (MINPP) activity to aggregate soil phytase activity, and they identity Acinetobacter spp. as harbouring this prevalent soil phytase activity. Previously, among bacteria MINPP was described exclusively as an activity of gut commensals. HPLC methods have also identified, in a facile manner, a known commercially successful histidine (acid) phosphatase enzyme. The methods described afford opportunity for isolation of phytases for biotechnological use from other environments. They reveal the position of attack on phytate by diverse histidine phosphatases, something that other methods lack.

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.

Both d- and l-Glucose Polyphosphates Mimic d-myo-Inositol 1,4,5-Trisphosphate: New Synthetic Agonists and Partial Agonists at the Ins(1,4,5)P3 Receptor.

Chiral sugar derivatives are potential cyclitol surrogates of the Ca2+-mobilizing intracellular messenger d-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. Six novel polyphosphorylated analogues derived from both d- and l-glucose were synthesized. Binding to Ins(1,4,5)P3 receptors [Ins(1,4,5)P3R] and the ability to release Ca2+ from intracellular stores via type 1 Ins(1,4,5)P3Rs were investigated. ß-d-Glucopyranosyl 1,3,4-tris-phosphate, with similar phosphate regiochemistry and stereochemistry to Ins(1,4,5)P3, and α-d-glucopyranosyl 1,3,4-tris-phosphate are full agonists, being equipotent and 23-fold less potent than Ins(1,4,5)P3, respectively, in Ca2+-release assays and similar to Ins(1,4,5)P3 and 15-fold weaker in binding assays. They can be viewed as truncated analogues of adenophostin A and refine understanding of structure-activity relationships for this Ins(1,4,5)P3R agonist. l-Glucose-derived ligands, methyl α-l-glucopyranoside 2,3,6-trisphosphate and methyl α-l-glucopyranoside 2,4,6-trisphosphate, are also active, while their corresponding d-enantiomers, methyl α-d-glucopyranoside 2,3,6-trisphosphate and methyl α-d-glucopyranoside 2,4,6-trisphosphate, are inactive. Interestingly, both l-glucose-derived ligands are partial agonists: they are among the least efficacious agonists of Ins(1,4,5)P3R yet identified, providing new leads for antagonist development.

Regioisomeric Family of Novel Fluorescent Substrates for SHIP2.

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, while maintaining the regiochemical properties of bona fide inositide substrates.

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.

Quantum Blue Reduces the Severity of Woody Breast Myopathy via Modulation of Oxygen Homeostasis-Related Genes in Broiler Chickens.

The incidence of woody breast (WB) is increasing on a global scale representing a significant welfare problem and economic burden to the poultry industry and for which there is no effective treatment due to its unknown etiology. In this study, using diffuse reflectance spectroscopy (DRS) coupled with iSTAT portable clinical analyzer, we provide evidence that the circulatory- and breast muscle-oxygen homeostasis is dysregulated [low oxygen and hemoglobin (HB) levels] in chickens with WB myopathy compared to healthy counterparts. Molecular analysis showed that blood HB subunit Mu (HBM), Zeta (HBZ), and hephaestin (HEPH) expression were significantly down regulated; however, the expression of the subunit rho of HB beta (HBBR) was upregulated in chicken with WB compared to healthy counterparts. The breast muscle HBBR, HBE, HBZ, and hypoxia-inducible factor prolyl hydroxylase 2 (PHD2) mRNA abundances were significantly down regulated in WB-affected compared to normal birds. The expression of HIF-1α at mRNA and protein levels was significantly induced in breasts of WB-affected compared to unaffected birds confirming a local hypoxic status. The phosphorylated levels of the upstream mediators AKT at Ser473 site, mTOR at Ser2481 site, and PI3K P85 at Tyr458 site, as well as their mRNA levels were significantly increased in breasts of WB-affected birds. In attempt to identify a nutritional strategy to reduce WB incidence, male broiler chicks (Cobb 500, n = 576) were randomly distributed into 48 floor pens and subjected to six treatments (12 birds/pen; 8 pens/treatment): a nutrient adequate control group (PC), the PC supplemented with 0.3% myo-inositol (PC + MI), a negative control (NC) deficient in available P and Ca by 0.15 and 0.16%, respectively, the NC fed with quantum blue (QB) at 500 (NC + 500 FTU), 1,000 (NC + 1,000 FTU), or 2,000 FTU/kg of feed (NC + 2,000 FTU). Although QB-enriched diets did not affect growth performances (FCR and FE), it did reduce the severity of WB by 5% compared to the PC diet. This effect is mediated by reversing the expression profile of oxygen homeostasis-related genes; i.e., significant down regulation of HBBR and upregulation of HBM, HBZ, and HEPH in blood, as well as a significant upregulation of HBA1, HBBR, HBE, HBZ, and PHD2 in breast muscle compared to the positive control.

Bacteria are important dimethylsulfoniopropionate producers in coastal sediments.

Dimethylsulfoniopropionate (DMSP) and its catabolite dimethyl sulfide (DMS) are key marine nutrients1,2 that have roles in global sulfur cycling2, atmospheric chemistry3, signalling4,5 and, potentially, climate regulation6,7. The production of DMSP was previously thought to be an oxic and photic process that is mainly confined to the surface oceans. However, here we show that DMSP concentrations and/or rates of DMSP and DMS synthesis are higher in surface sediment from, for example, saltmarsh ponds, estuaries and the deep ocean than in the overlying seawater. A quarter of bacterial strains isolated from saltmarsh sediment produced DMSP (up to 73 mM), and we identified several previously unknown producers of DMSP. Most DMSP-producing isolates contained dsyB8, but some alphaproteobacteria, gammaproteobacteria and actinobacteria used a methionine methylation pathway independent of DsyB that was previously only associated with higher plants. These bacteria contained a methionine methyltransferase gene (mmtN)-a marker for bacterial synthesis of DMSP through this pathway. DMSP-producing bacteria and their dsyB and/or mmtN transcripts were present in all of the tested seawater samples and Tara Oceans bacterioplankton datasets, but were much more abundant in marine surface sediment. Approximately 1 × 108 bacteria g-1 of surface marine sediment are predicted to produce DMSP, and their contribution to this process should be included in future models of global DMSP production. We propose that coastal and marine sediments, which cover a large part of the Earth's surface, are environments with high levels of DMSP and DMS productivity, and that bacteria are important producers of DMSP and DMS within these environments.

Effect of phytase on intestinal phytate breakdown, plasma inositol concentrations, and glucose transporter type 4 abundance in muscle membranes of weanling pigs1.

The objective of this present 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 4 treatments for a 7-wk study. Depending on the feeding phase, diets differed in dietary calcium (Ca) and phosphorus (P) levels (positive control [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 2,000 FTU/kg (NC + 2,000 FTU) phytase units/kg. Blood was collected after fasting (day 48) or feeding (day 49) for measurement of plasma inositol concentrations. On day 49, 2 pigs per pen were euthanized, and duodenal and ileal digesta samples were collected to determine inositol phosphates (InsP6-2) concentrations. High phytase supplementation increased BW on days 21, 35, and 49 (P < 0.05). Over the entire feeding period, ADG, ADFI, and feed efficiency were increased by NC + 2,000 FTU compared with the other treatments (P < 0.05). Postprandial plasma inositol concentration was increased in NC + 2,000 (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 (day 49) were not different among treatments. Duodenal digesta InsP5 and InsP6 concentrations were similar in PC and NC, but higher in these 2 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 with NC, phytase supplementation resulted in greater cumulative InsP6-2 disappearance (93.6% vs. 72.8% vs. 25.0%, for NC + 2,000 FTU, NC + 500 FTU and NC, respectively, P < 0.01) till the distal ileum. Longissimus dorsi muscle plasma membrane GLUT4 concentration was increased by NC + 2,000 FTU (P < 0.01) compared with 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 whether this is associated with enhanced cellular glucose uptake and utilization.

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.

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 zebrafish. 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.