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The mechanistic target of rapamycin complex 1 (mTORC1) kinase regulates cell growth by setting the balance between anabolic and catabolic processes. To be active, mTORC1 requires the environmental presence of amino acids and glucose. While a mechanistic understanding of amino acid sensing by mTORC1 is emerging, how glucose activates mTORC1 remains mysterious. Here, we used metabolically engineered human cells lacking the canonical energy sensor AMP-activated protein kinase to identify glucose-derived metabolites required to activate mTORC1 independent of energetic stress. We show that mTORC1 senses a metabolite downstream of the aldolase and upstream of the GAPDH-catalysed steps of glycolysis and pinpoint dihydroxyacetone phosphate (DHAP) as the key molecule. In cells expressing a triose kinase, the synthesis of DHAP from DHA is sufficient to activate mTORC1 even in the absence of glucose. DHAP is a precursor for lipid synthesis, a process under the control of mTORC1, which provides a potential rationale for the sensing of DHAP by mTORC1. Levels of the glycolytic intermediate metabolite dihydroxyacetone phosphate are shown to signal cellular glucose availability to the mTORC1 complex through an AMPK-independent route.

Triosephosphate isomerase (TIM) is a perfectly evolved enzyme which very fast interconverts dihydroxyacetone phosphate and d-glyceraldehyde-3-phosphate. Its catalytic site is at the dimer interface, but the four catalytic residues, Asn11, Lys13, His95 and Glu167, are from the same subunit. Glu167 is the catalytic base. An important feature of the TIM active site is the concerted closure of loop-6 and loop-7 on ligand binding, shielding the catalytic site from bulk solvent. The buried active site stabilises the enediolate intermediate. The catalytic residue Glu167 is at the beginning of loop-6. On closure of loop-6, the Glu167 carboxylate moiety moves approximately 2 Å to the substrate. The dynamic properties of the Glu167 side chain in the enzyme substrate complex are a key feature of the proton shuttling mechanism. Two proton shuttling mechanisms, the classical and the criss-cross mechanism, are responsible for the interconversion of the substrates of this enolising enzyme.

Diabetic kidney disease (DKD) can lead to accumulation of glucose upstream metabolites due to dysfunctional glycolysis. But the effects of accumulated glycolysis metabolites on podocytes in DKD remain unknown. The present study examined the effect of dihydroxyacetone phosphate (DHAP) on high glucose induced podocyte pyroptosis. By metabolomics, levels of DHAP, GAP, glucose‐6‐phosphate and fructose 1, 6‐bisphosphate were significantly increased in glomeruli of db/db mice. Furthermore, the expression of LDHA and PKM2 were decreased. mRNA sequencing showed upregulation of pyroptosis‐related genes (Nlrp3, Casp1, etc.). Targeted metabolomics demonstrated higher level of DHAP in HG‐treated podocytes. In vitro, ALDOB expression in HG‐treated podocytes was significantly increased. siALDOB‐transfected podocytes showed less DHAP level, mTORC1 activation, reactive oxygen species (ROS) production, and pyroptosis, while overexpression of ALDOB had opposite effects. Furthermore, GAP had no effect on mTORC1 activation, and mTORC1 inhibitor rapamycin alleviated ROS production and pyroptosis in HG‐stimulated podocytes. Our findings demonstrate that DHAP represents a critical metabolic product for pyroptosis in HG‐stimulated podocytes through regulation of mTORC1 pathway. In addition, the results provide evidence that podocyte injury in DKD may be treated by reducing DHAP.

Metabolic engineering often involves the addition of enzymes, redirection of metabolic flux or elimination of undesirable endpoints and thus requires laborious optimization of numerous parameters. A new method to derive 'blueprints' from real-time measurements of metabolic networks significantly accelerates this process as demonstrated with the production of dihydroxyacetone phosphate. Recruiting complex metabolic reaction networks for chemical synthesis has attracted considerable attention but frequently requires optimization of network composition and dynamics to reach sufficient productivity. As a design framework to predict optimal levels for all enzymes in the network is currently not available, state-of-the-art pathway optimization relies on high-throughput phenotype screening. We present here the development and application of a new in vitro real-time analysis method for the comprehensive investigation and rational programming of enzyme networks for synthetic tasks. We used this first to rationally and rapidly derive an optimal blueprint for the production of the fine chemical building block dihydroxyacetone phosphate (DHAP) via Escherichia coli 's highly evolved glycolysis. Second, the method guided the three-step genetic implementation of the blueprint, yielding a synthetic operon with the predicted 2.5-fold–increased glycolytic flux toward DHAP. The new analytical setup drastically accelerates rational optimization of synthetic multienzyme networks.

Carbon-carbon bond formation is one of the most challenging reactions in synthetic organic chemistry, and aldol reactions catalysed by dihydroxyacetone phosphate-dependent aldolases provide a powerful biocatalytic tool for combining C-C bond formation with the generation of two new stereo-centres, with access to all four possible stereoisomers of a compound. Dihydroxyacetone phosphate (DHAP) is unstable so the provision of DHAP for DHAP-dependent aldolases in biocatalytic processes remains complicated. Our research has investigated the efficiency of several different enzymatic cascades for the conversion of glycerol to DHAP, including characterising new candidate enzymes for some of the reaction steps. The most efficient cascade for DHAP production, comprising a one-pot four-enzyme reaction with glycerol kinase, acetate kinase, glycerophosphate oxidase and catalase, was coupled with a DHAP-dependent fructose-1,6-biphosphate aldolase enzyme to demonstrate the production of several rare chiral sugars. The limitation of batch biocatalysis for these reactions and the potential for improvement using kinetic modelling and flow biocatalysis systems is discussed.

Central carbon metabolism is a basic and exhaustively analyzed pathway. However, the intrinsic robustness of the pathway might still conceal uncharacterized reactions. To test this hypothesis, we constructed systematic multiple‐knockout mutants involved in central carbon catabolism in Escherichia coli and tested their growth under 12 different nutrient conditions. Differences between in silico predictions and experimental growth indicated that unreported reactions existed within this extensively analyzed metabolic network. These putative reactions were then confirmed by metabolome analysis and in vitro enzymatic assays. Novel reactions regarding the breakdown of sedoheptulose‐7‐phosphate to erythrose‐4‐phosphate and dihydroxyacetone phosphate were observed in transaldolase‐deficient mutants, without any noticeable changes in gene expression. These reactions, triggered by an accumulation of sedoheptulose‐7‐phosphate, were catalyzed by the universally conserved glycolytic enzymes ATP‐dependent phosphofructokinase and aldolase. The emergence of an alternative pathway not requiring any changes in gene expression, but rather relying on the accumulation of an intermediate metabolite may be a novel mechanism mediating the robustness of these metabolic networks. Synopsis Systematic phenotype analysis of gene‐deletion mutants, combined with in silico predictions from genome‐scale metabolic network models, has been used to identify new genetic interactions and previously unknown gene functions in model microorganisms. As this approach depends on a predicted or observed phenotype, genetic robustness limits the availability of gene candidates showing some phenotype under the conditions tested. Such robustness could, in part, originate from redundancy such as the presence of an isozyme or another pathway with a duplicate function. In addition, the specialized functions of many genes for specific growth conditions, such as the availability of different carbon sources, could contribute to overall robustness. Systematic deletion of two or more genes, and fitness tests of the mutants under many conditions, would be powerful systems for the discovery of new gene functions. Using a new method employing a P1 phage derivative, we created systematic double‐deletion mutants in the central carbon metabolism of E. coli . The mutants were created by combining 31 single‐gene deletions (first deletion) with deletions in seven key reactions (second deletion). The seven key reactions were selected to represent each of the following pathways: glycolysis (two reactions), the pentose phosphate pathway (two reactions), the anaplerotic pathway (two reactions), and the glyoxylate shunt (one reaction). The resulting strains were then tested for growth capabilities under various nutrient conditions, including rich medium, minimal medium with 10 different carbon sources, and medium containing a combination of two carbon sources (Figure 1 ). At the same time, we performed model‐based prediction of the growth phenotypes of these mutants using genome‐scale metabolic models. By contrasting the simulation result with the experimental result, we aimed to elucidate previously unknown reactions within this exhaustively analyzed pathway in one of the best‐studied organisms. Among 2177 double mutant experiments from which we obtained both experimental and predicted growth phenotypes, we found 39 cases in which model‐based analysis predicted double mutant‐specific slow‐growth phenotypes, although experimental results indicated growth comparable with that of the parental single‐knockout mutants. Out of the 39 cases, we were most interested in eight cases that carried one of their deletions in transaldolase ( talA talB ). Xylose was used as a carbon source in five of these eight cases. Further examination of metabolic pathways indicated that transaldolase must be essential for xylose utilization through currently known reactions in central carbon metabolism (Figure 4A and B ). Although one known pathway could potentially serve as a bypass for utilizing xylose in transaldolase mutants, this bypass could not explain the normal growth of several double‐knockout strains such as fbp ‐ talAB , tpiA ‐ talAB , deoC ‐ talAB , and deoB ‐ talAB (Figure 4C ). Thus, we decided to focus on this discrepancy to find new reaction(s). First, we performed microarray analysis to find specifically induced genes in the transaldolase mutant growing on xylose minimal medium. However, it revealed no notable changes in the mRNA levels of genes involved in related metabolic pathways and did not suggest interesting candidates for the novel pathway. Next, we performed metabolome analysis by CE‐MS, which revealed greater than 40‐fold accumulation of S7P in the talAB ‐knockout cells and several‐fold accumulation of pentose phosphates, but otherwise less than twofold change in the levels of metabolites in related pathways (Figure 4E ). We also discovered accumulation of an unidentified metabolite, postulated to be S1,7P, which was previously not considered to be present in E. coli. Combined with another experimental result from the phenotype analysis that pfkA is necessary for the growth of transaldolase mutants on xylose, we hypothesized that S7P is utilized through S1,7P and degraded to DHAP and E4P in transaldolase mutants (red reactions in Figure 4F ). To test this hypothesis, we performed MFA of wild‐type and talAB ‐knockout strains using 1‐ 13 C‐xylose as the sole carbon source and measured the isotopomer distribution of intermediate metabolites by CE‐MS. The wild‐type and talAB ‐knockout strains clearly showed distinct 13 C isotopomer distributions for many metabolites, and the differences were explained by the presence of the hypothesized new reactions in the talAB knockout, but not in the wild type (Figure 4F ). Finally, we validated these novel reactions at the level of enzymatic activity. Using purified recombinant PfkA, the candidate enzyme for converting S7P to S1,7P, and FbaA, the candidate enzyme for converting S1,7P to DHAP and E4P, we confirmed the conversion from S7P and ATP to (putative) S1,7P by PfkA and then to DHAP and E4P by addition of FbaA. Thus, consistent with our hypothesis, S7P must be converted to S1,7P and then to DHAP and E4P by sequential action of the glycolytic enzyme PfkA (phosphofructokinase) and FbpA (fructose‐bisphosphate‐aldorase) in transaldolase‐deficient cells. The discovery of new reactions, in addition to proving the potency of a strategy combining experimental and computational phenotype analysis of large‐scale multiple‐knockout mutants, has two substantially important implications. First, although the novel reactions seemed to be present only in transaldolase mutants in E. coli , other organisms might also possess these reactions. The most probable candidate organism might be another bacterium, L. lactis , which does not seem to encode transaldolase in its genome, but is known to utilize xylose through glycolysis and the pentose phosphate pathway. In higher eukaryote, some mammalian tissues known to lack transaldolase, and associated with liver cirrhosis, represent another possible candidate having the novel reactions. Second, emergence of these alternative reactions does not require any change in gene expression, but rather relies on the accumulation of an intermediate metabolite, S7P. The emergence of an alternative pathway that does not require any change in gene expression, but rather relies on the accumulation of an intermediate metabolite, may be a novel mechanism that mediates the robustness of metabolic networks. 7A; The production of new methods employing P1 phage derivatives enabled systematic construction of many double and triple mutants of E. coli . 7A; By contrasting experimentally obtained growth phenotypes of multiple knockout mutants in central carbon metabolism with phenotypes that were predicted using reconstructed genome‐scale metabolic models, we predicted novel reactions in the central carbon metabolism of transaldolase mutants of E. coli . 7A; Employing metabolome and metabolic flux analyses, as well as in vitro enzymatic assays, we confirmed the existence of the predicted reactions, from sedoheptulose 7‐phosphate to erythrose 4‐phosphate and dihydroxyacetone phosphate, catalyzed by the sequential action of ATP‐dependent phosphofructokinase and aldolase. 7A; The emergence of an alternative pathway that does not require any change in gene expression may be a novel mechanism that mediates the robustness of these metabolic networks.

Global histone acetylation varies with changes in the nutrient and cell cycle phases; however, the mechanisms connecting these variations are not fully understood. Herein, we report that nutrient-related and cell-cycle-regulated nuclear acetate regulates global histone acetylation. Histone deacetylation-generated acetate accumulates in the nucleus and induces histone hyperacetylation. The nuclear acetate levels were controlled by glycolytic enzyme triosephosphate isomerase 1 (TPI1). Cyclin-dependent kinase 2 (CDK2), which is phosphorylated and activated by nutrient-activated mTORC1, phosphorylates TPI1 Ser 117 and promotes nuclear translocation of TPI1, decreases nuclear dihydroxyacetone phosphate (DHAP) and induces nuclear acetate accumulation because DHAP scavenges acetate via the formation of 1-acetyl-DHAP. CDK2 accumulates in the cytosol during the late G1/S phases. Inactivation or blockade of nuclear translocation of TPI1 abrogates nutrient-dependent and cell-cycle-dependent global histone acetylation, chromatin condensation, gene transcription and DNA replication. These results identify the mechanism of maintaining global histone acetylation by nutrient and cell cycle signals. Zhang et al. identify a nuclear role for the glycolytic enzyme TPI1 in connecting nutrient status and cell cycle status to global histone acetylation.

During postgerminative seedling establishment, reserves stored during seed filling are mobilized to provide energy and carbon for the growing seedling until autotrophic growth is possible. A plastid isoform of triose phosphate isomerase (pdTPI) plays a crucial role in this transition from heterotrophic to autotrophic growth. A T-DNA insertion in Arabidopsis thaliana pdTPI resulted in a fivefold reduction in transcript, reduced TPI activity, and a severely stunted and chlorotic seedling that accumulated dihydroxyacetone phosphate (DHAP), glycerol, and glycerol-3-phosphate. Methylglyoxal (MG), a by-product of DHAP, also accumulated in the pdtpi mutant. Wild-type seed sown in the presence of any of these four metabolites resulted in a phenocopy of this pdtpi mutant, although MG and DHAP were the most effective based upon dosage. These metabolites (except MG) are by-products of triacylglycerol mobilization and precursors for glycerolipid synthesis, suggesting that lipid metabolism may also be affected. Lipid profiling revealed lower monogalactosyl but higher digalactosyl lipids. It is unclear whether the change in lipid composition is a direct or indirect consequence of the pdtpi mutation, as ribulose-1,5-bis-phosphate carboxylase/oxygenase expression, chloroplast morphology, and starch synthesis are also defective in this mutant. We propose that DHAP and MG accumulation in developing plastids delays the transition from heterotrophic to autotrophic growth, possibly due to MG toxicity.

Stereoselective carbon-carbon bond formation with aldolases has become an indispensable tool in preparative synthetic chemistry. In particular, the dihydroxyacetone phosphate (DHAP)-dependent aldolases are attractive because four different types are available that allow access to a complete set of diastereomers of vicinal diols from achiral aldehyde acceptors and the DHAP donor substrate. While the substrate specificity for the acceptor is rather relaxed, these enzymes show only very limited tolerance for substituting the donor. Therefore, access to DHAP is instrumental for the preparative exploitation of these enzymes, and several routes for its synthesis have become available. DHAP is unstable, so chemical synthetic routes have concentrated on producing a storable precursor that can easily be converted to DHAP immediately before its use. Enzymatic routes have concentrated on integrating the DHAP formation with upstream or downstream catalytic steps, leading to multi-enzyme arrangements with up to seven enzymes operating simultaneously. While the various chemical routes suffer from either low yields, complicated work-up, or toxic reagents or catalysts, the enzymatic routes suffer from complex product mixtures and the need to assemble multiple enzymes into one reaction scheme. Both types of routes will require further improvement to serve as a basis for a scalable route to DHAP.

5′-Methyl-thioadenosine (MTA) is a dead-end, sulfur-containing metabolite and cellular inhibitor that arises from S- adenosyl- l -methionine-dependent reactions. Recent studies have indicated that there are diverse bacterial m ethionine s alvage p athways (MSPs) for MTA detoxification and sulfur salvage. Here, via a combination of gene deletions and directed metabolite detection studies, we report that under aerobic conditions the facultatively anaerobic bacterium Rhodopseudomonas palustris employs both an MTA-isoprenoid shunt identical to that previously described in Rhodospirillum rubrum and a second novel MSP, both of which generate a methanethiol intermediate. The additional R. palustris aerobic MSP, a dihydroxyacetone phosphate (DHAP)-methanethiol shunt, initially converts MTA to 2-(methylthio)ethanol and DHAP. This is identical to the initial steps of the recently reported anaerobic ethylene-forming MSP, the DHAP-ethylene shunt. The aerobic DHAP-methanethiol shunt then further metabolizes 2-(methylthio)ethanol to methanethiol, which can be directly utilized by O-acetyl- l -homoserine sulfhydrylase to regenerate methionine. This is in contrast to the anaerobic DHAP-ethylene shunt, which metabolizes 2-(methylthio)ethanol to ethylene and an unknown organo-sulfur intermediate, revealing functional diversity in MSPs utilizing a 2-(methylthio)ethanol intermediate. When MTA was fed to aerobically growing cells, the rate of volatile methanethiol release was constant irrespective of the presence of sulfate, suggesting a general housekeeping function for these MSPs up through the methanethiol production step. Methanethiol and dimethyl sulfide (DMS), two of the most important compounds of the global sulfur cycle, appear to arise not only from marine ecosystems but from terrestrial ones as well. These results reveal a possible route by which methanethiol might be biologically produced in soil and freshwater environments. IMPORTANCE Biologically available sulfur is often limiting in the environment. Therefore, many organisms have developed methionine salvage pathways (MSPs) to recycle sulfur-containing by-products back into the amino acid methionine. The metabolically versatile bacterium Rhodopseudomonas palustris is unusual in that it possesses two RuBisCOs and two RuBisCO-like proteins. While RuBisCO primarily serves as the carbon fixation enzyme of the Calvin cycle, RuBisCOs and certain RuBisCO-like proteins have also been shown to function in methionine salvage. This work establishes that only one of the R. palustris RuBisCO-like proteins functions as part of an MSP. Moreover, in the presence of oxygen, to salvage sulfur, R. palustris employs two pathways, both of which result in production of volatile methanethiol, a key compound of the global sulfur cycle. When total available sulfur was plentiful, methanethiol was readily released into the environment. However, when sulfur became limiting, methanethiol release decreased, presumably due to methanethiol utilization to regenerate needed methionine. Biologically available sulfur is often limiting in the environment. Therefore, many organisms have developed methionine salvage pathways (MSPs) to recycle sulfur-containing by-products back into the amino acid methionine. The metabolically versatile bacterium Rhodopseudomonas palustris is unusual in that it possesses two RuBisCOs and two RuBisCO-like proteins. While RuBisCO primarily serves as the carbon fixation enzyme of the Calvin cycle, RuBisCOs and certain RuBisCO-like proteins have also been shown to function in methionine salvage. This work establishes that only one of the R. palustris RuBisCO-like proteins functions as part of an MSP. Moreover, in the presence of oxygen, to salvage sulfur, R. palustris employs two pathways, both of which result in production of volatile methanethiol, a key compound of the global sulfur cycle. When total available sulfur was plentiful, methanethiol was readily released into the environment. However, when sulfur became limiting, methanethiol release decreased, presumably due to methanethiol utilization to regenerate needed methionine.