The inositol-3-phosphate synthase biosynthetic enzyme has distinct catalytic and metabolic roles

Inositol levels, maintained by the biosynthetic enzyme inositol-3-phosphate synthase (Ino1), are altered in a range of disorders including bipolar disorder and Alzheimer's disease. To date, most inositol studies have focused on the molecular and cellular effects of inositol depletion without considering Ino1 levels. Here we employ a simple eukaryote, Dictyostelium, to demonstrate distinct effects of loss of Ino1 and inositol depletion. We show that loss of Ino1 results in inositol auxotrophy that can only be partially rescued by exogenous inositol. Removal of inositol supplementation from the ino1- mutant results in a rapid 56% reduction in inositol levels, triggering the induction of autophagy, reduced cytokinesis and substrate adhesion. Inositol depletion also caused a dramatic generalised decrease in phosphoinositide levels that was rescued by inositol supplementation. However, loss of Ino1 triggered broad metabolic changes consistent with the induction of a catabolic state that was not rescued by inositol supplementation. These data suggest a metabolic role for Ino1 independent of inositol biosynthesis. To characterise this role, an Ino1 binding partner containing SEL1L1 domains (Q54IX5) was identified with homology to mammalian macromolecular complex adaptor proteins. Our findings therefore identify a new role for Ino1, independent of inositol biosynthesis, with broad effects on cell metabolism.

Coriolis perturbations in the infrared spectrum of ethylene

Rotational structure has been resolved and analyzed in two of the infrared‐active perpendicular bands of C2H4 vapor: the Type b fundamental band, ν10, at 826 cm—1, and the Type c fundamental band, ν7, at 949 cm—1. Many of the individual PP and RR branch lines have been observed. The analysis has been confined to values of the quantum number K≥3, for which energy levels ethylene shows no detectable deviations from a symmetric‐top rotational structure. The analysis reveals a Coriolis interaction between ν7 and ν10, and between ν4 and ν10, and values of the Coriolis constants ζ7,10z and ζ4,10y are obtained; these are related to normal coordinate calculations for the appropriate symmetry species, and force constants are derived to fit the observed zeta constants. The band center of ν10 has been revised from the original figure of 810 cm—1 to the new value, 826 cm—1, and the inactive frequency ν4 is estimated to lie at 1023±3 cm—1, in good agreement with the previous estimate of 1027 cm—1. The change in the value of ν10 leads to a suggested change in the value of the Raman‐active fundamental ν6 from 1236 to 1222 cm—1. New combination bands have been observed at 2174 cm—1, assigned as ν3+ν10; and at 2252 cm—1, assigned as ν4+ν6; also rotational structure has been resolved and analyzed in the ν6+ν10 band at 2048 cm—1. The new data obtained for the C2H4 molecule are summarized in Table XII, with all of the other data presently available on the vibrational and rotational constants.

Sugars and related compounds as flavour precursors in meat

Sugars and related substances, namely sugar phosphates and ribonucleotides, are important meat flavour precursors. In particular, ribose and ribose 5-phosphate have been shown to be important in aroma development in heated model systems. There are few quantitative data on the concentrations and the variations of sugars and related substances in meat. This paper will report on the analysis of glucose, fructose, ribose, ribose 5-phosphate, fructose 6-phosphate, glucose 6-phosphate and inosine 5'-monophosphate (IMP) in aged beef. Sugars and related compounds were extracted from lean meat and derivatised to the corresponding TMS ethers. Analysis and quantitation of the sugars and sugar phosphates were performed using GC and GC/MS, while IMP analysis was performed using capillary electrophoresis (CE).

Bifidobacterium scardovii sp. nov., from human sources

Five strains of an unusual catalase-negative Gram-positive asporogenous rod-shaped bacterium from human sources were subjected to a polyphasic taxonomic study. The presence of fructose-6-phosphate phosphoketolase, a key enzyme of bifidobacterial hexose metabolism, indicated the strains were members of the genus Bifidobacterium but they did not correspond to any of the recognized species of this genus on the basis of biochemical profiles and whole-cell protein analyses. Comparative 16S rRNA gene sequencing confirmed the placement of the isolates in the genus Bifidobacterium, and demonstrated they represent a hitherto unknown subline within the genus displaying > 5% sequence divergence with recognized species. Based on both phenotypic and phylogenetic criteria, it is proposed that the isolates recovered from human sources be classified as a new species, Bifidobacterium scardovii sp. nov.; the type strain is CCUG 13008T (= DSM 13734T).

Twists and turns: a tale of two shikimate-pathway enzymes

We are studying two enzymes from the shikimate pathway, dehydroquinate synthase (DHQS) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Both enzymes have been the subject of numerous studies to elucidate their reaction mechanisms. Crystal structures of DHQS and EPSPS in the presence and absence of substrates, cofactors and/or inhibitors are now available. These structures reveal movements of domains, rearrangements of loops and changes in side-chain positions necessary for the formation of a catalytically competent active site. The potential for using complementary small-angle X-ray scattering (SAXS) studies to confirm the presence of these structural differences in solution has also been explored. Comparative analysis of crystal structures, in the presence and absence of ligands, has revealed structural features critical for substrate-binding and catalysis. We have also analysed these structures by generating GRID energy maps to detect favourable binding sites. The combination of X-ray crystallography, SAXS and computational techniques provides an enhanced analysis of structural features important for the function of these complex enzymes.

Variation in root-associated phosphatase activities in wheat contributes to the utilization of organic P substrates in vitro, but does not explain differences in the P-nutrition of plants when grown in soils

To understand whether genotypic variation in root-associated phosphatase activities in wheat impacts on its ability to acquire phosphorus (P), various phosphatase activities of roots were measured in relation to the utilization of organic P substrates in agar, and the P-nutrition of plants was investigated in a range of soils. Root-associated phosphatase activities of plants grown in hydroponics were measured against different organic P substrates. Representative genotypes were then grown in both agar culture and in soils with differing organic P contents and plant biomass and P uptake were determined. Differences in the activities of both root-associated and exuded phosphodiesterase and phosphomonoesterase were observed, and were related to the P content of plants supplied with either ribonucleic acid or glucose 6-phosphate, respectively, as the sole form of P. When the cereal lines were grown in different soils, however, there was little relationship between any root-associated phosphatase activity and plant P uptake. This indicates that despite differences in phosphatase activities of cereal roots, such variability appears to play no significant role in the P-nutrition of the plant grown in soil, and that any benefit derived from the hydrolysis of soil organic P is common to all genotypes.

A baseline study of vicine–convicine levels in faba bean (Vicia faba L.) germplasm

Vicine and convicine are anti-nutritional compounds that accumulate in the cotyledons of faba beans. When humans consume beans with high levels of these compounds, it can cause a condition called favism in individuals harbouring a deficiency in the activity of their glucose-6-phosphate dehydrogenase. When faba beans are used in animal feeds, there can be effects on performance. These concerns have resulted in increasing interest within plant breeding in developing low vicine and convicine faba bean germplasm. In order to facilitate this objective, we developed a rapid and robust screening method for vicine and convicine, capable of distinguishing between faba beans that are either high (wild type) or low in vicine and convicine. In the absence of reliable commercial reference materials, we report an adaptation of a previously published method where a biochemical assay and spectral data were used to confirm the identity of our analytes, vicine and convicine. This method could be readily adopted in other facilities and open the way to the efficient exploitation of diverse germplasm in regions where faba beans play a significant role in human nutrition. We screened a collection of germplasm of interest to a collaborative plant breeding programme developing between the National Institute for Agricultural Botany in the UK and L'Institut Nationale d'Agronomie de Tunisie in Tunisia. We report the results obtained and discuss the prospects for developing molecular markers for the low vicine and convicine trait.

Hydrothermal synthesis of [C6H16N2][In2Se3(Se2)]: a new one-dimensional indium selenide

A new organically templated indium selenide, [C6H16N2][In2Se3(Se2)], has been prepared hydrothermally from the reaction of indium, selenium and trans-1,4-diaminocyclohexane in water at 170 °C. This material was characterised by single-crystal and powder X-ray diffraction, thermogravimetric analysis, UV–vis diffuse reflectance spectroscopy, FT-IR and elemental analysis. The compound crystallises in the monoclinic space group C2/c (a=12.0221(16) Å, b=11.2498(15) Å, c=12.8470(17) Å, β=110.514(6)°). The crystal structure of [C6H16N2][In2Se3(Se2)] contains anionic chains of stoichiometry [In2Se3(Se2)]2−, which are aligned parallel to the [1 0 1] direction, and separated by diprotonated trans-1,4-diaminocyclohexane cations. The [In2Se3(Se2)]2− chains, which consist of alternating four-membered [In2Se2] and five-membered [In2Se3] rings, contain perselenide (Se2)2− units. UV–vis diffuse reflectance spectroscopy indicates that [C6H16N2][In2Se3(Se2)] has a band gap of 2.23(1) eV

Rational conversion of substrate and product specificity in a Salvia monoterpene synthase: structural insights into the evolution of terpene synthase function

Terpene synthases are responsible for the biosynthesis of the complex chemical defense arsenal of plants and microorganisms. How do these enzymes, which all appear to share a common terpene synthase fold, specify the many different products made almost entirely from one of only three substrates? Elucidation of the structure of 1,8-cineole synthase from Salvia fruticosa (Sf-CinS1) combined with analysis of functional and phylogenetic relationships of enzymes within Salvia species identified active-site residues responsible for product specificity. Thus, Sf-CinS1 was successfully converted to a sabinene synthase with a minimum number of rationally predicted substitutions, while identification of the Asn side chain essential for water activation introduced 1,8-cineole and alpha-terpineol activity to Salvia pomifera sabinene synthase. A major contribution to product specificity in Sf-CinS1 appears to come from a local deformation within one of the helices forming the active site. This deformation is observed in all other mono- or sesquiterpene structures available, pointing to a conserved mechanism. Moreover, a single amino acid substitution enlarged the active-site cavity enough to accommodate the larger farnesyl pyrophosphate substrate and led to the efficient synthesis of sesquiterpenes, while alternate single substitutions of this critical amino acid yielded five additional terpene synthases.

High-resolution crystal structure of the intramolecular d(TpA) thymine-adenine photoadduct and its mechanistic implications

A high-resolution crystal structure is reported for d(TpA)*, the intramolecular thymine-adenine photoadduct that is produced by direct ultraviolet excitation of the dinucleoside monophosphate d(TpA). It confirms the presence of a central 1,3-diazacyclooctatriene ring linking the remnants of the T and A bases, as previously deduced from heteronuclear NMR measurements by Zhao et al. (The structure of d(TpA)*, the major photoproduct of thymidylyl-(3'-5')-deoxyadenosine. Nucleic Acids Res., 1996, 24, 1554-1560). Within the crystal, the d(TpA)* molecules exist as zwitterions with a protonated amidine fragment of the eight-membered ring neutralizing the charge of the internucleotide phosphate monoanion. The absolute configuration at the original thymine C5 and C6 atoms is determined as 5S,6R. This is consistent with d(TpA)* arising by valence isomerization of a precursor cyclobutane photoproduct with cis-syn stereochemistry that is generated by [2 + 2] photoaddition of the thymine 5,6-double bond across the C6 and C5 positions of adenine. This mode of photoaddition should be favoured by the stacked conformation of adjacent T and A bases in B-form DNA. It is probable that the primary photoreaction is mechanistically analogous to pyrimidine dimerization despite having a much lower quantum yield.

Synthesis and crystal structure of a 3-D zinc phosphate, [C5N2H14][Zn-2(PO3(OH))(3)], containing (4.8) net sheets

A new 3-D zinc phosphate, [C5N2H14][Zn-2(PO3(OH))(3)], has been synthesised under solvothermal conditions in the presence of 1-methylpiperazine. The structure, determined by single-crystal X-ray diffraction at 293 K (RMM = 520.9, orthorhombic, space group P2(1)2(1)2(1); a = 10.0517(2) &ANGS;, b = 10.4293(2) &ANGS; and c = 14.9050(5) &ANGS;; V = 1562.52 &ANGS;(3); Z = 4; R(F) = 2.60%, wR(F) = 2.93%), consists of vertex linked ZnO4 and PO3(OH) tetrahedra assembled into (4.8) net sheets which in turn are linked through further PO3(OH) units to generate a 3-D framework. 1-Methylpiperazinium cations reside within the 3-D channel system, held in place by a strong network of hydrogen bonds. The (4.8) net sheets occur in a number of zeolite structures e.g. ABW and GIS and related zinc phosphate phases. © 2004 Academie des sciences. Published by Elsevier SAS. All rights reserved.

Characterization of the layered structure in main chain dibenzo-18-crown-6 ether polymers by simultaneous WAXS/MAXS-SAXS/DSC measurements

The structure and thermal properties of polymers containing dibenzo-18-crown-6 ether units in the main chain linked to an aliphatic spacer of different lengths (C10-C14) is reported. X-ray diffraction patterns of all the studied samples exhibit a peak in the medium angle region, revealing the existence of a lamellar structure. Simultaneous calorimetry and small, medium (SAXS-MAXS) and wide (WAXS) X-ray measurements during cooling and subsequent heating of the samples reveal that a layer phase is formed upon cooling. In the case of the homopolymers, this phase is almost simultaneously accompanied by the appearance of some reflections in the wide angle region as an indication of lateral crystallization. However, by copolymerization, the formation of the layer phase is decoupled from lateral crystallization, being stable in a wide temperature region.

Synthesis and crystal structure of a 3-D zinc phosphate, [C5N2H14][Zn2(PO3(OH))3], containing (4.8) net sheets

A new 3-D zinc phosphate, [C5N2H14][Zn-2(PO3(OH))(3)], has been synthesised under solvothermal conditions in the presence of 1-methylpiperazine. The structure, determined by single-crystal X-ray diffraction at 293 K (RMM = 520.9, orthorhombic, space group P2(1)2(1)2(1); a = 10.0517(2) &ANGS;, b = 10.4293(2) &ANGS; and c = 14.9050(5) &ANGS;; V = 1562.52 &ANGS;(3); Z = 4; R(F) = 2.60%, wR(F) = 2.93%), consists of vertex linked ZnO4 and PO3(OH) tetrahedra assembled into (4.8) net sheets which in turn are linked through further PO3(OH) units to generate a 3-D framework. 1-Methylpiperazinium cations reside within the 3-D channel system, held in place by a strong network of hydrogen bonds. The (4.8) net sheets occur in a number of zeolite structures e.g. ABW and GIS and related zinc phosphate phases. © 2004 Academie des sciences. Published by Elsevier SAS. All rights reserved.