Structural analysis of the subunits of the trehalose-6-phosphate synthase/phosphatase complex in Saccharomyces cerevisiae and their function during heat shock

Synthesis of trehalose in the yeast Saccharomyces cerevisiae is catalysed by the trehalose-6-phosphate (Tre6 P ) synthase/phosphatase complex, which is composed of at least three different subunits encoded by the genes TPS1 , TPS2 , and TSL1 . Previous studies indicated that Tps1 and Tps2 carry the catalytic activities of trehalose synthesis, namely Tre6 P synthase (Tps1) and Tre6 P phosphatase (Tps2), while Tsl1 was suggested to have regulatory functions. In this study two different approaches have been used to clarify the molecular composition of the trehalose synthase complex as well as the functional role of its potential subunits. Two-hybrid analyses of the in vivo interactions of Tps1, Tps2, Tsl1, and Tps3, a protein with high homology to Tsl1, revealed that both Tsl1 and Tps3 can interact with Tps1 and Tps2; the latter two proteins also interact with each other. In addition, trehalose metabolism upon heat shock was analysed in a set of 16 isogenic yeast strains carrying deletions of TPS1 , TPS2 , TSL1 , and TPS3 in all possible combinations. These results not only confirm the previously suggested roles for Tps1 and Tps2, but also provide, for the first time, evidence that Tsl1 and Tps3 may share a common function with respect to regulation and/or structural stabilization of the Tre6 P synthase/phosphatase complex in exponentially growing, heat-shocked cells.     

An unexpected plethora of trehalose biosynthesis genes in Arabidopsis thaliana.

Trehalose accumulation has been documented in many organisms, such as bacteria and fungi, where it serves a storage and stress-protection role. Although conspicuously absent in most plants, trehalose biosynthesis genes were discovered recently in higher plants. We have uncovered a family of 11 TPS genes in Arabidopsis thaliana, one of which encodes a trehalose-6-phosphate (Tre6P) synthase, and a subfamily of which might encode the still elusive Tre6P phosphatases. A regulatory role in carbon metabolism is likely but might not be restricted to the TPS control of hexokinase activity as documented for yeast. Incompatibility between high trehalose levels and chaperone-assisted protein folding might be a reason why plants have evolved to accumulate some alternative stress-protection compounds to trehalose.     

Reconstitution of ethanolic fermentation in permeabilized spheroplasts of wild-type and trehalose-6-phosphate synthase mutants of the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, TPS1-encoded trehalose-6-phosphate synthase (TPS) exerts an essential control on the influx of glucose into glycolysis, presumably by restricting hexokinase activity. Deletion of TPS1 results in severe hyperaccumulation of sugar phosphates and near absence of ethanol formation. To investigate whether trehalose 6-phosphate (Tre6P) is the sole mediator of hexokinase inhibition, we have reconstituted ethanolic fermentation from glucose in permeabilized spheroplasts of the wild-type, tps1Delta and tps2Delta (Tre6P phosphatase) strains. For the tps1Delta strain, ethanol production was significantly lower and was associated with hyperaccumulation of Glu6P and Fru6P. A tps2Delta strain shows reduced accumulation of Glu6P and Fru6P both in intact cells and in permeabilized spheroplasts. These results are not consistent with Tre6P being the sole mediator of hexokinase inhibition. Reconstitution of ethanolic fermentation in permeabilized spheroplasts with glycolytic intermediates indicates additional target site(s) for the Tps1 control. Addition of Tre6P partially shifts the ethanol production rate and the metabolite pattern in permeabilized tps1Delta spheroplasts to those of the wild-type strain, but only with glucose as substrate. This is observed at a very high ratio of glucose to Tre6P. Inhibition of hexokinase activity by Tre6P is less efficiently counteracted by glucose in permeabilized spheroplasts compared to cell extracts, and this effect is largely abolished by deletion of TPS2 but not TPS1. In permeabilized spheroplasts, hexokinase activity is significantly lower in a tps2Delta strain compared to a wild-type strain and this difference is strongly reduced by additional deletion of TPS1. These results indicate that Tps1-mediated protein-protein interactions are important for control of glucose influx into yeast glycolysis, that Tre6P inhibition of hexokinase might not be competitive with respect to glucose in vivo and that also Tps2 appears to play a role in the control of hexokinase activity.     

A secondary function of trehalose-6-phosphate synthase is required for resistance to oxidative and desiccation stress in Fusarium verticillioides

The disaccharide trehalose has long been recognized for its role as a stress solute, but in recent years some of the protective effects previously ascribed to trehalose have been suggested to arise from a function of the trehalose biosynthesis enzyme trehalose-6-phosphate (T6P) synthase that is distinct from its catalytic activity. In this study, we use the maize pathogenic fungus Fusarium verticillioides as a model to explore the relative contributions of trehalose itself and a putative secondary function of T6P synthase in protection against stress as well as to understand why, as shown in a previous study, deletion of the TPS1 gene coding for T6P synthase reduces pathogenicity against maize. We report that a TPS1-deletion mutant of F. verticillioides is compromised in its ability to withstand exposure to oxidative stress meant to simulate the oxidative burst phase of maize defense and experiences more ROS-induced lipid damage than the wild-type strain. Eliminating T6P synthase expression also reduces resistance to desiccation, but not resistance to phenolic acids. Expression of catalytically-inactive T6P synthase in the TPS1-deletion mutant leads to a partial rescue of the oxidative and desiccation stress-sensitive phenotypes, suggesting the importance of a T6P synthase function that is independent of its role in trehalose synthesis.     

Uncoupling of the glucose growth defect and the deregulation of glycolysis in Saccharomyces cerevisiae Tps1 mutants expressing trehalose-6-phosphate-insensitive hexokinase from Schizosaccharomyces pombe

In the yeast Saccharomyces cerevisiae inactivation of trehalose-6-phosphate (Tre6P) synthase (Tps1) encoded by the TPS1 gene causes a specific growth defect in the presence of glucose in the medium. The growth inhibition is associated with deregulation of the initial part of glycolysis. Sugar phosphates, especially fructose-1,6-bisphosphate (Fru1,6bisP), hyperaccumulate while the levels of ATP, Pi and downstream metabolites are rapidly depleted. This was suggested to be due to the absence of Tre6P inhibition on hexokinase. Here we show that overexpression of Tre6P (as well as glucose-6-phosphate (Glu6P))-insensitive hexokinase from Schizosaccharomyces pombe in a wild-type strain does not affect growth on glucose but still transiently enhances initial sugar phosphate accumulation. We have in addition replaced the three endogenous glucose kinases of S. cerevisiae by the Tre6P-insensitive hexokinase from S. pombe. High hexokinase activity was measured in cell extracts and growth on glucose was somewhat reduced compared to an S. cerevisiae wild-type strain but expression of the Tre6P-insensitive S. pombe hexokinase never caused the typical tps1Delta phenotype. Moreover, deletion of TPS1 in this strain expressing only the Tre6P-insensitive S. pombe hexokinase still resulted in a severe drop in growth capacity on glucose as well as sensitivity to millimolar glucose levels in the presence of excess galactose. In this case, poor growth on glucose was associated with reduced rather than enhanced glucose influx into glycolysis. Initial glucose transport was not affected. Apparently, deletion of TPS1 causes reduced activity of the S. pombe hexokinase in vivo. Our results show that Tre6P inhibition of hexokinase is not the major mechanism by which Tps1 controls the influx of glucose into glycolysis or the capacity to grow on glucose. In addition, they show that a Tre6P-insensitive hexokinase can still be controlled by Tps1 in vivo.     

Enhanced thermotolerance for ethanol fermentation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain by overexpression of the gene coding for trehalose-6-phosphate synthase

The effect of overexpression of the trehalose-6-phosphate (T6P) synthase gene (TPS1) on ethanol fermentation of Saccharomyces cerevisiae has been studied at 30 and 38°C. The activity of T6P synthase and the accumulation of trehalose during ethanol fermentation were significantly improved by overexpression of TPS1, and especially at 38°C. Ethanol produced by transformants with and without TPS1 gene overexpression at 38°C was approx. 60 and 37 g/l, respectively. The fermentation efficiency of transformants with TPS1 gene overexpression at 38°C was similar to that at 30°C. The critical growth temperature was increased from 36 to 42°C by TPS1 gene overexpression. These results indicated that overexpression of the TPS1 gene had a beneficial effect on the fermentation capacity of the title yeast strain at high temperatures.     

Enhanced freeze tolerance of baker’s yeast by overexpressed trehalose-6-phosphate synthase gene (TPS1) and deleted trehalase genes in frozen dough

Several recombinant strains with overexpressed trehalose-6-phosphate synthase gene (TPS1) and/or deleted trehalase genes were obtained to elucidate the relationships between TPS1, trehalase genes, content of intracellular trehalose and freeze tolerance of baker’s yeast, as well as improve the fermentation properties of lean dough after freezing. In this study, strain TL301_TPS1 overexpressing TPS1 showed 62.92 % higher trehalose-6-phosphate synthase (Tps1) activity and enhanced the content of intracellular trehalose than the parental strain. Deleting ATH1 exerted a significant effect on trehalase activities and the degradation amount of intracellular trehalose during the first 30 min of prefermentation. This finding indicates that acid trehalase (Ath1) plays a role in intracellular trehalose degradation. NTH2 encodes a functional neutral trehalase (Nth2) that was significantly involved in intracellular trehalose degradation in the absence of the NTH1 and/or ATH1 gene. The survival ratio, freeze-tolerance ratio and relative fermentation ability of strain TL301_TPS1 were approximately twice as high as those of the parental strain (BY6-9α). The increase in freeze tolerance of strain TL301_TPS1 was accompanied by relatively low trehalase activity, high Tps1 activity and high residual content of intracellular trehalose. Our results suggest that overexpressing TPS1 and deleting trehalase genes are sufficient to improve the freeze tolerance of baker’s yeast in frozen dough. The present study provides guidance for the commercial baking industry as well as the research on the intracellular trehalose mobilization and freeze tolerance of baker’s yeast.     

A yeast gene for trehalose-6-phosphate synthase and its complementation of an Escherichia coli otsA mutant

Abstract A Saccharomyces cerevisiae gene for trehalose-6-phosphate synthase (TPS1) was sequenced. The gene appeared to code for a protein of 495 amino acid residues, giving the protein a molecular mass of 56 kDa. The TPS1 gene was able to restore both osmotolerance and trehalose accumulation during salt stress in an Escherichia coli strain mutated in the otsA gene encoding trehalose-6-phosphate synthase. Complementation studies with E. coli galU mutants showed that the TPS1-encoded trehalose-6-phosphate synthase is UDP-glucose-dependent. Sequence analysis and data base searches showed that TPS1 is allelic to GGS1, byp1, cif1 and fdp1 . A possible gene for trehalose-6-phosphate synthase in Methanobacterium thermoautotrophicum was identified.     

Roles of trehalose phosphate synthase in yeast glycogen metabolism and sporulation

Trehalose is a major storage carbohydrate in budding yeast, Saccharomyces cerevisiae. Alterations in trehalose synthesis affect carbon source-dependent growth, accumulation of glycogen and sporulation. Trehalose is synthesized by trehalose phosphate synthase (TPS), which is a complex of at least four proteins. In this work, we show that the Tps1p subunit protein catalyses trehalose phosphate synthesis in the absence of other TPS components. The tps1-H223Y allele (glc6-1) that causes a semidominant decrease in glycogen accumulation exhibits greater enzyme activity than wild-type TPS1 because, unlike the wild-type enzyme, TPS activity in tps1-H223Y cells is not inhibited by phosphate. Poor sporulation in tps1 null diploids is caused by reduced expression of meiotic inducers encoded by IME1, IME2 and MCK1. Furthermore, high-copy MCK1 or heterozygous hxk2 mutations can suppress the tps1 sporulation trait. These results suggest that the trehalose-6-phosphate inhibition of hexokinase activity is required for full induction of MCK1 in sporulating yeast cells.     

Trehalose biosynthesis in Thermus thermophilus RQ-1: biochemical properties of the trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase

The genes for trehalose synthesis in Thermus thermophilus RQ-1, namely otsA [trehalose-phosphate synthase (TPS)], otsB [trehalose-phosphate phosphatase (TPP)], and treS [trehalose synthase (maltose converting) (TreS)] genes are structurally linked. The TPS/TPP pathway plays a role in osmoadaptation, since mutants unable to synthesize trehalose via this pathway were less osmotolerant, in trehalose-deprived medium, than the wild-type strain. The otsA and otsB genes have now been individually cloned and overexpressed in Escherichia coli and the corresponding recombinant enzymes purified. The apparent molecular masses of TPS and TPP were 52 and 26 kDa, respectively. The recombinant TPS utilized UDP-glucose, TDP-glucose, ADP-glucose, or GDP-glucose, in this order as glucosyl donors, and glucose-6-phosphate as the glucosyl acceptor to produce trehalose-6-phosphate (T6P). The recombinant TPP catalyzed the dephosphorylation of T6P to trehalose. This enzyme also dephosphorylated G6P, and this activity was enhanced by NDP-glucose. TPS had an optimal activity at about 98°C and pH near 6.0; TPP had a maximal activity near 70°C and at pH 7.0. The enzymes were extremely thermostable: at 100°C, TPS had a half-life of 31 min, and TPP had a half-life of 40 min. The enzymes did not require the presence of divalent cations for activity; however, the presence of Co²⁺ and Mg²⁺ stimulates both TPS and TPP. This is the first report of the characterization of TPS and TPP from a thermophilic organism.     

Trehalose synthesis inhibitor: A molecular in silico drug design

Infectious diseases are serious public health problems, affecting a large portion of the world's population. A molecule that plays a key role in pathogenic organisms is trehalose and recently has been an interest in the metabolism of this molecule for drug development. The trehalose-6-phosphate synthase (TPS1) is an enzyme responsible for the biosynthesis of trehalose-6-phosphate (T6P) in the TPS1/TPS2 pathway, which results in the formation of trehalose. Studies carried out by our group demonstrated the inhibitory capacity of T6P in the TPS1 enzyme from Saccharomyces cerevisiae, preventing the synthesis of trehalose. By in silico techniques, we compiled sequences and experimentally determined structures of TPS1. Sequence alignments and molecular modeling were performed. The generated structures were submitted in validation of algorithms, aligned structurally and analyzed evolutionarily. Molecular docking methodology was applied to analyze the interaction between T6P and TPS1 and ADMET properties of T6P were analyzed. The results demonstrated the models created presented sequence and structural similarities with experimentally determined structures. With the molecular docking, a cavity in the protein surface was identified and the molecule T6P was interacting with the residues TYR-40, ALA-41, MET-42, and PHE-372, indicating the possible uncompetitive inhibition mechanism provided by this ligand, which can be useful in directing the molecular design of inhibitors. In ADMET analyses, T6P had acceptable risk values compared with other compounds from World Drug Index. Therefore, these results may present a promising strategy to explore to develop a broad-spectrum antibiotic of this specific target with selectivity, potency, and reduced side effects, leading to a new way to treat infectious diseases like tuberculosis and candidiasis.     

TREHALOSE SYNTHESIS IN EUGLENA GRACILIS (EUGLENOPHYCEAE) OCCURS THROUGH AN ENZYME COMPLEX1

The aim of this study was to isolate and characterize a trehalose‐synthesizing enzyme from Euglena gracilis Klebs. After purification by anion exchange chromatography, gel filtration, isoelectric focusing, and native electrophoresis, trehalose‐6‐phosphate synthase (TPS, EC 2.4.1.15) and trehalose‐6‐phosphate phosphatase (TPP, EC 3.1.3.12) activities could not be separated. Consequently, a TPS/TPP enzyme complex of about 250 kDa was suggested as responsible for trehalose synthesis in E. gracilis. The TPS activity was shown to be highly specific for glucose‐6‐P, and UDP‐Glc was the preferred glucose donor, but GDP‐Glc and CDP‐Glc could also act as TPS substrates. The TPP activity was highly specific for trehalose‐6‐P. In vitro phosphorylation assays revealed rapid decreases in TPS and TPP activities. These changes corresponded to variations in the elution profile of gel filtration chromatography after the phosphorylation treatment. Taken together, these results suggest that the proposed TPS/TPP complex might be regulated through a protein phosphorylation/dephosphorylation‐mediated mechanism that could affect the association state of the complex. Such a regulatory mechanism might lead to a rapid change in trehalose synthesis in response to variations in environmental conditions.     

Trehalose phosphate synthase overexpression in Parachlorella kessleri improves growth and photosynthetic performance under high light conditions

Parachlorella kessleri is a promising oil-bearing marine alga which shows decreased growth under high light stress. Osmolytes are known to relieve stress by protecting the cell membrane, proteins, and enzymes. Enhanced production of osmolyte (trehalose) was thus used to relieve stress in P. kessleri by overexpression of trehalose phosphate synthase (TPS) gene. Transformed P. kessleri was grown under different light regimes to study the effect of trehalose overproduction on growth. Study of one of the TPS transformants showed increased trehalose as well as increased biomass and decreased pigments, reactive oxygen species, and lipid peroxidation of cell membrane. The improved photosynthetic performance of the transformant was also signified by pulse-amplitude-modulated fluorometric analysis. All of these factors reveal improved stress tolerance under high light conditions by increased trehalose accumulation due to TPS overexpression in P. kessleri.     

Metabolism control over growth: a case for trehalose-6-phosphate in plants

How plants relate their requirements for energy with the reducing power necessary to fuel growth is not understood. The activated glucose forms and NADPH are key precursors in pathways yielding, respectively, energy and reducing power for anabolic metabolism. Moreover, they are substrates or allosteric regulators of trehalose-phosphate synthase (TPS1) in fungi and probably also in plants. TPS1 synthesizes the signalling metabolite trehalose-6-phosphate (T6P) and, therefore, has the potential to relate reducing power with energy metabolism to fuel growth. A working model is discussed where trehalose-6-phosphate (T6P) inhibition of SnRK1 is part of a growth-regulating loop in young and metabolically active heterotrophic plant tissues. SnRK1 is the Snf1 Related Kinase 1 and the plant homologue of the AMP-dependent protein kinase of animals, a central energy gauge. T6P accumulation in response to high sucrose levels in a cell inhibits SnRK1 activity, thus promoting anabolic processes and growth. When T6P levels drop due to low glucose-6-phosphate, uridine-diphosphoglucose, and altered NADPH or due to restricted TPS1 activity, active SnRK1 promotes catabolic processes required to respond to energy and carbon deprivation. The model explains why too little or too much T6P has been found to be growth inhibitory: Arabidopsis thaliana embryos and seedlings without TPS1 are growth arrested and Arabidopsis seedlings accumulating T6P on a trehalose medium are growth arrested. Finally, the insight gained with respect to the possible role of T6P metabolism, where it is known to alter developmental and environmental responses of plants, is discussed.     

Trehalose-6-phosphate synthase/phosphatase complex from bakers' yeast: purification of a proteolytically activated form.

A protein of about 800 kDa with trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) activity was purified from bakers' yeast. This TPS/P complex contained 57, 86 and 93 kDa polypeptides. The 86 and 93 kDa polypeptides both appeared to be derived from a polypeptide of at least 115 kDa in the native enzyme. A TPS-activator (a dimer of 58 kDa subunits) was also purified. It decreased the Michaelis constants for both UDP-glucose (three-fold) and glucose 6-phosphate (G6P) (4.5-fold), and increased TPS activity at 5 mM-UDP-glucose/10 mM-G6P about three-fold. It did not affect TPP activity. The purification of TPS/P included an endogenous proteolytic step that increased TPS activity about three-fold and abolished its requirement for TPS-activator, but did not change TPP activity. This activation was accompanied by a decrease of some 20 kDa in the molecular mass of a cluster of SDS-PAGE bands at about 115 kDa recognized by antiserum to pure TPS/P, but by no change in the 57 kDa band. Phosphate inhibited TPS activity (Ki about 5 mM), but increased TPP activity about six-fold (Ka about 4 mM). Phosphate (6 mM) stimulated the synthesis of trehalose from G6P and UDP-glucose and decreased the accumulation of trehalose 6-phosphate.     

Aggregation dependent enhancement of trehalose-6-phosphate synthase activity in Saccharomyces cerevisiae

Trehalose-6-phosphate synthase (TPS) is one of the key subunits of the trehalose synthase complex, responsible for synthesis of trehalose in Saccharomyces cerevisiae. Different laboratories have tried to purify TPS, but have been unable to separate it from the complex. During the present study, active TPS has been isolated from the trehalose synthase complex as a free 59 kDa protein. A 158 fold purification was achieved with over 84% recovery of active TPS. N-terminal sequence confirmed the 59 kDa protein to be TPS. It was revealed to be a highly hydrophobic protein by amino acid analysis data. Activity of TPS was identified to be governed by association-dissociation of protein components. TPS activity of the isolated enzyme was highly unstable due to dissociation of the protein from the complex. Aggregation of active molecules was also seen to enhance as well as stabilize enzyme activity. This aggregation was concentration dependent and activity was seen to be enhanced by increasing the number of active molecules and fell with dilution. The association of the active complex was also found to be governed by ionic interactions.     

Aggregation dependent enhancement of trehalose-6-phosphate synthase activity in Saccharomyces cerevisiae

Trehalose-6-phosphate synthase (TPS) is one of the key subunits of the trehalose synthase complex, responsible for synthesis of trehalose in Saccharomyces cerevisiae. Different laboratories have tried to purify TPS, but have been unable to separate it from the complex. During the present study, active TPS has been isolated from the trehalose synthase complex as a free 59kDa protein. A 158 fold purification was achieved with over 84% recovery of active TPS. N-terminal sequence confirmed the 59kDa protein to be TPS. It was revealed to be a highly hydrophobic protein by amino acid analysis data. Activity of TPS was identified to be governed by association–dissociation of protein components. TPS activity of the isolated enzyme was highly unstable due to dissociation of the protein from the complex. Aggregation of active molecules was also seen to enhance as well as stabilize enzyme activity. This aggregation was concentration dependent and activity was seen to be enhanced by increasing the number of active molecules and fell with dilution. The association of the active complex was also found to be governed by ionic interactions.