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The rare sugar d-tagatose is a safe natural product used as a commercial food ingredient. Here, we show that d-tagatose controls a wide range of plant diseases and focus on downy mildews to analyze its mode of action. It likely acts directly on the pathogen, rather than as a plant defense activator. Synthesis of mannan and related products of d-mannose metabolism are essential for development of fungi and oomycetes; d-tagatose inhibits the first step of mannose metabolism, the phosphorylation of d-fructose to d-fructose 6-phosphate by fructokinase, and also produces d-tagatose 6-phosphate. d-Tagatose 6-phosphate sequentially inhibits phosphomannose isomerase, causing a reduction in d-glucose 6-phosphate and d-fructose 6-phosphate, common substrates for glycolysis, and in d-mannose 6-phosphate, needed to synthesize mannan and related products. These chain-inhibitory effects on metabolic steps are significant enough to block initial infection and structural development needed for reproduction such as conidiophore and conidiospore formation of downy mildew., Mochizuki, Fukumoto, Ohara, et al. study the protective role of the rare sugar d-tagatose against plant disease. They focus on its effects against the downy mildews and delineate the metabolic pathway that blocks the initial infection. Their work paves the way for the development of safe fungicidal agrochemicals using natural products.

Myxococcus xanthus is a model bacterium to study social behavior. At the cellular level, the different social behaviors of M. xanthus involve extensive cell–cell contacts. Here, we used bioinformatics, genetics, heterologous expression and biochemical experiments to identify and characterize the key enzymes in M. xanthus implicated in O-antigen and lipopolysaccharide (LPS) biosynthesis and examined the role of LPS O-antigen in M. xanthus social behaviors. We identified WbaPMx (MXAN_2922) as the polyisoprenyl-phosphate hexose-1-phosphate transferase responsible for priming O-antigen synthesis. In heterologous expression experiments, WbaPMx complemented a Salmonella enterica mutant lacking the endogenous WbaP that primes O-antigen synthesis, indicating that WbaPMx transfers galactose-1-P to undecaprenyl-phosphate. We also identified WaaLMx (MXAN_2919), as the O-antigen ligase that joins O-antigen to lipid A-core. Our data also support the previous suggestion that WzmMx (MXAN_4622) and WztMx (MXAN_4623) form the Wzm/Wzt ABC transporter. We show that mutations that block different steps in LPS O-antigen synthesis can cause pleiotropic phenotypes. Also, using a wbaPMx deletion mutant, we revisited the role of LPS O-antigen and demonstrate that it is important for gliding motility, conditionally important for type IV pili-dependent motility and required to complete the developmental program leading to the formation of spore-filled fruiting bodies.

Archaeal DNA polymerases from the B-family (polB) have found essential applications in biotechnology. In addition, some of their variants can accept a wide range of modified nucleotides or xenobiotic nucleotides, such as 1,5-anhydrohexitol nucleic acid (HNA), which has the unique ability to selectively cross-pair with DNA and RNA. This capacity is essential to allow the transmission of information between different chemistries of nucleic acid molecules. Variants of the archaeal polymerase from Thermococcus gorgonarius, TgoT, that can either generate HNA from DNA (TgoT_6G12) or DNA from HNA (TgoT_RT521) have been previously identified. To understand how DNA and HNA are recognized and selected by these two laboratory-evolved polymerases, we report six X-ray structures of these variants, as well as an in silico model of a ternary complex with HNA. Structural comparisons of the apo form of TgoT_6G12 together with its binary and ternary complexes with a DNA duplex highlight an ensemble of interactions and conformational changes required to promote DNA or HNA synthesis. MD simulations of the ternary complex suggest that the HNA-DNA hybrid duplex remains stable in the A-DNA helical form and help explain the presence of mutations in regions that would normally not be in contact with the DNA if it were not in the A-helical form. One complex with two incorporated HNA nucleotides is surprisingly found in a one nucleotide-backtracked form, which is new for a DNA polymerase. This information can be used for engineering a new generation of more efficient HNA polymerase variants. ispartof: BIOMOLECULES vol:10 issue:12 ispartof: location:Switzerland status: published

The isoform of glucose-6-phosphatase in liver, G6PC1, has a major role in whole-body glucose homeostasis, whereas G6PC3 is widely distributed among organs but has poorly-understood functions. A recent, elegant analysis of neutrophil dysfunction in G6PC3-deficient patients revealed G6PC3 is a neutrophil metabolite repair enzyme that hydrolyzes 1,5-anhydroglucitol-6-phosphate, a toxic metabolite derived from a glucose analog present in food. These patients exhibit a spectrum of phenotypic characteristics and some have learning disabilities, revealing a potential linkage between cognitive processes and G6PC3 activity. Previously-debated and discounted functions for brain G6PC3 include causing an ATP-consuming futile cycle that interferes with metabolic brain imaging assays and a nutritional role involving astrocyte-neuron glucose-lactate trafficking. Detailed analysis of the anhydroglucitol literature reveals that it competes with glucose for transport into brain, is present in human cerebrospinal fluid, and is phosphorylated by hexokinase. Anhydroglucitol-6-phosphate is present in rodent brain and other organs where its accumulation can inhibit hexokinase by competition with ATP. Calculated hexokinase inhibition indicates that energetics of brain and erythrocytes would be more adversely affected by anhydroglucitol-6-phosphate accumulation than heart. These findings strongly support the paradigm-shifting hypothesis that brain G6PC3 removes a toxic metabolite, thereby maintaining brain glucose metabolism- and ATP-dependent functions, including cognitive processes.

Neutropenia and neutrophil dysfunction cause serious infections and inflammatory bowel disease in glycogen storage disease type Ib (GSD-Ib). Our discovery that accumulating 1,5-anhydroglucitol-6-phosphate (1,5AG6P) caused neutropenia in a glucose-6-phosphatase 3 (G6PC3)–deficient mouse model and in 2 rare diseases (GSD-Ib and G6PC3 deficiency) led us to repurpose the widely used antidiabetic drug empagliflozin, an inhibitor of the renal glucose cotransporter sodium glucose cotransporter 2 (SGLT2). Off-label use of empagliflozin in 4 GSD-Ib patients with incomplete response to granulocyte colony-stimulating factor (GCSF) treatment decreased serum 1,5AG and neutrophil 1,5AG6P levels within 1 month. Clinically, symptoms of frequent infections, mucosal lesions, and inflammatory bowel disease resolved, and no symptomatic hypoglycemia was observed. GCSF could be discontinued in 2 patients and tapered by 57% and 81%, respectively, in the other 2. The fluctuating neutrophil numbers in all patients were increased and stabilized. We further demonstrated improved neutrophil function: normal oxidative burst (in 3 of 3 patients tested), corrected protein glycosylation (2 of 2), and normal neutrophil chemotaxis (1 of 1), and bactericidal activity (1 of 1) under treatment. In summary, the glucose-lowering SGLT2 inhibitor empagliflozin, used for type 2 diabetes, was successfully repurposed for treating neutropenia and neutrophil dysfunction in the rare inherited metabolic disorder GSD-Ib without causing symptomatic hypoglycemia. We ascribe this to an improvement in neutrophil function resulting from the reduction of the intracellular concentration of 1,5AG6P.

Sorbitol-6-phosphatase (EC 3.1.3.50) catalyzes sorbitol production from sorbitol-6-phosphate in certain organisms, but has not been identified unequivocally. We screened the activity of the haloacid dehalogenase-like hydrolases (HAD) superfamily and identified four HAD proteins from Escherichia coli as sorbitol-6-phosphatase. Of these proteins, HAD2 (YfbT) exhibited catalytic activity (kcat/Km) that was better than that of the previously reported "preferred" substrate. HAD1 (YniC) and HAD2 exhibited higher sorbitol-6-phosphatase activity than that of HAD12 (YbiV) and HAD13 (YidA). Therefore, genes of HAD may be useful for metabolic engineering of effective sorbitol production.

Fine structure of the O-polysaccharide chain of the lipopolysaccharide (O-antigen) defines the serospecificity of bacterial cells, which is the basis for O-serotyping of medically and agriculturally important gram-negative bacteria including Escherichia coli. In order to obtain the O-polysaccharide for structural analysis, the lipopolysaccharide was isolated from cells of E. coli O84a by phenol/water extraction and degraded with mild acid. However, the O-polysaccharide was cleaved at a highly acid-labile β-l-fucopyranosyl phosphate (β-l-Fucp-1-P) linkage to give mainly a pentasaccharide that corresponded to the O-polysaccharide repeat. Therefore, the lipopolysaccharide and the pentasaccharide as well as their O-deacylated derivatives were studied using sugar analysis, NMR spectroscopy, and (for oligosaccharides) ESI HR MS, and the O84-polysaccharide structure was established. The O-polysaccharide is distinguished by the presence of β-l-Fucp-1-P and randomly di-O-acetylated 6-deoxy-d-talose, which are found for the first time in natural carbohydrates. The gene cluster for the O84-antigen biosynthesis was analysed and its content was found to be consistent with the O-polysaccharide structure.

Analysis of nucleotide sugars, nucleoside di- and triphosphates and sugar-phosphates is an essential step in the process of understanding enzymatic pathways. A facile and rapid separation method was developed to analyze these compounds present in an enzymatic reaction mixture utilized to produce nucleotide sugars. The Primesep SB column explored in this study utilizes hydrophobic interactions as well as electrostatic interactions with the phosphoric portion of the nucleotide sugars. Ammonium formate buffer was selected due to its compatibility with mass spectrometry. Negative ion mode mass spectrometry was adopted for detection of the sugar phosphate (fucose-1-phophate), as the compound is not amenable to UV detection. Various mobile phase conditions such as pH, buffer concentration and organic modifier were explored. The semi-preparative separation method was developed to prepare 30mg of the nucleotide sugar. (19)F NMR was utilized to determine purity of the purified fluorinated nucleotide sugar. The collected nucleotide sugar was found to be 99% pure.

Futile cycling between free sugars and hexose phosphates occurring under phosphate deficiency could be involved in the maintenance of a threshold level of free cellular phosphate to preserve respiratory metabolism. We studied the metabolic response of potato cell cultures growing in Pi sufficient (2.5 mM, +Pi) or deficient (125 µM, −Pi) conditions. Under Pi deficiency, cellular growth was severely affected, however −Pi cells were able to maintain a low but steady level of free Pi. We surveyed the activities of 33 primary metabolic enzymes during the course of a 12 days Pi deficiency period. Our results show that many of these enzymes had higher specific activity in –Pi cells. Among these, we found typical markers of Pi deficiency such as phosphoenolpyruvate phosphatase and phosphoenolpyruvate carboxylase as well as enzymes involved in the biosynthesis of organic acids. Intriguingly, several ATP-consuming enzymes such as hexokinase (HK) and phosphofructokinase also displayed increased activity in –Pi condition. For HK, this was associated with an increase in the steady state of a specific HK polypeptide. Quantification of glycolytic intermediates showed a pronounced decrease in phosphate esters under Pi deficiency. Adenylate levels also decreased in –Pi cells, but the Adenylate Energy Charge was not affected by the treatment. To investigate the significance of HK induction under low Pi, [U-14C]-glucose tracer studies were conducted. We found in vivo evidence of futile cycling between pools of hexose phosphates and free sugars under Pi deficiency. Our study suggests that the futile cycling between hexose phosphates and free sugars which is active under +Pi conditions is sustained under Pi deficiency. The possibility that this process represents a metabolic adaptation to Pi deficiency is discussed with respect to Pi homeostasis in Pi-deficient conditions.

Sorbitol-6-phosphate 2-dehydrogenases (S6PDH) catalyze the interconversion of d -sorbitol 6-phosphate to d -fructose 6-phosphate. In the plant pathogen Erwinia amylovora the S6PDH SrlD is used by the bacterium to utilize sorbitol, which is used for carbohydrate transport in the host plants belonging to the Amygdaloideae subfamily (e.g., apple, pear, and quince). We have determined the crystal structure of S6PDH SrlD at 1.84 A resolution, which is the first structure of an EC 1.1.1.140 enzyme. Kinetic data show that SrlD is much faster at oxidizing d -sorbitol 6-phosphate than in reducing d -fructose 6-phosphate, however, equilibrium analysis revealed that only part of the d -sorbitol 6-phosphate present in the in vitro environment is converted into d -fructose 6-phosphate. The comparison of the structures of SrlD and Rhodobacter sphaeroides sorbitol dehydrogenase showed that the tetrameric quaternary structure, the catalytic residues and a conserved aspartate residue that confers specificity for NAD+ over NADP+ are preserved. Analysis of the SrlD cofactor and substrate binding sites identified residues important for the formation of the complex with cofactor and substrate and in particular the role of Lys42 in selectivity towards the phospho-substrate. The comparison of SrlD backbone with the backbone of 302 short-chain dehydrogenases/reductases showed the conservation of the protein core and identified the variable parts. The SrlD sequence was compared with 500 S6PDH sequences selected by homology revealing that the C-terminal part is more conserved than the N-terminal, the consensus of the catalytic tetrad (Y[SN]AGXA) and a not previously described consensus for the NAD(H) binding.

Fucose metabolism pathways are present in many bacterial species and typically contain the central fucose-processing enzymes fucose isomerase (FcsI), fuculose kinase (FcsK), and fuculose-1-phosphate aldolase (FcsA). Fucose initially undergoes isomerization by FcsI producing fuculose, which is then phosphorylated by FcsK. FcsA cleaves the fuculose-1-phosphate product into lactaldehyde and dihydroxyacetone phosphate, which can be incorporated into central metabolism allowing the bacterium to use fucose as an energy source. Streptococcus pneumoniae has fucose-processing operons containing homologs of FcsI, FcsK, and FcsA; however, this bacterium appears unable to utilize fucose as an energy source. To investigate this contradiction, we performed biochemical and structural studies of the S. pneumoniae fucose-processing enzymes SpFcsI, SpFcsK, and SpFcsA. These enzymes are demonstrated to act in a sequential manner to ultimately produce dihydroxyacetone phosphate and have structural features entirely consistent with their observed biochemical activities. Analogous to the regulation of the Escherichia coli fucose utilization operon, fuculose-1-phosphate appears to act as an inducing molecule for activation of the S. pneumoniae fucose operon. Despite our evidence that S. pneumoniae appears to have the appropriate regulatory and biochemical machinery for fucose metabolism, we confirmed the inability of the S. pneumoniae TIGR4 strain to grow on fucose or on the H-disaccharide, which is the probable substrate of the transporter for the pathway. On the basis of these observations, we postulate that the S. pneumoniae fucose-processing pathway has a non-metabolic role in the interaction of this bacterium with its human host.

The facultative human pathogen Vibrio cholerae transits between the gastrointestinal tract of its host and aquatic reservoirs. V. cholerae adapts to different situations by the timely coordinated expression of genes during its life cycle. We recently identified a subclass of genes that are induced at late stages of infection. Initial characterization demonstrated that some of these genes facilitate the transition of V. cholerae from host to environmental conditions. Among these genes are uptake systems lacking detailed characterization or correct annotation. In this study, we comprehensively investigated the function of the VCA0682-to-VCA0687 gene cluster, which was previously identified as in vivo induced. The results presented here demonstrate that the operon encompassing open reading frames VCA0685 to VCA0687 encodes an ABC transport system for hexose-6-phosphates with K m values ranging from 0.275 to 1.273 μM for glucose-6P and fructose-6P, respectively. Expression of the operon is induced by the presence of hexose-6P controlled by the transcriptional activator VCA0682, representing a UhpA homolog. Finally, we provide evidence that the operon is essential for the utilization of hexose-6P as a C and P source. Thereby, a physiological role can be assigned to hexose-6P uptake, which correlates with increased fitness of V. cholerae after a transition from the host into phosphate-limiting environments.

International audience; The dairy population of Carnobacterium maltaromaticum is characterized by a high diversity suggesting a high diversity of the genetic traits linked to the dairy process. As lactose is the main carbon source in milk, the genetics of lactose metabolism was investigated in this LAB. Comparative genomic analysis revealed that the species C. maltaromaticum exhibits genes related to the Leloir and the tagatose-6-phosphate (Tagatose-6P) pathways. More precisely, strains can bear genes related to one or both pathways and several strains apparently do not contain homologs related to these pathways. Analysis at the population scale revealed that the Tagatose-6P and the Leloir encoding genes are disseminated in multiple phylogenetic lineages of C. maltaromaticum: genes of the Tagatose-6P pathway are present in the lineages I, II and III, and genes of the Leloir pathway are present in the lineages I, III and IV. These data suggest that these genes evolved thanks to horizontal transfer, genetic duplication and translocation. We hypothesize that the lac and gal genes evolved in C. maltaromaticum according to a complex scenario that mirrors the high population diversity.

Glucitol, also known as sorbitol, is a major photosynthetic product in plants from the Rosaceae family. This sugar alcohol is synthesized from glucose-6-phosphate by the combined activities of aldose-6-phosphate reductase (Ald6PRase) and glucitol-6-phosphatase. In this work we show the purification and characterization of recombinant Ald6PRase from peach leaves. The recombinant enzyme was inhibited by glucose-1-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate and orthophosphate. Oxidizing agents irreversibly inhibited the enzyme and produced protein precipitation. Enzyme thiolation with oxidized glutathione protected the enzyme from insolubilization caused by diamide, while incubation with NADP+ (one of the substrates) completely prevented enzyme precipitation. Our results suggest that Ald6PRase is finely regulated to control carbon partitioning in peach leaves. Fil: Hartman, Matias Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Figueroa, Carlos Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Arias, Diego Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina

Fuculose-1-phosphate aldolase (FucA) catalyses the reversible cleavage of L-fuculose 1-phosphate to dihydroxyacetone phosphate (DHAP) and L-lactaldehyde. This enzyme from mesophiles and thermophiles has been extensively studied; however, there is no report on this enzyme from a psychrophile. In this study, the gene encoding FucA fromGlaciozyma antarcticaPI12 (GaFucA) was cloned and the enzyme was overexpressed inEscherichia coli, purified and crystallized. The tetrameric structure of GaFucA was determined to 1.34 Å resolution. The overall architecture of GaFucA and its catalytically essential histidine triad are highly conserved among other fuculose aldolases. Comparisons of structural features between GaFucA and its mesophilic and thermophilic homologues revealed that the enzyme has typical psychrophilic attributes, indicated by the presence of a high number of nonpolar residues at the surface and a lower number of arginine residues.

Flux measurements through metabolic pathways generate insights into the integration of metabolism, and there is increasing interest in using such measurements to quantify the metabolic effects of mutation and genetic manipulation. Isotope labelling provides a powerful approach for measuring metabolic fluxes, and it gives rise to several distinct methods based on either dynamic or steady-state experiments. We discuss the application of these methods to photosynthetic and non-photosynthetic plant tissues, and we illustrate the different approaches with an analysis of the pathways interconverting hexose phosphates and triose phosphates. The complicating effects of the pentose phosphate pathway and the problems arising from the extensive compartmentation of plant cell metabolism are considered. The non-trivial nature of the analysis is emphasised by reference to invalid deductions in earlier work. It is concluded that steady-state isotopic labelling experiments can provide important information on the fluxes through primary metabolism in plants, and that the combination of stable isotope labelling with detection by nuclear magnetic resonance is particularly informative.

Residual lactose and galactose in fermented dairy foods leads to several industrial and health concerns. There is very little information pertaining to manufacture of fermented dairy foods that are low in lactose and galactose. In the present study, comparative genomic survey demonstrated the constant presence of chromosome-encoded tagatose-6-phosphate (T6P) pathway in Lactobacillus casei group. Lactose/galactose utilization tests and β-galactosidase assay suggest that PTSGal system, PTSLac system and T6P pathway are major contributors for lactose/galactose catabolism in this group of organisms. In addition, it was found than lactose catabolism by Lb. casei group accumulated very limited galactose in the MRS-lactose medium and in reconstituted skim milk, whereas Streptococcus thermophilus and Lb. delbrueckii subsp. bulgaricus (Lb. bulgaricus) strains secreted high amount of galactose extracellularly. Moreover, co-culturing Lb. casei group with Str. thermophilus showed significant reduction in galactose content, while co-culturing Lb. casei group with Lb. bulgaricus showed significant reduction in lactose content but significant increase in galactose content in milk. Overall, the present study highlighted the potential of Lb. casei group for reducing galactose accumulation in fermented milks due to its species-specific T6P pathway.

Fuculose-1-phosphate aldolase (FucA) has been produced in Escherichia coli as active inclusion bodies (IBs) in batch cultures. The activity of insoluble FucA has been modulated by a proper selection of producing strain, culture media, and process conditions. In some cases, when an optimized defined medium was used, FucA IBs were more active (in terms of specific activity) than the soluble protein version obtained in the same process with a conventional defined medium, supporting the concept that solubility and conformational quality are independent protein parameters. FucA IBs have been tested as biocatalysts, either directly or immobilized into Lentikat beads, in an aldolic reaction between DHAP and (S)-Cbz-alaninal, obtaining product yields ranging from 65 to 76%. The production of an active aldolase as IBs, the possibility of tailoring IBs properties by both genetic and process approaches, and the reusability of IBs by further entrapment in appropriate matrices fully support the principle of using self-assembled enzymatic clusters as tunable mechanically stable and functional biocatalysts.

Bacterial microcompartments (BMCs) are proteinaceous organelles encapsulating enzymes that catalyze sequential reactions of metabolic pathways. BMCs are phylogenetically widespread; however, only a few BMCs have been experimentally characterized. Among them are the carboxysomes and the propanediol- and ethanolamine-utilizing microcompartments, which play diverse metabolic and ecological roles. The substrate of a BMC is defined by its signature enzyme. In catabolic BMCs, this enzyme typically generates an aldehyde. Recently, it was shown that the most prevalent signature enzymes encoded by BMC loci are glycyl radical enzymes, yet little is known about the function of these BMCs. Here we characterize the glycyl radical enzyme-associated microcompartment (GRM) loci using a combination of bioinformatic analyses and active-site and structural modeling to show that the GRMs comprise five subtypes. We predict distinct functions for the GRMs, including the degradation of choline, propanediol, and fuculose phosphate. This is the first family of BMCs for which identification of the signature enzyme is insufficient for predicting function. The distinct GRM functions are also reflected in differences in shell composition and apparently different assembly pathways. The GRMs are the counterparts of the vitamin B 12 -dependent propanediol- and ethanolamine-utilizing BMCs, which are frequently associated with virulence. This study provides a comprehensive foundation for experimental investigations of the diverse roles of GRMs. Understanding this plasticity of function within a single BMC family, including characterization of differences in permeability and assembly, can inform approaches to BMC bioengineering and the design of therapeutics.

A significant rise in antibody against pneumococcal types 3, 6A and 25 after pneumococcal vaccination could be demonstrated in 26 of 30 individuals (87%) using an enzyme-linked immunosorbent assay. Similar geometric mean antibody fold increases were found in splenectomized children, non-splenectomized children and healthy adults for types 6A and 25. Splenectomized children exhibited a somewhat lower, but not statistically significant, geometric mean antibody fold increase against type 3 (p = 0.10), as compared to non-splenectomized children. For the whole group a negative correlation could be demonstrated between prevaccination antibody concentration and antibody fold increase after vaccination. After vaccination an enhanced serum opsonic activity against type 3 was found by granulocyte glucose-l-14C oxidation for the whole group of 30 patients (p less than 0.05) but nine individual patients (30%) failed to exhibit an increase. No differences between the three patient groups were demonstrable. Changes in serum opsonic activity could not be detected by a pneumococcal bactericidal assay.

Apple leaf cytosolic phosphoglucose isomerase (PGI, EC 5.3.1.9) was purified to an apparent homogeneity with a specific activity of 2456 units/mg protein, and chloroplastic PGI was partially purified to a specific activity of 72 units/mg protein to characterize their biochemical properties. These two isoforms showed differential responses to heat treatment; incubation at 50 degrees C for 10 min resulted in a complete loss of the chloroplastic PGI activity, whereas the cytosolic PGI only lost 50% of its activity. Apple cytosolic PGI is a dimeric enzyme with a molecular mass of 66 kDa for each monomer. The activity of both isoforms was strongly inhibited by erythrose 4-phosphate (E4P) with a K(i) of 1.2 and 3.0 microM for the cytosolic PGI and chloroplastic PGI, respectively. Sorbitol 6-phosphate (Sor6P), an intermediate in sorbitol biosynthesis, was found to be a competitive inhibitor for both cytosolic and chloroplastic PGIs with a K(i) of 61 and 40 microM, respectively. PGIs from both spinach and tomato leaves were also inhibited by Sor6P in a similar manner. The possible physiological significance of this finding is discussed.

An infant, suspected of having galactosemia following a positive screening test on dried blood spots, was shown to be a Duarte-transferase deficiency compound heterozygote through studies of electrophoretic mobility of the transferase enzyme in blood from the patient and family members. No rise in blood glucose was seen following oral ingestion of galactose. At the same time, galactose rose in plasma and was excreted in the urine; galactose-1-phosphate accumulated in erythrocytes. A galactose-free diet was considered the prudent course in the presence of the patient's inability to metabolize galactose completely.

Transaldolase B (TalB) and D-fructose-6-phosphate aldolase A (FSAA) fromEscherichia coliare C—C bond-forming enzymes. Using kinetic inhibition studies and mass spectrometry, it is shown that enzyme variants of FSAA and TalB that exhibit D-fructose-6-phosphate aldolase activity are inhibited covalently and irreversibly by D-tagatose 6-phosphate (D-T6P), whereas no inhibition was observed for wild-type transaldolase B fromE. coli. The crystal structure of the variant TalBF178Ywith bound sugar phosphate was solved to a resolution of 1.46 Å and revealed a novel mode of covalent inhibition. The sugar is bound covalentlyviaits C2 atom to the ∊-NH2group of the active-site residue Lys132. It is neither bound in the open-chain form nor as the closed-ring form of D-T6P, but has been converted to β-D-galactofuranose 6-phosphate (D-G6P), a five-membered ring structure. The furanose ring of the covalent adduct is formedviaa Heyns rearrangement and subsequent hemiacetal formation. This reaction is facilitated by Tyr178, which is proposed to act as acid–base catalyst. The crystal structure of the inhibitor complex is compared with the structure of the Schiff-base intermediate of TalBE96Qformed with the substrate D-fructose 6-phosphate determined to a resolution of 2.20 Å. This comparison highlights the differences in stereochemistry at the C4 atom of the ligand as an essential determinant for the formation of the inhibitor adduct in the active site of the enzyme.

We report the first enzymatic synthesis of D-tagatose-1-phosphate (Tag-1P) by the multicomponent phosphoenolpyruvate:sugar phosphotransferase system (PEP-PTS) present in tagatose-grown cells of Klebsiella pneumoniae. Physicochemical characterization by (31)P and (1)H nuclear magnetic resonance spectroscopy reveals that, in solution, this derivative is primarily in the pyranose form. Tag-1P was used to characterize the putative tagatose-1-phosphate kinase (TagK) of the Bacillus licheniformis PTS-mediated D-tagatose catabolic pathway (Bli-TagP). For this purpose, a soluble protein fusion was obtained with the 6 His-tagged trigger factor (TF(His6)) of Escherichia coli. The active fusion enzyme was named TagK-TF(His6). Tag-1P and D-fructose-1-phosphate are substrates for the TagK-TF(His6) enzyme, whereas the isomeric derivatives D-tagatose-6-phosphate and D-fructose-6-phosphate are inhibitors. Studies of catalytic efficiency (kcat/Km) reveal that the enzyme specificity is markedly in favor of Tag-1P as the substrate. Importantly, we show in vivo that the transfer of the phosphate moiety from PEP to the B. licheniformis tagatose-specific Enzyme II in E. coli is inefficient. The capability of the PTS general cytoplasmic components of B. subtilis, HPr and Enzyme I to restore the phosphate transfer is demonstrated.

Human brain relies on a steady supply of glucose as the source of fuel, and type I hexokinase is the major isozyme governing the introduction of glucose to glycolysis in the brain. One unique regulatory property associated with type I isozyme is the alleviation of product inhibition by inorganic phosphate which binds to the N-terminal half, and the conformational change induced by inorganic phosphate must be propagated to the active site in the C-terminal half. With a single interdomain alpha-helix as the only covalent connection between the N- and C-terminal halves, the question arises as what role the interdomain alpha-helix plays at the interdomain signal transduction. Two mutants were constructed in an attempt to answer this question. The first mutant, A464P/E465G, with a helix breaker embedded in the interdomain alpha-helix had a smaller magnitude of phosphate alleviation than the wild type. The second mutant, with an insertion of seven additional residues between Gln 466 and His 467, had this phosphate relief property further diminished. Neither mutant showed dramatic changes nor the other kinetic properties. It is speculated that the interdomain alpha-helix is important for keeping the proper non-covalent contact so that transmission of the conformational changes across the N- and C-terminal half boundary can be achieved.

One of the early steps in the biosynthesis of coenzyme F 420 in Methanocaldococcus jannaschii requires generation of 2-phospho- l -lactate, which is formed by the phosphorylation of l -lactate. Preliminary studies had shown that l -lactate in M. jannaschii is not derived from pyruvate, and thus an alternate pathway(s) for its formation was examined. Here we report that l -lactate is formed by the NAD + -dependent oxidation of l -lactaldehyde by the MJ1411 gene product. The lactaldehyde, in turn, was found to be generated either by the NAD(P)H reduction of methylglyoxal or by the aldol cleavage of fuculose-1-phosphate by fuculose-1-phosphate aldolase, the MJ1418 gene product.

Modification of tuber carbohydrate metabolism by the tuber-specific expression of a yeast invertase targeted to the cytosol or apoplast has previously been demonstrated to have diverse effects on tuber growth and metabolism. In the current study, we generated plants exhibiting tuber-specific expression of the same enzyme targeted to the vacuole. Enzymatic analysis of the carbohydrate levels of the tuber revealed dramatic decreases in sucrose content coupled with large increases in the levels of glucose and hexose phosphates, but unaltered starch content in the transformants. Analysis of the key enzyme of glycolysis suggests that this pathway is down-regulated in the transformants. Despite these changes in metabolite pools and enzyme activity, few consistent changes could be observed in the estimated metabolic fluxes following incubation of isolated tuber discs in labelled glucose. The analysis of the relative levels of a wide range of metabolites using a gas chromatography-mass spectrometry (GC-MS)-based metabolite profiling method revealed large changes in the levels of fructose and decreases in a range of other sugars, but very few changes in the contents of organic and amino acids. This metabolic profile is remarkably consistent with that obtained following expression of the invertase in the apoplastic compartment, providing circumstantial evidence for the endocytotic trafficking of sugars within potato tuber parenchyma. Finally, the results of this study are compared with those from other plant species and the relative roles of the vacuolar isoform of the enzyme are contrasted.

D-Sorbitol-6-phosphate 2-dehydrogenase catalyzes the NADH-dependent conversion of D-fructose 6-phosphate to D-sorbitol 6-phosphate and improved production and purification of the enzyme from Escherichia coli is reported. Preliminary inhibition studies of the enzyme revealed 5-phospho-D-arabinonohydroxamic acid and 5-phospho-D-arabinonate as new substrate analogue inhibitors of the F6P catalyzed reduction with IC50 values of (40 +/- 1) microM and (48 +/- 3) microM and corresponding Km/IC50 ratio values of 14 and 12, respectively. Furthermore, we report here the phosphomannose isomerase substrate D-mannose 6-phosphate as the best inhibitor of E. coli D-sorbitol-6-phosphate 2-dehydrogenase yet reported with an IC50 = 7.5 +/- 0.4 microM and corresponding Km/IC50 ratio = about 76.

Recent work has raised a question as to the involvement of erythrose-4-phosphate, a product of the pentose phosphate pathway, in the metabolism of the methanogenic archaea (R. H. White, Biochemistry 43: 7618-7627, 2004). To address the possible absence of erythrose-4-phosphate in Methanocaldococcus jannaschii , we have assayed cell extracts of this methanogen for the presence of this and other intermediates in the pentose phosphate pathway and have determined and compared the labeling patterns of sugar phosphates derived metabolically from [6,6- 2 H 2 ]- and [U- 13 C]-labeled glucose-6-phosphate incubated with cell extracts. The results of this work have established the absence of pentose phosphate pathway intermediates erythrose-4-phosphate, xylose-5-phosphate, and sedoheptulose-7-phosphate in these cells and the presence of d - arabino -3-hexulose-6-phosphate, an intermediate in the ribulose monophosphate pathway. The labeling of the d - ara-bino -3-hexulose-6-phosphate, as well as the other sugar-Ps, indicates that this hexose-6-phosphate was the precursor to ribulose-5-phosphate that in turn was converted into ribose-5-phosphate by ribose-5-phosphate isomerase. Additional work has demonstrated that ribulose-5-phosphate is derived by the loss of formaldehyde from d - arabino -3-hexulose-6-phosphate, catalyzed by the protein product of the MJ1447 gene.

Sorbitol 6-phosphate (S6P) is one of two major sugars (another is trehalose) in the larval hemolymph of Bombyx mori, and its amount dramatically decreases concomitantly with the onset of prepupal period. In the last (fifth) instar larvae, the amount of S6P is approximately 30 micromol/larva at its maximum and decreases to less than 1 micromol at the wandering stage. Incubation of fat bodies of wandering larvae with S6P generates sorbitol in the medium, while S6P in the medium decreases, indicating that fat body possesses sorbitol 6-phosphatase (S6Pase) activity. S6Pase activity in the fat body remains low during the feeding period, abruptly increases at the wandering and decreases to a low level after gut purge. 20-Hydroxyecdysone (20E) increases S6Pase activity in the fat body of feeding larvae, and the activation is dose-dependent. Cell fractionation studies show that S6Pase is mainly associated with the membrane and the optimal pH for membrane-bound S6Pase is 5.5, which is different from that for soluble acid phosphatase (pH 4). Present findings indicate that the S6Pase responsible for a decrease in hemolymph S6P is membrane-bound, and its activity is controlled by a rise of hemolymph ecdysteroid titer at the onset of the wandering stage.

Regulation of glucose-induced oscillations in intracellular Ca2+ concentration ([Ca2+]i) was investigated by using a novel technique, electroporation from an electrolyte-filled capillary, to deliver energy metabolites to the intracellular compartment of mouse islets. Intracellular application of ATP resulted in a nifedipine-sensitive increase in [Ca2+]i, consistent with a KATP-channel dependent mechanism of Ca2+ influx. [Ca2+]i in islets exposed to 10 mM glucose oscillated with a period of approximately 3 min, often superimposed with faster oscillations. Electroporation of ATP blocked all types of oscillations and elevated [Ca2+]i while delivery of ADP had no effect on oscillations. Intracellular delivery of glucose-6-phosphate or fructose-1,6-bisphosphate tended to transform slow oscillations to fast oscillations. These results demonstrate that modulation of ATP concentrations and glycolytic flux are important in development of slow oscillations.

International audience; Pre-diabetic subjects with high insulin secretory capacity have double risk of cardiovascular disease compared with subjects who do not develop insulin-resistance. It is well established that the ability of the myocardium to increase its glycolytic ATP production plays a crucial role in determining cell survival under conditions of ischemia. Up to now, whether the pre-diabetic state reduces the tolerance of the heart to ischemia by affecting its ability to increase its energy production through glycolysis remains unknown. The aim of the present study was to assess whether insulin resistance affects the ability of the myocardium to increase glycolysis under ischemic conditions. Male Wistar rats were fed for 8 weeks a fructose-enriched (33%) diet to induce a pre-diabetic state. Hearts were isolated and subjected to ex-vivo low-flow (2%) ischemia for 30 min. The fructose diet increased sarcolemmal GLUT4 localisation in myocardial cells under basal conditions compared with controls. This effect was not accompanied by increased glucose utilisation. Ischemia induced the translocation of GLUT4 to the plasma membrane in controls but did not significantly modify the distribution of these transporters in pre-diabetic hearts. Glycolytic flux under ischemic conditions was significantly lower in fructose-fed rat hearts compared with controls. The reduction of glycolytic flux during ischemia in fructose-fed rat hearts was not due to metabolic inhibition downstream hexokinase II since no cardiac accumulation of glucose-6-phosphate was detected. In conclusion, our results suggest that the pre-diabetic state reduces the tolerance of the myocardium to ischemia by decreasing glycolytic flux adaptation.

Phosphoglucose isomerase (PGI; E.C. 5.3.1.9) is a cytosolic enzyme that catalyzes the reversible isomerization of G6P and F6P. Its activity is important for glycolysis; gluconeogenesis; the pentose phosphate pathway; and the glycosylation of proteins, lipids, and other molecules. Recently, attention to PGI has grown because PGI is a moonlighting protein with a second function outside of the cell. PGI is the same protein as the extracellular growth factors neuroleukin (Chaput et al. 1988; Faik et al. 1988), autocrine motility factor (Watanabe et al. 1996), and differentiation and maturation mediator (Xu et al. 1996). It stimulates antibody secretion, promotes the survival of embryonic spinal neurons in vitro (Gurney et al. 1986a,b), affects an increase in tumor cell motility, and causes the differentiation of human myelogenous leukemia cells (Chiao et al. 1999) and a human myelocytic cell line (HL-60 cells) to terminal monocytic cells (Leung and Chiao 1985; Abolhassani et al. 1990). Recently, PGI also has been found to be the self-antigen in a mouse model of rheumatoid arthritis (Matsumoto et al. 1999; Schaller et al. 2001). One lab has identified antibodies to PGI in patients suffering from rheumatoid arthritis (Schaller et al. 2001). The enzymatic activity of PGI was first described over 70 years ago (Lohmann 1933). Biochemical characterization of PGI enzyme activity by several labs led to a proposed multistep mechanism involving general acid–base catalysis (Rose and O’Connell 1961; Rose 1975, 1981; Willem et al. 1990). Both G6P and F6P are substrates for PGI activity. Because G6P and F6P exist predominantly in their cyclic forms in solution (Swenson and Barker 1971), it is believed that first the cyclic form of G6P or F6P binds to the active site. PGI catalyzes ring opening of the substrate to yield the open chain form. The reaction then proceeds through the isomerization step using acid–base catalysis with proton transfer. PGI is a dimer with 557 amino acids in each subunit and has two equivalent active sites. X-ray crystal structures have been reported of PGI without bound ligand or with a substrate (F6P) or competitive inhibitors of catalytic activity bound in the active site pocket. X-ray crystal structures of mammalian (rabbit, pig, and human) PGI have been solved with 6PGA (Jeffery et al. 2000), 5PAA (Jeffery et al. 2001; Davies and Muirhead 2002; Davies et al. 2003), 5PAH (Arsenieva et al. 2002), or F6P (Lee et al. 2001), or with no bound ligand (Arsenieva and Jeffery 2002; Davies and Muirhead 2002, 2003). A crystal structure of human PGI with a sulfate ion was also reported (Read et al. 2001). Other crystal structures of PGI included two structures from a bacterium, Bacillus stearothermophilus, with no bound ligand (Sun et al. 1999) or complexed with 5PAA (Chou et al. 2000). Three crystal structures of PGI enzyme from Pyrococcus furiosus were also solved with no bound ligand (Berrisford et al. 2003), with 5PAA, and with 6PGA (Swan et al. 2003). Interestingly, the PGI enzyme from P. furiosus does not share sequence or structural homology with either mammalian or B. stearothermophilus PGI, and its activity is probably the result of convergent evolution. The X-ray crystal structures of PGI have helped to identify active site amino acids, ordered water molecules, and conformational changes involved in the ligand binding, ring opening, and isomerization steps of the multistep catalytic mechanism. Comparison of a crystal structure of rabbit PGI without a bound ligand to structures with ligands provided information about induced fit of the enzyme upon substrate binding. A crystal structure of rabbit PGI complexed with F6P provided information about the roles of Glu216, His388, Lys518, and an ordered water molecule in the ring opening step (Lee et al. 2001). Crystal structures of rabbit PGI complexed with 5PAA and 5PAH were used to identify the role of Glu357 as the active site amino acid residue that transfers a proton between C1 and C2 during the isomerization step (Jeffery et al. 2001) and the role of a water molecule in transferring a proton between the hydroxyl groups on C1 and C2 (Arsenieva et al. 2002). Arg272 was proposed to have a role in helping stabilize the cis-enedio-l(ate) intermediate. Comparison of the PGI/5PAA, PGI/5PAH, and PGI/F6P complexes also indicated that the substrate undergoes a rotation about the C3–C4 bond between the ring opening and isomerization steps to position C1 and C2 near to Glu357. Also, a helix containing amino acid residues 512–520 moves in toward the substrate to help hold it in place for the isomerization step. However, less clear is the state of the active site and substrate between the ring opening and isomerization steps. To complete the details of the catalytic mechanism of PGI, a structure of PGI complexed with an open chain substrate, or an analog, is required. Here, we report an X-ray crystal structure of rabbit PGI complexed with an analog of the open chain form of G6P (S6P), bound in the active site. This structure was solved at 2.0 A resolution.

d-Mannose is an essential monosaccharide constituent of glycoproteins and glycolipids. However, it is unknown how plasma mannose is supplied. The aim of this study was to explore the source of plasma mannose. Oral administration of glucose resulted in a significant decrease of plasma mannose concentration after 20 min in fasted normal rats. However, in fasted type 2 diabetes model rats, plasma mannose concentrations that were higher compared with normal rats did not change after the administration of glucose. When insulin was administered intravenously to fed rats, it took longer for plasma mannose concentrations to decrease significantly in diabetic rats than in normal rats (20 and 5 min, respectively). Intravenous administration of epinephrine to fed normal rats increased the plasma mannose concentration, but this effect was negated by fasting or by administration of a glycogen phosphorylase inhibitor. Epinephrine increased mannose output from the perfused liver of fed rats, but this effect was negated in the presence of a glucose-6-phosphatase inhibitor. Epinephrine also increased the hepatic levels of hexose 6-phosphates, including mannose 6-phosphate. When either lactate alone or lactate plus alanine were administered as gluconeogenic substrates to fasted rats, the concentration of plasma mannose did not increase. When lactate was used to perfuse the liver of fasted rats, a decrease, rather than an increase, in mannose output was observed. These findings indicate that hepatic glycogen is a source of plasma mannose.

Long-term steroid treatment and UVB exposure are well-known cataractogenic factors. The purpose of this study was to investigate metabolic changes in the rabbit lens after long-term dexamethasone treatment in combination with UVB exposure, using high-resolution magic angle spinning proton nuclear magnetic resonance (HR-MAS (1)H NMR) spectroscopy to analyse intact lens tissues.Rabbits received topical doses of 0.1% dexamethasone or 0.9% saline (50 microl) four times daily for 36 days. On day 37, the eyes were exposed to UVB radiation (2.05 J/cm(2)). Twenty-four hours later the animals were killed, and HR-MAS (1)H NMR spectra of lens tissues were obtained.More than 15 major metabolites were assigned in NMR spectra of rabbit lenses. The combined treatment with dexamethasone and UVB induced large reductions in the concentration of reduced glutathione, inositols, taurine and lactate compared with normal lenses. Concurrently, the levels of glucose, sorbitol and sorbitol-3-phosphate were increased. After exposure to UVB radiation only, the most significant finding was a decrease in the concentration of lactate. No lens opacities were detected.HR-MAS (1)H NMR spectroscopy was found to be an efficient tool for analysis of intact lens tissues. High-resolution NMR spectra of intact lens tissue enabled metabolic changes to be quantified. Long-term treatment with dexamethasone combined with UVB exposure induced substantial metabolic changes, dominated by osmolytic regulation processes and loss of glutathione.

UDP-sugars, activated forms of monosaccharides, are synthesized through de novo and salvage pathways and serve as substrates for the synthesis of polysaccharides, glycolipids, and glycoproteins in higher plants. A UDP-sugar pyrophosphorylase, designated PsUSP, was purified about 1,200-fold from pea (Pisum sativum L.) sprouts by conventional chromatography. The apparent molecular mass of the purified PsUSP was 67,000 Da. The enzyme catalyzed the formation of UDP-Glc, UDP-Gal, UDP-glucuronic acid, UDP-l-arabinose, and UDP-xylose from respective monosaccharide 1-phosphates in the presence of UTP as a co-substrate, indicating that the enzyme has broad substrate specificity toward monosaccharide 1-phosphates. Maximum activity of the enzyme occurred at pH 6.5-7.5, and at 45 degrees C in the presence of 2 mm Mg(2+). The apparent K(m) values for Glc 1-phosphate and l-arabinose 1-phosphate were 0.34 and 0.96 mm, respectively. PsUSP cDNA was cloned by reverse transcriptase-PCR. PsUSP appears to encode a protein with a molecular mass of 66,040 Da (600 amino acids) and possesses a uridine-binding site, which has also been found in a human UDP-N-acetylhexosamine pyrophosphorylase. Phylogenetic analysis revealed that PsUSP can be categorized in a group together with homologues from Arabidopsis and rice, which is distinct from the UDP-Glc and UDP-N-acetylhexosamine pyrophosphorylase groups. Recombinant PsUSP expressed in Escherichia coli catalyzed the formation of UDP-sugars from monosaccharide 1-phosphates and UTP with efficiency similar to that of the native enzyme. These results indicate that the enzyme is a novel type of UDP-sugar pyrophosphorylase, which catalyzes the formation of various UDP-sugars at the end of salvage pathways in higher plants.

A cDNA encoding sorbitol-6-phosphate dehydrogenase (S6PDH), which is a key enzyme in sorbitol biosynthesis in Rosaceae, was introduced into the Japanese persimmon (Diospyros kaki) to increase the environmental stress tolerance. Resultant transformants exhibited salt-tolerance with dwarfing phenotypes. Therefore, we studied two transgenic lines to understand the physiological mechanism of this dwarfism: lines PS1 and PS6 accumulated high and moderate levels of sorbitol, respectively. The average length of shoots was significantly shorter as compared with the wild-type in line PS1, while no such decrease was observed in line PS6. The myo-inositol and glucose 6-phosphate (G6P) contents were measured in the transgenic lines because previous work with tobacco transformed with S6PDH had suggested that growth inhibition was due to depletion of these metabolites. Although the myo-inositol content was decreased in PS1 plants, the decrease was much smaller than that observed in transgenic tobacco that accumulates sorbitol. The G6P contents were the same in PS1 plants and phenotypically normal PS6 plants. The level of indole-3-acetic acid (IAA), which affects stem elongation, in line PS1 was similar to the levels in the other lines. A decrease in gibberellin (GA) content generally induces dwarfism in plants. However, GA was not decreased in PS1 plants compared with wild-type or control plants. Therefore, we focused on sorbitol accumulation as the most remarkable feature of PS1 plants. As one possibility, the observed growth inhibition was likely caused by an osmotic imbalance between the cytosol and vacuole.

This review provides a chronological survey of over fifty fluorescent chemosensors for carbohydrates from the period between 1992 to the present. The survey contains only those sensors that are synthetic or chemosensory, utilize boronic acids and display a fluorescence response in the form of intensity changes or shifts in wavelength. With each compound listed, a description of the saccharide probe is given with regard to concentration, excitation and emission wavelengths, pH and solvent mixture proportions. In addition, the selectivity of each chemosensor is provided as well as the trends in binding constants. Where possible, a description of the fluorescence signaling mechanism is given as well as commentary on the probe's unique features within this class of sensors.

The inhibition of glucokinase by rat and Xenopus GKRPs (glucokinase regulatory protein) is well documented. We report a comparison of the effects of human and rat GKRPs on glucokinase activity. Human GKRP is a more potent inhibitor of glucokinase than rat GKRP in the absence of fructose 6-phosphate or sorbitol 6-phosphate, and has a higher affinity for these ligands. However, human and rat GKRPs have similar affinities for fructose 1-phosphate and chloride. Residues that are not conserved between the rodent and human proteins affect both the affinity for fructose 6-phosphate and sorbitol 6-phosphate and the inhibitory potency of GKRP on glucokinase in the absence of these ligands.

In previous studies, we have found that negatively charged, but not uncharged, amino acids and sugars block sea urchin fertilization. These studies were developed from modeling work in non-living systems using derivatized agarose beads that suggested that charge-charge bonding may control at least some adhesive interactions. In the present study, the effects of positively charged, negatively charged and uncharged molecules were examined in the sea urchin sperm-egg system in over 300 individual trials. The results indicate that depending on the specific molecules utilized, both sperm and egg are exquisitely sensitive to charged but not uncharged molecules and to pH changes in sea water caused by some of the charged molecules. It is shown that egg activation, as well as sperm motility and sperm-egg interactions, can be affected by charged molecules. One compound, fructose-1-phosphate blocked fertilization in S. purpuratus sea urchins but not in Lytechinus pictus sea urchins. These findings indicate that charge alone cannot explain all the results. In this case, the presence of a ketone instead of an aldehyde group indicates that species-specific components may control fertilization reactions. The present study is a comprehensive survey of the effects of charge, pH and molecular structure on the fertilization activation continuum in a model system of sea urchins.

Nylon filtration alters the morphology of human granulocytes and slightly reduces their phagocytic ability as measured by activation of the hexose monophosphate shunt (HMS) pathway of glucose metabolism. In addition, the ability of rat granulocytes to circulate in granulocytopenic rats is reduced, and this is not attributed to HMS activation during filtration. The activation of the hexose monophosphate shunt is related to the size of the particles rather than to the nature of the material.

The crystal structure of the hypothetical protein MJ1247 from Methanococccus jannaschii at 2 A resolution, a detailed sequence analysis, and biochemical assays infer its molecular function to be 3-hexulose-6-phosphate isomerase (PHI). In the dissimilatory ribulose monophosphate (RuMP) cycle, ribulose-5-phosphate is coupled to formaldehyde by the 3-hexulose-6-phosphate synthase (HPS), yielding hexulose-6-phosphate, which is then isomerized to fructose-6-phosphate by the enzyme 3-hexulose-6-phosphate isomerase. MJ1247 is an alpha/beta structure consisting of a five-stranded parallel beta sheet flanked on both sides by alpha helices, forming a three-layered alpha-beta-alpha sandwich. The fold represents the nucleotide binding motif of a flavodoxin type. MJ1247 is a tetramer in the crystal and in solution and each monomer has a folding similar to the isomerase domain of glucosamine-6-phosphate synthase (GlmS).

A huge variety of chemically modified oligonucleotide derivatives has been synthesized for possible antisense applications. One such derivative, hexitol nucleic acid (HNA), is a DNA analogue containing the standard nucleoside bases, but with a phosphorylated 1',5'-anhydrohexitol backbone. Hexitol nucleic acids are some of the strongest hybridizing antisense compounds presently known, but HNA duplexes are even more stable. We present here the first high-resolution structure of a double helical nucleic acid with all sugars being hexitols. Although designed to have a restricted conformational flexibility, the hexitol oligomer h(GTGTACAC) is able to crystallize in two different double helical conformations. Both structures display a high x-displacement, normal Watson-Crick base pairing, similar base stacking patterns, and a very deep major groove together with a minor groove with increased hydrophobicity. One of the conformations displays a major groove which is wide enough to accommodate a second HNA double helix resulting in the formation of a double helix of HNA double helices. Both structures show most similarities with the A-type helical structure, the anhydrohexitol chair conformation thereby acting as a good mimic for the furanose C3'-endo conformation observed in RNA. As compared to the quasi-linear structure of homo-DNA, the axial position of the base in HNA allows efficient base stacking and hence double helix formation.

In a 13C experiment for metabolic flux analysis (13C MFA), we examined isotope discrimination by measuring the labeling of glucose, amino acids, and hexose monophosphates via mass spectrometry. When Escherichia coli grew in a mix of 20% fully labeled and 80% naturally labeled glucose medium, the cell metabolism favored light isotopes and the measured isotopic ratios (δ13C) were in the range of −35 to −92. Glucose transporters might play an important role in such isotopic fractionation. Flux analysis showed that both isotopic discrimination and isotopic impurities in labeled substrates could affect the solution of 13C MFA.

The sorbitol-6-phosphate dehydrogenase (S6PDH) is a key enzyme for sorbitol synthesis and plays an important role in the alleviation of salinity stress in plants. Despite the huge significance, the structure and the mode of action of this enzyme are still not known. In the present study, sequence analysis, cloning, expression, activity assays and enzyme kinetics using various substrates (glucose-6-phosphate, sorbitol-6-phosphate and mannose-6-phosphate) were performed to establish the functional role of S6PDH protein from rice (Oryza sativa). For the structural analysis of the protein, a comparative homology model was prepared on the basis of percentage sequence identity and substrate similarity using the crystal structure of human aldose reductase in complex with glucose-6-phosphate and NADP+ (PDB ID: 2ACQ) as a template. Molecular docking was performed for studying the structural details of substrate binding and possible enzyme mechanism. The cloned sequence resulted into an active recombinant protein when expressed into a bacterial expression system. The purified recombinant protein was found to be active with glucose-6-phosphate and sorbitol-6-phosphate; however, activity against mannose-6-phosphate was not found. The K m values for glucose-6-phosphate and sorbitol-6-phosphate were found to be 15.9 ± 0.2 and 7.21 ± 0.5 mM, respectively. A molecular-level analysis of the active site of OsS6PDH provides valuable information about the enzyme mechanism and requisite enantioselectivity for its physiological substrates. Thus, the fundamental studies of structure and function of OsS6PDH could serve as the basis for the future studies of bio-catalytic applications of this enzyme.