Interactions among sites responsible for trehalose 6-phosphate and trehalose-activated glucose 6-phosphate hydrolysis on trehalose phosphatase isolated from Phormia regina.
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Correlation of trehalose content and heat resistance in yeast mutants altered in the RAS/adenylate cyclase pathway: is trehalose a thermoprotectant?
Trehalose content and thermotolerance were closely correlated in wild type yeast (Saccharomyces cerevisiae) and in cyr1-2 and bcy1-1 mutants both during exponential growth at 27 degrees C and during heat shock at 40 degrees C. Trehalose levels were high when heat shock proteins (hsps) were expected to be induced and low when hsps were presumably absent. It was tried to uncouple trehalose biosynthesis and hsp-induction. Various non-heat stresses affected trehalose levels of wild type cells in a similar way as they would have affected hsps. However, no trehalose was accumulated when cells were treated with canavanine, a well-known inducer of hsps but not of the thermotolerant state.
Adenylyl Cyclases↗
Screening of synthetic trehalose 6,6'-diesters and trehalose 6-monoesters as potential immunoreactants for the serodiagnosis of tuberculosis.
The absence of serological cross-reactivity between trehalose 2,3-diester (DAT, formerly SL-IV) and synthetic trehalose 6,6'-diesters and trehalose 6-monoesters was established by ELISA testing using polyclonal immune sera raised in rabbits sensitized with "DAT". From the screening of fifteen synthetic trehalose 6,6'- and 6-esters, "mirror" pseudo cord factor no. 1, "mirror" amides no. 5 and 6, cord factor analogues 7 and 8 and trehalose 6-monoesters 10 and 11 were selected for future, more extensive serological analysis. Paired comparisons of analogues among these fifteen substances showed that serodiagnostic discrimination power was more a function of the carbon chain length of their substituent groups--as well as of their position--than of the "mirror" constitution of the molecules. More exhaustive testing of these seven compounds is needed to select the synthetic product most efficient in the ELISA serodiagnosis of tuberculosis.
Animals↗
Truncation of Arabidopsis thaliana and Selaginella lepidophylla trehalose-6-phosphate synthase unlocks high catalytic activity and supports high trehalose levels on expression in yeast.
Plants, such as Arabidopsis thaliana and Selaginella lepidophylla, contain genes homologous with the trehalose-6-phosphate synthase (TPS) genes of bacteria and fungi. Most plants do not accumulate trehalose with the desert resurrection plant S. lepidophylla, being a notable exception. Overexpression of the plant genes in a Saccharomyces cerevisiae tps1 mutant results in very low TPS-catalytic activity and trehalose accumulation. We show that truncation of the plant-specific N-terminal extension in the A. thaliana AtTPS1 and S. lepidophylla SlTPS1 homologues results in 10-40-fold higher TPS activity and 20-40-fold higher trehalose accumulation on expression in yeast. These results show that the plant TPS enzymes possess a high-potential catalytic activity. The growth defect of the tps1 strain on glucose was restored, however, the proper homoeostasis of glycolytic flux was not restored, indicating that the plant enzymes were unable to substitute for the yeast enzyme in the regulation of hexokinase activity. Further analysis of the N-terminus led to the identification of two conserved residues, which after mutagenesis result in strongly enhanced trehalose accumulation upon expression in yeast. The plant-specific N-terminal region may act as an inhibitory domain allowing modulation of TPS activity.
Alleles↗
Trehalose-6-phosphate synthase 1, which catalyses the first step in trehalose synthesis, is essential for Arabidopsis embryo maturation.
Despite the recent discovery that trehalose synthesis is widespread in higher plants very little is known about its physiological significance. Here we report on an Arabidopsis mutant (tps1), disrupted in a gene encoding the first enzyme of trehalose biosynthesis (trehalose-6-phosphate synthase). The tps1 mutant is a recessive embryo lethal. Embryo morphogenesis is normal but development is retarded and stalls early in the phase of cell expansion and storage reserve accumulation. TPS1 is transiently up-regulated at this same developmental stage and is required for the full expression of seed maturation marker genes (2S2 and OLEOSN2). Sucrose levels also increase rapidly in seeds during the onset of cell expansion. In Saccharomyces cerevisiae trehalose-6-phosphate (T-6-P) is required to regulate sugar influx into glycolysis via the inhibition of hexokinase and a deficiency in TPS1 prevents growth on sugars (Thevelein and Hohmann, 1995). The growth of Arabidopsis tps1-1 embryos can be partially rescued in vitro by reducing the sucrose level. However, T-6-P is not an inhibitor of AtHXK1 or AtHXK2. Nor does reducing hexokinase activity rescue tps1-1 embryo growth. Our data establish for the first time that an enzyme of trehalose metabolism is essential in plants and is implicated in the regulation of sugar metabolism/embryo development via a different mechanism to that reported in S. cerevisiae.
Arabidopsis↗
Trehalose-based oligosaccharides isolated from the cytoplasm of Mycobacterium smegmatis. Relation to trehalose-based oligosaccharides attached to lipid.
A series of trehalose-based oligosaccharides were isolated from the cytoplasmic fraction of Mycobacterium smegmatis and purified by gel-filtration and paper chromatography and TLC. Their structures were determined by HPLC and GLC to determine sugar composition and ratios, MALDI-TOF MS to measure molecular mass, methylation analysis to determine linkages, (1)H-NMR to obtain anomeric configurations of glycosidic linkages, and exoglycosidase digestions followed by TLC to determine sequences and anomeric configurations of the monosaccharides. Six different oligosaccharides were identified all with trehalose as the basic structure and additional glucose or galactose residues attached in various linkages. One of these oligosaccharides is the disaccharide trehalose (Glcalpha1-1alphaGlc), which is present in substantial amounts in these cells and also in other mycobacteria. Two other oligosaccharides, the tetrasaccharides Glcalpha1-4Glcalpha1-1alphaGlc6-1alphaGal and Galalpha1-6Galalpha1-6Glcalpha1-1alphaGlc, have not previously been isolated from natural sources or synthesized chemically. The fourth oligosaccharide, Glcbeta1-6Glcbeta1-6Glcalpha1-1alphaGlc, has been isolated from corynebacteria, but not reported in other organisms. Two other oligosaccharides, Glcalpha1-4Glcalpha1-1alphaGlc, which has been synthesized chemically and isolated from insects but not previously reported in mycobacteria, and Glcbeta1-6Glcalpha1-1alphaGlc, which was previously isolated from Mycobacterium fortuitum and yeast, were also characterized. Another trisaccharide found in the cytosol has been partially characterized as arabinosyl-1-4trehalose, but neither the anomeric configuration nor the D or L configuration of the arabinose is known. In analogy with sucrose and its higher homologs, raffinose and stachyose, which may act as protective agents during maturation drying in plants, these trehalose homologs may also have a protective role in mycobacteria, perhaps during latency.
Carbohydrate Conformation↗
Trehalose accumulation in a high-trehalose-accumulating mutant of Saccharomycopsis fibuligera sdu does not respond to stress treatments.
The isolation of high-trehalose-accumulating mutant A11 from Saccharomycopsis fibuligera sdu has been previously described. In this paper, accumulation of trehalose under various stress conditions in S. fibuligera A11 was investigated. Neither activation of trehalose-6-phosphate synthase (SfTps1) nor change in trehalose content was observed under stress exposure of S. fibuligera A11 cells. A fragment of the Sftps1 gene in this strain was also cloned by degenerate PCR using the CoDeHOP strategy and multiply-aligned Tps1 sequences. This sequence allowed us to investigate the expression of the Sftps1 gene, which was also kept constant under the various stress conditions. Altogether, these results indicate that trehalose metabolism in S. fibuligera A11 in response to stress conditions clearly differs from that of Saccharomyces cerevisiae and most other fungi. The expression of the Sftps1 gene was not responsive to different stress treatments.
Amino Acid Sequence↗
Crystal transformation from anhydrous alpha-maltose to hydrous beta-maltose and from anhydrous trehalose to hydrous trehalose.
Anhydrous sugars such as maltose and trehalose are useful for making dry powder of foods and liquids. The crystal-transformation rate of maltose and trehalose were investigated under humid conditions and by kneading. The enthalpy for solubilization was 7.0 kJ/mol for the anhydrous maltose. The crystal-transformation rate of anhydrous alpha-maltose to hydrous beta-maltose depended on the temperature at 75% humidity. However, that of anhydrous trehalose did not depend on the temperature, and transformation was very rapid. An anomeric change to maltose and no such change to trehalose might have caused this. The activation energy of crystal transformation was 79 kJ/mol for maltose and zero for trehalose. The rate of crystal transformation of anhydrous maltose while kneading depended on the purity of the anhydrous alpha-maltose and the amount of water present. This crystal transformation rate fitted the Avrami equation.
Crystallization↗
Acceptor specificity of trehalose phosphorylase from Thermoanaerobacter brockii: production of novel nonreducing trisaccharide, 6-O-alpha-D-galactopyranosyl trehalose.
We investigated the acceptor specificity of a thermostable trehalose phosphorylase from Thermoanaerobacter brockii ATCC 35047 (TbTP) was examined using beta-D-glucose-1-phosphate (beta-G1P) as a glucosyl donor and oligosaccharides as the acceptor. Oligosaccharides with a reducing-end glucose residue as the C-6 substituent (e.g., isomaltose, gentiobiose, melibiose, isomaltotriose, and isopanose) were found to be successful acceptors. The transfer products of isomaltose, gentiobiose, and melibiose were isolated and characterized as 6-O-alpha-D-glucopyranosyl trehalose (alpha-GlcTre), 6-O-beta-D-glucopyranosyl trehalose (beta-GlcTre), and 6-O-alpha-D-galactopyranosyl trehalose (alpha-GalTre), respectively. To produce alpha-GalTre, a novel nonreducing trisaccharide, the reaction conditions of alpha-GalTre were examined using trehalose as a glucosyl donor. As a result, the yield of alpha-GalTre reached 40.5%.
Enzyme Activation↗
Cloning and expression of a trehalose synthase from Pseudomonas stutzeri CJ38 in Escherichia coli for the production of trehalose.
A novel strain was isolated, Pseudomonas stutzeri CJ38, that enabled direct transformation of maltose to trehalose. In comparison with others reported to date, CJ38 provided a novel trehalose synthase (TSase) without any byproduct, including glucose. Activity analysis, using either maltose or trehalose as a substrate, showed a reversible reaction. There was also no detectable activity of related enzymes with liquid starch and maltooligosaccharides as substrates. Using a malPQ-negative host and MacConkey medium, the TSase gene was cloned in Escherichia coli from CJ38. The resulting sequence contained an open reading frame consisted of 689 amino acids with a calculated molecular mass of 76 kDa. A search for related sequences in various gene and protein data banks revealed a novel family of enzymes that was predicted putatively as a glycosidase or TSase family, with no biochemical evidence. The recombinant enzyme exhibited a high activity toward the substrate maltose, about 50-fold higher than the parent strain and resulted in a high conversion yield (72%) at a relatively high substrate concentration (20%). These results provided the possibility that the strain was effectively used as a potential biocatalyst for the production of trehalose from maltose in a one-step reaction.
Escherichia coli↗
Mechanistic study of the intramolecular conversion of maltose to trehalose by Thermus caldophilus GK24 trehalose synthase.
This paper questions what types of molecular transformation are involved in the conversion of maltose to trehalose by trehalose synthase from Thermus caldophilus GK24. The reverse reaction pathway has been examined with the aid of alpha,alpha-(2,4,6,6',2',4',6",6"'-(2)H(8))trehalose (1). The mass data of the isolated reaction products clearly indicate that deuterated glucose is confined only to substrate molecules, and thus the reversible enzymatic conversion of trehalose into maltose proceeds through an intramolecular pathway.
Disaccharides↗
Dicorynomycoloyl trehalose activity: comparison of the activity of alpha,alpha'- and beta,beta'-trehalose derivatives on mitochondrial oxidative phosphorylation.
6,6'-Dicorynomycoloyl esters of alpha,alpha'- or beta;beta'-trehalose were synthesized and tested on isolated rat liver mitochondria. In contrast to the well known site-II-specific uncoupling effect of the alpha,alpha'-trehalose derivative, the beta,beta'-trehalose derivative exhibited only non-specific inhibition of active respiration in the presence of glutamate. It is proposed that this unexpected difference between the two isomers could arise from conformational differences in the carbohydrate moiety of the glycolipids, which would render the beta,beta' isomer unable to recognize targets specific to the alpha,alpha'-trehalose glycolipid.
Animals↗
Cloning and characterization of genes encoding trehalose-6-phosphate synthase (TPS1) and trehalose-6-phosphate phosphatase (TPS2) from Zygosaccharomyces rouxii.
In many organisms, trehalose protects against several environmental stresses, such as heat, desiccation, and salt, probably by stabilizing protein structures and lipid membranes. Trehalose synthesis in yeast is mediated by a complex of trehalose-6-phosphate synthase (TPS1) and trehalose-6-phosphate phosphatase (TPS2). In this study, genes encoding TPS1 and TPS2 were isolated from Zygosaccharomyces rouxii (designated ZrTPS1 and ZrTPS2, respectively). They were functionally identified by their complementation of the tps1 and tps2 yeast deletion mutants, which are unable to grow on glucose medium and with heat, respectively. Full-length ZrTPS1 cDNA is composed of 1476 nucleotides encoding a protein of 492 amino acids with a molecular mass of 56 kDa. ZrTPS2 cDNA consists of 2843 nucleotides with an open reading frame of 2700 bp, which encodes a polypeptide of 900 amino acids with a molecular mass of 104 kDa. The amino acid sequence encoded by ZrTPS1 has relatively high homology with TPS1 of Saccharomyces cerevisiae and Schizosaccharomyces pombe, compared with TPS2. Western blot analysis showed that the antibody against S. cerevisiae TPS1 recognizes ZrTPS1. Under normal growth conditions, ZrTPS1 and ZrTPS2 were highly and constitutively expressed, unlike S. cerevisiae TPS1 and TPS2. Salt stress and heat stress reduced the expression of the ZrTPS1 and ZrTPS2 genes, respectively.
Amino Acid Sequence↗
Trehalose Toxicity in Cuscuta reflexa: Cell Wall Synthesis Is Inhibited upon Trehalose Feeding.
alpha,alpha-Trehalose induced a rapid blackening of the terminal 2.5-centimeter region of excised Cuscuta reflexa Roxb. vine. The incorporation of radioactivity from [(14)C]glucose into alkali-insoluble fraction of shoot tip was markedly inhibited by 12 hours of trehalose feeding to an excised vine. This inhibition was confined to the apical segment of the vine in which cell elongation occurred. The rate of blackening of shoot tip explants was hastened by the addition of gibberellic acid A(3), which promoted elongation growth of isolated Cuscuta shoot tips. The symptom of trehalose toxicity was duplicated by 2-deoxyglucose, which has been shown to be a potent inhibitor of cell wall synthesis in yeast. The observations suggest that trehalose interferes with the synthesis of cell wall polysaccharides, the chief component of which was presumed to be cellulose.
Journal Article↗
D-trehalose/D-maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis: the binding of trehalose and maltose results in different protein conformational states.
In this work, we used fluorescence spectroscopy, molecular dynamics simulation, and Fourier transform infrared spectroscopy for investigating the effect of trehalose binding and maltose binding on the structural properties and the physical parameters of the recombinant D-trehalose/D-maltose binding protein (TMBP) from the hyperthermophilic archaeon Thermococcus litoralis. The binding of the two sugars to TMBP was studied in the temperature range 20 degrees-100 degrees C. The results show that TMBP possesses remarkable temperature stability and its secondary structure does not melt up to 90 degrees C. Although both the secondary structure itself and the sequence of melting events were not significantly affected by the sugar binding, the protein assumes different conformations with different physical properties depending whether maltose or trehalose is bound to the protein. At low and moderate temperatures, TMBP possesses a structure that is highly compact both in the absence and in the presence of two sugars. At about 90 degrees C, the structure of the unliganded TMBP partially relaxes whereas both the TMBP/maltose and the TMBP/trehalose complexes remain in the compact state. In addition, Fourier transform infrared results show that the population of alpha-helices exposed to the solvent was smaller in the absence than in the presence of the two sugars. The spectroscopic results are supported by molecular dynamics simulations. Our data on dynamics and stability of TMBP can contribute to a better understanding of transport-related functions of TMBP and constitute ground for targeted modifications of this protein for potential biotechnological applications.
Carrier Proteins↗
Analysis of the otsBA operon for osmoregulatory trehalose synthesis in Escherichia coli and homology of the OtsA and OtsB proteins to the yeast trehalose-6-phosphate synthase/phosphatase complex.
The Escherichia coli otsBA operon, located at min 42, was sequenced and shown to encode a 29.1-kDa trehalose-6-phosphate phosphatase (OtsB) and a 53.6-kDa trehalose-6-phosphate synthase (OtsA). Both proteins display sequence homology with subunits of the Saccharomyces cerevisiae trehalose-6-phosphate synthase/phosphatase complex, which is made up of the subunits TPS1, TPS2 and TPS3 (TSL1). OtsA has homology to the full-length TPS1, the N-terminal part of TPS2 and an internal region of TPS3 (TSL1). OtsB has homology to the C-terminal part of TPS2, but no homology to the other subunits. Primer extension analysis showed only one transcription start point upstream from otsB and one upstream from otsA, regardless of the growth conditions tested. The start codons of the otsB and otsA genes were established by N-terminal sequence determination of the proteins. The 3' end of the otsB coding region overlaps the 5' end of the otsA coding region by 23 nucleotides. The araH gene is located directly upstream from otsBA, and otsB may be identical to pexA.
Amino Acid Sequence↗
Infrared spectroscopic study on the properties of the anhydrous form II of trehalose. Implications for the functional mechanism of trehalose as a biostabilizer.
FTIR spectra were obtained for several different states of trehalose including dihydrate crystal, anhydrous form II (designated by Gil, A. M.; Belton, P. S.; Felix V. Spectrochim. Acta 1996, A52, 1649-1659), anhydrate crystal, dried melt, amorphous solid and aqueous solution. From the observation of the symmetric and antisymmetric stretch vibrations of the glycosidic linkage, it is found that this sugar assumes at least three types of backbone conformations. Among them, the conformation with C(2) symmetry is characterized as 'open state', which means that the sugar easily absorbs water molecules. The conformation of the sugars in anhydrous form II and in freeze-dried trehalose is shown to be in the open state. Next, the hygroscopic properties of the anhydrate, form II and the amorphous solid are compared based on their IR spectra. Interestingly, form II alone is converted to the original dihydrate in a week under mild environmental-like conditions: relative humidity of 40% and room temperature. These results suggest the possibility that form II plays a role in avoiding the devitrification of the sugar glass. Finally, we discuss the role of form II in preserving freeze-dried biomaterials.
Animals↗
Inhibitory effect of trehalose dimycolate (TDM) and its stereoisometric derivatives, trehalose dicorynomycolates (TDCMs), with low toxicity on lung metastasis of tumour cells in mice.
We examined the effect of semisynthetic trehalose-6,6 '-dimycolate (TDM) and its synthetic stereoisomeric derivatives (trehalose 6,6'-dicorynomycolates; TDCMs) prepared in oil-in-water (o/w) emulsion on inhibition of lung metastasis produced by highly metastatic murine tumour cells, colon 26-M3.1 carcinoma and B16-BL6 melanoma cells, using experimental and spontaneous metastasis models. Intravenous (i.v.) administration of TDM (100 microg/mouse) 1, 3 or 8 days before tumour inoculation significantly inhibited lung metastasis of colon 26-M3.1 cells, in a dose-dependent manner. Single administration of TDM 1 day after tumour inoculation also showed the therapeutic effect on experimental lung metastasis of colon 26-M3.1 cells. Similarly, multiple administrations of TDM after tumour inoculation resulted in a significant inhibition of spontaneous lung metastasis of B16-BL6 cells (on day 35), although it showed no effect on suppression of tumour growth (on day 21). In comparison of toxicity in vivo among TDM and four TDCMs such as TDCM(2R,3R), TDCM(2S,3R), TDCM(2R,3S) and TDCM(2S,3S), all of the TDCMs appeared to be less toxic than TDM itself. Furthermore, all of the TDCMs were prophylactically as well as therapeutically active for inhibition of lung metastasis of both colon 26-M3.1 and B16-BL6 tumour cells, showing higher inhibitory activity than that of TDM. In particular, TDCMs induced a marked suppression of the growth of B16-BL6 tumour cells in vivo. These results suggest that systemic administration of TDM as well as TDCMs led to inhibition of tumour metastasis and TDCMs are more potential to suppress tumour growth and inhibit tumour metastasis than TDM.
Adjuvants, Immunologic↗