Phosphofructokinases A and B from Mycobacterium tuberculosis Display Different Catalytic Properties and Allosteric Regulation.

Tuberculosis (TB) remains one of the major health concerns worldwide. Mycobacterium tuberculosis (Mtb), the causative agent of TB, can flexibly change its metabolic processes during different life stages. Regulation of key metabolic enzyme activities by intracellular conditions, allosteric inhibition or feedback control can effectively contribute to Mtb survival under different conditions. Phosphofructokinase (Pfk) is one of the key enzymes regulating glycolysis. Mtb encodes two Pfk isoenzymes, Pfk A/Rv3010c and Pfk B/Rv2029c, which are differently expressed upon transition to the hypoxia-induced non-replicating state of the bacteria. While pfkB gene and protein expression are upregulated under hypoxic conditions, Pfk A levels decrease. Here, we present biochemical characterization of both Pfk isoenzymes, revealing that Pfk A and Pfk B display different kinetic properties. Although the glycolytic activity of Pfk A is higher than that of Pfk B, it is markedly inhibited by an excess of both substrates (fructose-6-phosphate and ATP), reaction products (fructose-1,6-bisphosphate and ADP) and common metabolic allosteric regulators. In contrast, synthesis of fructose-1,6-bisphosphatase catalyzed by Pfk B is not regulated by higher levels of substrates, and metabolites. Importantly, we found that only Pfk B can catalyze the reverse gluconeogenic reaction. Pfk B thus can support glycolysis under conditions inhibiting Pfk A function.

Structural Studies of HNA Substrate Specificity in Mutants of an Archaeal DNA Polymerase Obtained by Directed Evolution.

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.

Hypothesis: A Novel Neuroprotective Role for Glucose-6-phosphatase (G6PC3) in Brain-To Maintain Energy-Dependent Functions Including Cognitive Processes.

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.

Identification of the lipopolysaccharide O-antigen biosynthesis priming enzyme and the O-antigen ligase in Myxococcus xanthus: critical role of LPS O-antigen in motility and development.

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.

Sustained substrate cycles between hexose phosphates and free sugars in phosphate-deficient potato (Solanum tuberosum) cell cultures.

MAIN CONCLUSION: 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.

Detection of active sorbitol-6-phosphate phosphatase in the haloacid dehalogenase-like hydrolase superfamily.

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.

Structural and functional analysis of Erwinia amylovora SrlD. The first crystal structure of a sorbitol-6-phosphate 2-dehydrogenase.

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 Šresolution, 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.

Inhibition of Recombinant Aldose-6-Phosphate Reductase from Peach Leaves by Hexose-Phosphates, Inorganic Phosphate and Oxidants.

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.

The potential of species-specific tagatose-6-phosphate (T6P) pathway in Lactobacillus casei group for galactose reduction in fermented dairy foods.

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 ß-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.

Crystal structure of fuculose aldolase from the Antarctic psychrophilic yeast Glaciozyma antarctica PI12.

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 from Glaciozyma antarctica PI12 (GaFucA) was cloned and the enzyme was overexpressed in Escherichia coli, purified and crystallized. The tetrameric structure of GaFucA was determined to 1.34 Šresolution. 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.

Bioinformatic characterization of glycyl radical enzyme-associated bacterial microcompartments.

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 B12-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.

Synthesis and Physicochemical Characterization of D-Tagatose-1-Phosphate: The Substrate of the Tagatose-1-Phosphate Kinase in the Phosphotransferase System-Mediated D-Tagatose Catabolic Pathway of Bacillus licheniformis.

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.

Identification and functional characterization of sorbitol-6-phosphate dehydrogenase protein from rice and structural elucidation by in silico approach.

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.

Structural and functional analysis of fucose-processing enzymes from Streptococcus pneumoniae.

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.

Crystal structure and substrate specificity of D-galactose-6-phosphate isomerase complexed with substrates.

D-Galactose-6-phosphate isomerase from Lactobacillus rhamnosus (LacAB; EC 5.3.1.26), which is encoded by the tagatose-6-phosphate pathway gene cluster (lacABCD), catalyzes the isomerization of D-galactose-6-phosphate to D-tagatose-6-phosphate during lactose catabolism and is used to produce rare sugars as low-calorie natural sweeteners. The crystal structures of LacAB and its complex with D-tagatose-6-phosphate revealed that LacAB is a homotetramer of LacA and LacB subunits, with a structure similar to that of ribose-5-phosphate isomerase (Rpi). Structurally, LacAB belongs to the RpiB/LacAB superfamily, having a Rossmann-like αßα sandwich fold as has been identified in pentose phosphate isomerase and hexose phosphate isomerase. In contrast to other family members, the LacB subunit also has a unique α7 helix in its C-terminus. One active site is distinctly located at the interface between LacA and LacB, whereas two active sites are present in RpiB. In the structure of the product complex, the phosphate group of D-tagatose-6-phosphate is bound to three arginine residues, including Arg-39, producing a different substrate orientation than that in RpiB, where the substrate binds at Asp-43. Due to the proximity of the Arg-134 residue and backbone Cα of the α6 helix in LacA to the last Asp-172 residue of LacB with a hydrogen bond, a six-carbon sugar-phosphate can bind in the larger pocket of LacAB, compared with RpiB. His-96 in the active site is important for ring opening and substrate orientation, and Cys-65 is essential for the isomerization activity of the enzyme. Two rare sugar substrates, D-psicose and D-ribulose, show optimal binding in the LacAB-substrate complex. These findings were supported by the results of LacA activity assays.

Bifunctional sucrose phosphate synthase/phosphatase is involved in the sucrose biosynthesis by Methylobacillus flagellatus KT.

The aerobic obligate methylotroph Methylobacillus flagellatus KT was shown to synthesize sucrose in the presence of 0.5-2% NaCl in the growth medium. In the genome of this bacterium, an open reading frame (ORF) encoding a predicted 84-kD polypeptide homologous to the plant and cyanobacterial sucrose phosphate synthases (SPSs) was found. Using heterologous expression of the putative sps gene in Escherichia coli, followed by affinity chromatography, pure recombinant protein SPS-His6 was obtained. The enzyme catalyzed two reactions: conversion of fructose 6-phosphate and UDP-glucose into sucrose 6-phosphate and hydrolysis of sucrose 6-phosphate to sucrose. The bifunctional sucrose phosphate synthase/phosphatase (SPS/SPP) was a 340 kDa homotetrameric Mg(2+) -dependent enzyme activated by fructose 1,6-phosphate2 and ATP but inhibited by glucose 6-phosphate, fructose 1-phosphate, AMP and inorganic phosphate. The amino acid sequence of the protein had a C-terminal domain homologous to SPPs. This correlated with the absence of the spp gene in the M. flagellatus chromosome. The ORFs homologous to the M. flagellatus SPS were found in the genomes of another obligate methylotroph Methylovorus glucosetrophus as well as the lithoautotrophic bacteria Acidithiobacillus ferrooxidans, Nitrosomonas europaea and Nitrosospira multiformis whose genomes lacked the spp genes. Thus, data extending the knowledge of biochemical properties of bacterial SPSs have been obtained.

Characterizing the hexose-6-phosphate transport system of Vibrio cholerae, a utilization system for carbon and phosphate sources.

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 Km 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.

The futile cycling of hexose phosphates could account for the fact that hexokinase exerts a high control on glucose phosphorylation but not on glycolytic rate in transgenic potato (Solanum tuberosum) roots.

The metabolism of potato (Solanum tuberosum) roots constitutively over- and underexpressing hexokinase (HK, EC 2.7.1.1) was examined. An 11-fold variation in HK activity resulted in altered root growth, with antisense roots growing better than sense roots. Quantification of sugars, organic acids and amino acids in transgenic roots demonstrated that the manipulation of HK activity had very little effect on the intracellular pools of these metabolites. However, adenylate and free Pi levels were negatively affected by an increase in HK activity. The flux control coefficient of HK over the phosphorylation of glucose was measured for the first time in plants. Its value varied with HK level. It reached 1.71 at or below normal HK activity value and was much lower (0.32) at very high HK levels. Measurements of glycolytic flux and O(2) uptake rates demonstrated that the differences in glucose phosphorylation did not affect significantly glycolytic and respiratory metabolism. We hypothesized that these results could be explained by the existence of a futile cycle between the pools of hexose-Ps and carbohydrates. This view is supported by several lines of evidence. Firstly, activities of enzymes capable of catalyzing these reactions were detected in roots, including a hexose-P phosphatase. Secondly, metabolic tracer experiments using (14)C-glucose as precursor showed the formation of (14)C-fructose and (14)C-sucrose. We conclude that futile cycling of hexose-P could be partially responsible for the differences in energetic status in roots with high and low HK activity and possibly cause the observed alterations in growth in transgenic roots. The involvement of HK and futile cycles in the control of glucose-6P metabolism is discussed.

GFAJ-1 is an arsenate-resistant, phosphate-dependent organism.

The bacterial isolate GFAJ-1 has been proposed to substitute arsenic for phosphorus to sustain growth. We have shown that GFAJ-1 is able to grow at low phosphate concentrations (1.7 µM), even in the presence of high concentrations of arsenate (40 mM), but lacks the ability to grow in phosphorus-depleted (<0.3 µM), arsenate-containing medium. High-resolution mass spectrometry analyses revealed that phosphorylated central metabolites and phosphorylated nucleic acids predominated. A few arsenylated compounds, including C6 sugar arsenates, were detected in extracts of GFAJ-1, when GFAJ-1 was incubated with arsenate, but further experiments showed they formed abiotically. Inductively coupled plasma mass spectrometry confirmed the presence of phosphorus in nucleic acid extracts, while arsenic could not be detected and was below 1 per mil relative to phosphorus. Taken together, we conclude that GFAJ-1 is an arsenate-resistant, but still a phosphate-dependent, bacterium.

Target metabolomics revealed complementary roles of hexose- and pentose-phosphates in the regulation of carbohydrate-dependent gene expression.

Carbohydrate response element-binding protein (ChREBP) is a transcription factor that mediates glucose signaling in mammalian liver, leading to the expression of different glycolytic and lipogenic genes, such as pyruvate kinase (L-PK) and fatty acid synthase (FAS). The current model for ChREBP activation in response to sugar phosphates holds that glucose metabolization to xylulose 5-phosphate (X-5-P) triggers the activation of protein phosphatase 2A, which dephosphorylates ChREBP and leads to its nuclear translocation and activation. However, evidence indicates that glucose 6-phosphate (G-6-P) is the most likely signal metabolite for the glucose-induced transcription of these genes. The glucose derivative that is responsible for carbohydrate-dependent gene expression remains to be identified. The difficulties in measuring G-6-P and X-5-P concentrations simultaneously and in changing them independently have hindered such identification. To discriminate between these possibilities, we adapted a liquid chromatography mass spectrometry method to identify and quantify sugar phosphates in human hepatocarcinoma cells (Hep G2) and rat hepatocytes in response to different carbon sources and in the presence/absence of a glucose-6-phosphate dehydrogenase inhibitor. We also used this method to demonstrate that these cells could not metabolize 2-deoxyglucose beyond 2-deoxyglucose-6-phosphate. The simultaneous quantification of sugar phosphates and FAS and L-PK expression levels demonstrated that both X-5-P and G-6-P play a role in the modulation of gene expression. In conclusion, this report presents for the first time a single mechanism that incorporates the effects of X-5-P and G-6-P on the enhancement of the expression of carbohydrate-responsive genes.