Differential heat-response characteristics of two plastid isoforms of triose phosphate isomerase in tomato.

Heat stress causes dysfunction of the carbon-assimilation metabolism. As a member of Calvin-Benson-Bassham (CBB) cycle, the chloroplast triose phosphate isomerases (TPI) catalyse the interconversion of glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP). The tomato (Solanum lycopersicum) genome contains two individual SlTPI genes, Solyc10g054870 and Solyc01g111120, which encode the chloroplast-located proteins SlTPI1 and SlTPI2, respectively. The tpi1 and tpi2 single mutants had no visible phenotypes, but the leaves of their double mutant lines tpi1tpi2 had obviously reduced TPI activity and displayed chlorotic variegation, dysplasic chloroplasts and lower carbon-assimilation efficiency. In addition to altering carbon metabolism, proteomic data showed that the loss of both SlTPI1 and SlTPI2 severely affected photosystem proteins, reducing photosynthetic capacity. None of these phenotypes was evident in the tpi1 or tpi2 single mutants, suggesting that SlTPI1 and SlTPI2 are functionally redundant. However, the two proteins differed in their responses to heat stress; the protein encoded by the heat-induced SlTPI2 showed a higher level of thermotolerance than that encoded by the heat-suppressed SlTPI1. Notably, heat-induced transcription factors, SlWRKY21 and SlHSFA2/7, which negatively regulated SlTPI1 expression and positively regulated SlTPI2 expression, respectively. Our findings thus reveal that SlTPI1 and SlTPI2 have different thermostabilities and expression patterns in response to heat stress, which have the potential to be applied in thermotolerance strategies in crops.

Suppression of chloroplast triose phosphate isomerase evokes inorganic phosphate-limited photosynthesis in rice.

The availability of inorganic phosphate (Pi) for ATP synthesis is thought to limit photosynthesis at elevated [CO2] when Pi regeneration via sucrose or starch synthesis is limited. We report here another mechanism for the occurrence of Pi-limited photosynthesis caused by insufficient capacity of chloroplast triose phosphate isomerase (cpTPI). In cpTPI-antisense transgenic rice (Oryza sativa) plants with 55%-86% reductions in cpTPI content, CO2 sensitivity of the rate of CO2 assimilation (A) decreased and even reversed at elevated [CO2]. The pool sizes of the Calvin-Benson cycle metabolites from pentose phosphates to 3-phosphoglycerate increased at elevated [CO2], whereas those of ATP decreased. These phenomena are similar to the typical symptoms of Pi-limited photosynthesis, suggesting sufficient capacity of cpTPI is necessary to prevent the occurrence of Pi-limited photosynthesis and that cpTPI content moderately affects photosynthetic capacity at elevated [CO2]. As there tended to be slight variations in the amounts of total leaf-N depending on the genotypes, relationships between A and the amounts of cpTPI were examined after these parameters were expressed per unit amount of total leaf-N (A/N and cpTPI/N, respectively). A/N at elevated [CO2] decreased linearly as cpTPI/N decreased before A/N sharply decreased, owing to further decreases in cpTPI/N. Within this linear range, decreases in cpTPI/N by 80% led to decreases up to 27% in A/N at elevated [CO2]. Thus, cpTPI function is crucial for photosynthesis at elevated [CO2].

Overexpression of Chloroplast Triosephosphate Isomerase Marginally Improves Photosynthesis at Elevated CO2 Levels in Rice.

We recently suggested that chloroplast triosephosphate isomerase (cpTPI) has moderate control over the rate of CO2 assimilation (A) at elevated CO2 levels via the capacity for triose phosphate utilization (TPU) in rice (Oryza sativa L.) from its antisense-suppression study. In the present study, the effects of cpTPI overexpression on photosynthesis were examined in transgenic rice plants overexpressing the gene encoding cpTPI. The amounts of cpTPI protein in the two lines of transgenic plants were 4.8- and 12.1-folds higher than in wild-type plants, respectively. The magnitude of the increase approximately corresponded to the increase in transcript levels of cpTPI. A at CO2 levels of 100 and 120 Pa increased by 6-9% in the transgenic plants, whereas those at ambient and low CO2 levels were scarcely affected. Similar increases were observed for TPU capacity estimated from the CO2 response curves of A. These results indicate that the overexpression of cpTPI marginally improved photosynthesis at elevated CO2 levels via improvement in TPU capacity in rice. However, biomass production at a CO2 level of 120 Pa did not increase in transgenic plants, suggesting that the improvement in photosynthesis by cpTPI overexpression was not sufficient to improve biomass production in rice.

Linear Free Energy Relationships for Enzymatic Reactions: Fresh Insight from a Venerable Probe.

Linear free energy relationships (LFERs) for substituent effects on reactions that proceed through similar transition states provide insight into transition state structures. A classical approach to the analysis of LFERs showed that differences in the slopes of Brønsted correlations for addition of substituted alkyl alcohols to ring-substituted 1-phenylethyl carbocations and to the ß-galactopyranosyl carbocation intermediate of reactions catalyzed by ß-galactosidase provide evidence that the enzyme catalyst modifies the curvature of the energy surface at the saddle point for the transition state for nucleophile addition. We have worked to generalize the use of LFERs in the determination of enzyme mechanisms. The defining property of enzyme catalysts is their specificity for binding the transition state with a much higher affinity than the substrate. Triosephosphate isomerase (TIM), orotidine 5'-monophosphate decarboxylase (OMPDC), and glycerol 3-phosphate dehydrogenase (GPDH) show effective catalysis of reactions of phosphorylated substrates and strong phosphite dianion activation of reactions of phosphodianion truncated substrates, with rate constants kcat/Km (M-1 s-1) and kcat/KdKHPi (M-2 s-1), respectively. Good linear logarithmic correlations, with a slope of 1.1, between these kinetic parameters determined for reactions catalyzed by five or more variant forms of each catalyst are observed, where the protein substitutions are mainly at side chains which function to stabilize the cage complex between the enzyme and substrate. This shows that the enzyme-catalyzed reactions of a whole substrate and substrate pieces proceed through transition states of similar structures. It provides support for the proposal that the dianion binding energy of whole phosphodianion substrates and of phosphite dianion is used to drive the conversion of these protein catalysts from flexible and entropically rich ground states to stiff and catalytically active Michaelis complexes that show the same activity toward catalysis of the reactions of whole and phosphodianion truncated substrates. There is a good linear correlation, with a slope of 0.73, between values of the dissociation constants log Ki for release of the transition state analog phosphoglycolate (PGA) trianion and log kcat/Km for isomerization of GAP for wild-type and variants of TIM. This correlation shows that the substituted amino acid side chains act to stabilize the complex between TIM and the PGA trianion and that ca. 70% of this stabilization is observed at the transition state for substrate deprotonation. The correlation provides evidence that these side chains function to enhance the basicity of the E165 side chain of TIM, which deprotonates the bound carbon acid substrate. There is a good linear correlation, with a slope of 0.74, between the values of ΔG‡ and ΔG° determined by electron valence bond (EVB) calculations to model deprotonation of dihydroxyacetone phosphate (DHAP) in water and when bound to wild-type and variant forms of TIM to form the enediolate reaction intermediate. This correlation provides evidence that the stabilizing interactions of the transition state for TIM-catalyzed deprotonation of DHAP are optimized by placement of amino acid side chains in positions that provide for the maximum stabilization of the charged reaction intermediate, relative to the neutral substrate.

A mutagenesis screen for essential plastid biogenesis genes in human malaria parasites.

Endosymbiosis has driven major molecular and cellular innovations. Plasmodium spp. parasites that cause malaria contain an essential, non-photosynthetic plastid-the apicoplast-which originated from a secondary (eukaryote-eukaryote) endosymbiosis. To discover organellar pathways with evolutionary and biomedical significance, we performed a mutagenesis screen for essential genes required for apicoplast biogenesis in Plasmodium falciparum. Apicoplast(-) mutants were isolated using a chemical rescue that permits conditional disruption of the apicoplast and a new fluorescent reporter for organelle loss. Five candidate genes were validated (out of 12 identified), including a triosephosphate isomerase (TIM)-barrel protein that likely derived from a core metabolic enzyme but evolved a new activity. Our results demonstrate, to our knowledge, the first forward genetic screen to assign essential cellular functions to unannotated P. falciparum genes. A putative TIM-barrel enzyme and other newly identified apicoplast biogenesis proteins open opportunities to discover new mechanisms of organelle biogenesis, molecular evolution underlying eukaryotic diversity, and drug targets against multiple parasitic diseases.

Boronic acid derivative activates pyruvate kinase M2 indispensable for redox metabolism in oral cancer cells.

PKM2is considered a desirable target as its enzymatic activation is expected to cause a diminution in tumorigenesis and prevent limitless replication in cancerous cells. However, considering the functional consequences of kinase inhibitors, the design of PKM2 activators has been an attractive strategy that has yielded potent anticancer molecules like DASA-58. Therefore, a new class of boronic acid derivate was developed to elucidate the possible mechanistic link between PKM2 activation and TPI1 activity, which has a significant role in the redox balance in cancer. The present in vitro study revealed that treatment with boronic acid-based compound 1 and DASA-58 was found to activate PKM2 with an AC50 of 25 nM and 52 nM, respectively. Furthermore, at the AC50 concentration of compound 1, we found a significant increase in TPI1 activity and a decrease in GSH and NADP+/NADPH ratio. We also found increased ROS levels and decreased lactate secretion with treatment. Together with these findings, we can presume that compound 1 affects the redox balance by activating PKM2 and TPI1 activity. Implementation of this treatment strategy may improve the effect of chemotherapy in the conditions of ROS induced cancer drug resistance. This study for the first time supports the link between PKM2 and the TPI1 redox balance pathway in oral cancer. Collectively, the study findings provide a novel molecule for PKM2 activation for the therapeutic intervention in oral cancer.

Highly Similar Sequence and Structure Yet Different Biophysical Behavior: A Computational Study of Two Triosephosphate Isomerases.

Homodimeric triosephosphate isomerases (TIMs) from Trypanosoma cruzi (TcTIM) and Trypanosoma brucei (TbTIM) have markedly similar amino-acid sequences and three-dimensional structures. However, several of their biophysical parameters, such as their susceptibility to sulfhydryl agents and their reactivation speed after being denatured, have significant differences. The causes of these differences were explored with microsecond-scale molecular dynamics (MD) simulations of three different TIM proteins: TcTIM, TbTIM, and a chimeric protein, Mut1. We examined their electrostatic interactions and explored the impact of simulation length on them. The same salt bridge between catalytic residues Lys 14 and Glu 98 was observed in all three proteins, but key differences were found in other interactions that the catalytic amino acids form. In particular, a cation-π interaction between catalytic amino acids Lys 14 and His 96 and both a salt bridge and a hydrogen bond between catalytic Glu 168 and residue Arg 100 were only observed in TcTIM. Furthermore, although TcTIM forms less hydrogen bonds than TbTIM and Mut1, its hydrogen bond network spans almost the entire protein, connecting the residues in both monomers. This work provides new insight into the mechanisms that give rise to the different behavior of these proteins. The results also show the importance of long simulations.

Osteoblasts induce glucose-derived ATP perturbations in chondrocytes through noncontact communication.

Cartilage and subchondral bone communicate with each other through material and signal exchanges. However, direct evidence provided by experimental studies on their interactions is insufficient. In the present study, we establish a noncontact co-culture model with a transwell chamber to explore the energetic perturbations in chondrocytes influenced by osteoblasts. Our results indicate that osteoblasts induce more ATP generation in chondrocytes through an energetic shift characterized by enhanced glycolysis and impaired mitochondrial tricarboxylic acid cycle. Enhanced glycolysis is shown by an increase of secreted lactate and the upregulation of glycolytic enzymes, including glucose-6-phosphate isomerase (Gpi), liver type ATP-dependent 6-phosphofructokinase (Pfkl), fructose-bisphosphate aldolase C (Aldoc), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), triosephosphate isomerase (Tpi1), and phosphoglycerate kinase 1 (Pgk1). Impaired mitochondrial tricarboxylic acid cycle is characterized by the downregulation of cytoplasmic aspartate aminotransferase (Got1) and mitochondrial citrate synthase (Cs). Osteoblasts induce the activation of Akt and P38 signaling to mediate ATP perturbations in chondrocytes. This study may deepen our understanding of the maintenance of metabolic homeostasis in the bone-cartilage unit.

Ligand-Based Virtual Screening and Molecular Docking of Benzimidazoles as Potential Inhibitors of Triosephosphate Isomerase Identified New Trypanocidal Agents.

Trypanosoma cruzi (T. cruzi) is a parasite that affects humans and other mammals. T. cruzi depends on glycolysis as a source of adenosine triphosphate (ATP) supply, and triosephosphate isomerase (TIM) plays a key role in this metabolic pathway. This enzyme is an attractive target for the design of new trypanocidal drugs. In this study, a ligand-based virtual screening (LBVS) from the ZINC15 database using benzimidazole as a scaffold was accomplished. Later, a molecular docking on the interface of T. cruzi TIM (TcTIM) was performed and the compounds were grouped by interaction profiles. Subsequently, a selection of compounds was made based on cost and availability for in vitro evaluation against blood trypomastigotes. Finally, the compounds were analyzed by molecular dynamics simulation, and physicochemical and pharmacokinetic properties were determined using SwissADME software. A total of 1604 molecules were obtained as potential TcTIM inhibitors. BP2 and BP5 showed trypanocidal activity with half-maximal lytic concentration (LC50) values of 155.86 and 226.30 µM, respectively. Molecular docking and molecular dynamics simulation analyzes showed a favorable docking score of BP5 compound on TcTIM. Additionally, BP5 showed a low docking score (-5.9 Kcal/mol) on human TIM compared to the control ligand (-7.2 Kcal/mol). Both compounds BP2 and BP5 showed good physicochemical and pharmacokinetic properties as new anti-T. cruzi agents.

Evolution of Enzyme Function and the Development of Catalytic Efficiency: Triosephosphate Isomerase, Jeremy R. Knowles, and W. John Albery.

Every reader knows that an enzyme accelerates a reaction by reducing the activation-energy barrier. However, understanding how this is achieved by the structure of the enzyme and its interactions with stable complexes and transition states and, then, using this to (re)design enzymes to catalyze novel reactions remain the "holy grail" of mechanistic enzymology. The necessary foundation is the free-energy profile that specifies the energies of the bound substate, product, and intervening intermediates as well as the transition states by which they are interconverted. When this free-energy profile is compared to that for the uncatalyzed reaction, strategies for establishing and enhancing catalysis can be identified. This Perspective reminds readers that the first free-energy profile determined for an enzyme-catalyzed reaction, that for triosephosphate isomerase, was published in Biochemistry in 1976 by Jeremy R. Knowles, W. John Albery, and co-workers. They used the profile to propose three steps of increasing "subtlety" that can be influenced by evolutionary pressure to increase the flux through the reaction coordinate: (1) "uniform binding" of the substrate, product, and intermediates; (2) "differential binding" of complexes so that these are isoenergetic (to minimize the energy of the intervening transition states); and (3) "catalysis of an elementary step" in which the transition state for the kinetically significant chemical step is stabilized so that flux can be determined by the rate of substrate binding or product dissociation. These papers continue to guide mechanistic studies of enzyme-catalyzed reactions and provide principles for the (re)design of novel enzymes.

Identification of protein quality control regulators using a Drosophila model of TPI deficiency.

Triosephosphate isomerase (TPI) deficiency (Df) is a rare recessive metabolic disorder that manifests as hemolytic anemia, locomotor impairment, and progressive neurodegeneration. Research suggests that TPI Df mutations, including the "common" TPIE105Dmutation, result in reduced TPI protein stability that appears to underlie disease pathogenesis. Drosophila with the recessive TPIsugarkill allele (a.k.a. sgk or M81T) exhibit progressive locomotor impairment, neuromuscular impairment and reduced longevity, modeling the human disorder. TPIsugarkill produces a functional protein that is degraded by the proteasome. Molecular chaperones, such as Hsp70 and Hsp90, have been shown to contribute to the regulation of TPIsugarkill degradation. In addition, stabilizing the mutant protein through chaperone modulation results in improved TPI deficiency phenotypes. To identify additional regulators of TPIsugarkill degradation, we performed a genome-wide RNAi screen that targeted known and predicted quality control proteins in the cell to identify novel factors that modulate TPIsugarkill turnover. Of the 430 proteins screened, 25 regulators of TPIsugarkill were identified. Interestingly, 10 proteins identified were novel, previously undescribed Drosophila proteins. Proteins involved in co-translational protein quality control and ribosome function were also isolated in the screen, suggesting that TPIsugarkill may undergo co-translational selection for polyubiquitination and proteasomal degradation as a nascent polypeptide. The proteins identified in this study may reveal novel pathways for the degradation of a functional, cytosolic protein by the ubiquitin proteasome system and define therapeutic pathways for TPI Df and other biomedically important diseases.

Crystal structure of a novel putative sugar isomerase from the psychrophilic bacterium Paenibacillus sp. R4.

Sugar isomerases (SIs) catalyze the reversible conversion of aldoses to ketoses. A novel putative SI gene has been identified from the genome sequence information on the psychrophilic bacterium Paenibacillus sp. R4. Here, we report the crystal structure of the putative SI from Paenibacillus sp. R4 (PbSI) at 2.98 Å resolution. It was found that the overall structure of PbSI adopts the triose-phosphate isomerase (TIM) barrel fold. PbSI was also identified to have two heterogeneous metal ions as its cofactors at the active site in the TIM barrel, one of which was confirmed as a Zn ion through X-ray anomalous scattering and inductively coupled plasma mass spectrometry analysis. Structural comparison with homologous SI proteins from mesophiles, hyperthermophiles, and a psychrophile revealed that key residues in the active site are well conserved and that dimeric PbSI is devoid of the extended C-terminal region, which tetrameric SIs commonly have. Our results provide novel structural information on the cold-adaptable SI, including information on the metal composition in the active site.

Candida albicans and Candida glabrata triosephosphate isomerase - a moonlighting protein that can be exposed on the candidal cell surface and bind to human extracellular matrix proteins.

BACKGROUND: Triosephosphate isomerase (Tpi1) is a glycolytic enzyme that has recently been reported also to be an atypical proteinaceous component of the Candida yeast cell wall. Similar to other known candidal "moonlighting proteins", surface-exposed Tpi1 is likely to contribute to fungal adhesion during the colonization and infection of a human host. The aim of our present study was to directly prove the presence of Tpi1 on C. albicans and C. glabrata cells under various growth conditions and characterize the interactions of native Tpi1, isolated and purified from the candidal cell wall, with human extracellular matrix proteins. RESULTS: Surface plasmon resonance measurements were used to determine the dissociation constants for the complexes of Tpi1 with host proteins and these values were found to fall within a relatively narrow range of 10- 8-10- 7 M. Using a chemical cross-linking method, two motifs of the Tpi1 molecule (aa 4-17 and aa 224-247) were identified to be directly involved in the interaction with vitronectin. A proposed structural model for Tpi1 confirmed that these interaction sites were at a considerable distance from the catalytic active site. Synthetic peptides with these sequences significantly inhibited Tpi1 binding to several extracellular matrix proteins suggesting that a common region on the surface of Tpi1 molecule is involved in the interactions with the host proteins. CONCLUSIONS: The current study provided structural insights into the interactions of human extracellular matrix proteins with Tpi1 that can occur at the cell surface of Candida yeasts and contribute to the host infection by these fungal pathogens.

The heat released during catalytic turnover enhances the diffusion of an enzyme.

Recent studies have shown that the diffusivity of enzymes increases in a substrate-dependent manner during catalysis. Although this observation has been reported and characterized for several different systems, the precise origin of this phenomenon is unknown. Calorimetric methods are often used to determine enthalpies from enzyme-catalysed reactions and can therefore provide important insight into their reaction mechanisms. The ensemble averages involved in traditional bulk calorimetry cannot probe the transient effects that the energy exchanged in a reaction may have on the catalyst. Here we obtain single-molecule fluorescence correlation spectroscopy data and analyse them within the framework of a stochastic theory to demonstrate a mechanistic link between the enhanced diffusion of a single enzyme molecule and the heat released in the reaction. We propose that the heat released during catalysis generates an asymmetric pressure wave that results in a differential stress at the protein-solvent interface that transiently displaces the centre-of-mass of the enzyme (chemoacoustic effect). This novel perspective on how enzymes respond to the energy released during catalysis suggests a possible effect of the heat of reaction on the structural integrity and internal degrees of freedom of the enzyme.

Structural basis for the modulation of plant cytosolic triosephosphate isomerase activity by mimicry of redox-based modifications.

Reactive oxidative species (ROS) and S-glutathionylation modulate the activity of plant cytosolic triosephosphate isomerases (cTPI). Arabidopsis thaliana cTPI (AtcTPI) is subject of redox regulation at two reactive cysteines that function as thiol switches. Here we investigate the role of these residues, AtcTPI-Cys13 and At-Cys218, by substituting them with aspartic acid that mimics the irreversible oxidation of cysteine to sulfinic acid and with amino acids that mimic thiol conjugation. Crystallographic studies show that mimicking AtcTPI-Cys13 oxidation promotes the formation of inactive monomers by reposition residue Phe75 of the neighboring subunit, into a conformation that destabilizes the dimer interface. Mutations in residue AtcTPI-Cys218 to Asp, Lys, or Tyr generate TPI variants with a decreased enzymatic activity by creating structural modifications in two loops (loop 7 and loop 6) whose integrity is necessary to assemble the active site. In contrast with mutations in residue AtcTPI-Cys13, mutations in AtcTPI-Cys218 do not alter the dimeric nature of AtcTPI. Therefore, modifications of residues AtcTPI-Cys13 and AtcTPI-Cys218 modulate AtcTPI activity by inducing the formation of inactive monomers and by altering the active site of the dimeric enzyme, respectively. The identity of residue AtcTPI-Cys218 is conserved in the majority of plant cytosolic TPIs, this conservation and its solvent-exposed localization make it the most probable target for TPI regulation upon oxidative damage by reactive oxygen species. Our data reveal the structural mechanisms by which S-glutathionylation protects AtcTPI from irreversible chemical modifications and re-routes carbon metabolism to the pentose phosphate pathway to decrease oxidative stress.

COX5A over-expression protects cortical neurons from hypoxic ischemic injury in neonatal rats associated with TPI up-regulation.

BACKGROUND: Neonatal hypoxic-ischemic encephalopathy (HIE) represents as a major cause of neonatal morbidity and mortality. However, the underlying molecular mechanisms in brain damage are still not fully elucidated. This study was conducted to determine the specific potential molecular mechanism in the hypoxic-ischemic induced cerebral injury. METHODS: Here, hypoxic-ischemic (HI) animal models were established and primary cortical neurons were subjected to oxygen-glucose deprivation (OGD) to mimic HIE model in vivo and in vitro. The HI-induced neurological injury was evaluated by Zea-longa scores, Triphenyte-trazoliumchloride (TTC) staining the Terminal Deoxynucleotidyl Transferased Utp Nick End Labeling (TUNEL) and immunofluorescent staining. Then the expression of Cytochrome c oxidase subunit 5a (COX5A) was determined by immunohistochemistry, western blotting (WB) and quantitative real time Polymerase Chain Reaction (qRT-PCR) techniques. Moreover, HSV-mediated COX5A over-expression virus was transducted into OGD neurons to explore the role of COX5A in vitro, and the underlying mechanism was predicted by GeneMANIA, then verified by WB and qRT-PCR. RESULTS: HI induced a severe neurological dysfunction, brain infarction, and cell apoptosis as well as obvious neuron loss in neonatal rats, in corresponding to the decrease on the expression of COX5A in both sides of the brain. What's more, COX5A over-expression significantly promoted the neuronal survival, reduced the apoptosis rate, and markedly increased the neurites length after OGD. Moreover, Triosephosephate isomerase (TPI) was predicted as physical interactions with COX5A, and COX5A over-expression largely increased the expressional level of TPI. CONCLUSIONS: The present findings suggest that COX5A plays an important role in promoting neurological recovery after HI, and this process is related to TPI up-regulation.

A Cytosolic Bypass and G6P Shunt in Plants Lacking Peroxisomal Hydroxypyruvate Reductase.

The oxygenation of ribulose 1,5-bisphosphate by Rubisco is the first step in photorespiration and reduces the efficiency of photosynthesis in C3 plants. Our recent data indicate that mutants in photorespiration have increased rates of photosynthetic cyclic electron flow around photosystem I. We investigated mutant lines lacking peroxisomal hydroxypyruvate reductase to determine if there are connections between 2-phosphoglycolate accumulation and cyclic electron flow in Arabidopsis (Arabidopsis thaliana). We found that 2-phosphoglycolate is a competitive inhibitor of triose phosphate isomerase, an enzyme in the Calvin-Benson cycle that converts glyceraldehyde 3-phosphate to dihydroxyacetone phosphate. This block in metabolism could be overcome if glyceraldehyde 3-phosphate is exported to the cytosol, where cytosolic triose phosphate isomerase could convert it to dihydroxyacetone phosphate. We found evidence that carbon is reimported as glucose-6-phosphate, forming a cytosolic bypass around the block of stromal triose phosphate isomerase. However, this also stimulates a glucose-6-phosphate shunt, which consumes ATP, which can be compensated by higher rates of cyclic electron flow.

The Photorespiratory Metabolite 2-Phosphoglycolate Regulates Photosynthesis and Starch Accumulation in Arabidopsis.

The Calvin-Benson cycle and its photorespiratory repair shunt are in charge of nearly all biological CO2 fixation on Earth. They interact functionally and via shared carbon flow on several levels including common metabolites, transcriptional regulation, and response to environmental changes. 2-Phosphoglycolate (2PG) is one of the shared metabolites and produced in large amounts by oxidative damage of the CO2 acceptor molecule ribulose 1,5-bisphosphate. It was anticipated early on, although never proven, that 2PG could also be a regulatory metabolite that modulates central carbon metabolism by inhibition of triose-phosphate isomerase. Here, we examined this hypothesis using transgenic Arabidopsis thaliana lines with varying activities of the 2PG-degrading enzyme, 2PG phosphatase, and analyzing the impact of this intervention on operation of the Calvin-Benson cycle and other central pathways, leaf carbohydrate metabolism, photosynthetic gas exchange, and growth. Our results demonstrate that 2PG feeds back on the Calvin-Benson cycle. It also alters the allocation of photosynthates between ribulose 1,5-bisphosphate regeneration and starch synthesis. 2PG mechanistically achieves this by inhibiting the Calvin-Benson cycle enzymes triose-phosphate isomerase and sedoheptulose 1,7-bisphosphate phosphatase. We suggest this may represent one of the control loops that sense the ratio of photorespiratory to photosynthetic carbon flux and in turn adjusts stomatal conductance, photosynthetic CO2 and photorespiratory O2 fixation, and starch synthesis in response to changes in the environment.

Increasing the pyruvate pool by overexpressing phosphoenolpyruvate carboxykinase or triosephosphate isomerase enhances phloroglucinol production in Escherichia coli.

OBJECTIVES: Acetyl-CoA is a precursor for phloroglucinol (PG), and pyruvate is one of the sources of intracellular acetyl-CoA. Therefore, enhancing intracellular pyruvate levels may help to improve the anabolic pathway of PG. RESULTS: In this study, the effects of phosphoenolpyruvate carboxykinase (PckA, encoded by pckA) or triosephosphate isomerase (TpiA, encoded by tpiA) overexpression on the production of PG were studied. Overexpression of pckA or tpiA could enhance the pyruvate anabolic pathway in shake-flask culture compared to the control strain, and the concentration of PG also increased by 44% and 92%, respectively. In addition, the acetate levels were all down regulated by the overexpression of the two genes to some extent and lower acetate level resulted in lower ATP pool and higher survival rate. CONCLUSIONS: These results indicate that overexpression of pckA or tpiA can enhance the pyruvate "pool" and PG production in Escherichia coli, which provides a new reference for further increasing the production of PG.

Therapeutic Targeting of Cancer Metabolism with Triosephosphate Isomerase.

The increase in glycolytic flux in cancer, known as aerobic glycolysis, is one of the most important hallmarks of cancer. Therefore, glycolytic enzymes have importance in understanding the molecular mechanism of cancer progression. Triosephosphate isomerase (TPI) is one of the key glycolytic enzymes. Furthermore, it takes a part in gluconeogenesis, pentose phosphate pathway and fatty acid biosynthesis. To date, it has been shown altered levels of TPI in various cancer types, especially in metastatic phenotype. According to other studies, TPI might be considered as a potential therapeutic target and a cancer-related biomarker in different types of cancer. However, its function in tumor formation and development has not been fully understood. Here, we reviewed the relationship between TPI and cancer for the first time.