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PubMed1986

Catalytic versatility of trehalase: synthesis of alpha-D-glucopyranosyl alpha-D-xylopyranoside from beta-D-glucosyl fluoride and alpha-D-xylose.

Trehalase was previously shown (see ref. 5) to hydrolyze alpha-D-glucosyl fluoride, forming beta-D-glucose, and to synthesize alpha, alpha-trehalose from beta-D-glucosyl fluoride plus alpha-D-glucose. Present observations further define the enzyme's separate cosubstrate requirements in utilizing these nonglycosidic substrates. alpha-D-Glucopyranose and alpha-D-xylopyranose were found to be uniquely effective in enabling Trichoderma reesei trehalase to catalyze reactions with beta-D-glucosyl fluoride. As little as 0.2mM added alpha-D-glucose (0.4mM alpha-D-xylose) substantially increased the rate of enzymically catalyzed release of fluoride from 25mM beta-D-glucosyl fluoride at 0 degrees. Digests of beta-D-glucosyl fluoride plus alpha-D-xylose yielded the alpha, alpha-trehalose analog, alpha-D-glucopyranosyl alpha-D-xylopyranoside, as a transient (i.e., subsequently hydrolyzed) transfer-product. The need for an aldopyranose acceptor having an axial 1-OH group when beta-D-glucosyl fluoride is the donor, and for water when alpha-D-glucosyl fluoride is the substrate, indicates that the catalytic groups of trehalose have the flexibility to catalyze different stereochemical reactions.

Disaccharides↗
PubMed1990

Time-dependent inhibition of porcine kidney trehalase by aminosugars.

The inhibitory effects of various nitrogen-containing sugars on porcine kidney trehalase were studied. Validamycin A, validoxylamine A and MDL 25,637 were found to be potent, time-dependent inhibitors of the enzyme in vitro. The validoxylamine A-inhibited enzyme showed slow but reversible reactivation over time (t1/2 = 1.2 h). To our knowledge, this is the first report of time-dependent inhibition exhibited by either these particular aminosugars or a trehalase.

Amino Sugars↗
PubMed1990

Renal trehalase: function and development.

1. Following injection of trehalose into the bloodstream, no trehalose was found in urine of rabbits until the concentration of trehalose in blood exceeded 0.6 mg/ml. 2. Absence of trehalose in urine of the rabbit when the concentration of the sugar in blood is elevated supports the hypothesis that renal trehalase functions as a digestive enzyme in kidney. 3. The rat does not possess renal trehalase, and excretion of trehalose was in direct relation to the concentration of trehalose in blood. 4. There are differences in expression of the disaccharidases in kidney and intestine although they share many structural and enzymatic characteristics.

Animals↗
PubMed1978

Comparative specificities of trehalases from various species.

1. Using derivatives or non-symmetrical analogs of alpha,alpha-trehalose, we studied the catalytic specificities of trehalases from various species: Pseudomonas fluorescens, Melolontha vulgaris, porcine and human kidneys. 2. alpha,Beta-trehalose, beta,beta-trehalose, 6,6'dideoxy alpha,alpha-trehalose, alpha-D-xylopyranosyl alpha-D-xylopyranoside were shown to be neither substrates nor inhibitors. 3. 6'deoxy alpha,alpha-trehalose, alpha-D-glucopyranosyl alpha-D-xylopyranoside, alpha-D-allopyranosyl alpha-D-glucopyranoside and alpha-D-galactosyl alpha-D-glucopyranoside, which all possess an intact alpha-D-glucopyranosyl residue, were split by all these trehalases. 4. alpha-D-glucopyranosyl alpha-D-mannopyranoside, alpha,alpha-trehalosamine are competitive inhibitors. 5. These results show the importance of the primary alcohol group at C-6, of the equatorial configuration of the OH groups at C-2, C-3 and C-4 and of the modification of the structure at C-2 of the substrate for the catalytic activity.

Animals↗
PubMed1998

Correlation between stationary phase survival and acid trehalase activity in yeast.

The levels of two trehalose hydrolysing enzymes, acid trehalase (AT) and neutral trehalase (NT), have been investigated in Candida utilis at different stages of growth; in complete contrast to Saccharomyces cerevisiae, significant AT activity appears to be absent at all stages of growth studied in C. utilis. In addition, presence of only very low amounts of iso-aspartyl methyl transferase (IMT) activity at the onset of stationary phase and lower survival ability in early stationary phase in contrast to that of S. cerevisiae lend support to the ideas that (a) lower degree of survival of C. utilis in the stationary phase may be a direct consequence of inability to mobilise stored trehalose due to absence of intracellular AT and reduced levels of IMT activities and (b) trehalose may have a dual role vis-à-vis stress resistance in yeasts.

Candida↗
PubMed1997

Possible role of isoaspartyl methyltransferase towards regulation of acid trehalase activity in Saccharomyces cerevisiae.

Logarithmically growing cells of S. cerevisiae contained high neutral trehalase (NT) activity while stationary-phase cells had high acid trehalase (AT) activity. Change in activity profile of AT and NT were different during growth under different conditions, particularly during growth in acetate medium and up to 1 h of germination period, but that for AT and isoaspartyl methyltransferase (IMT) were found to be almost identical. Concomitant increase in NT activity as well as increase in cAMP level was noticed at the onset of spore germination. Increase in AT and IMT activities as well as decrease in S-adenosyl-L-methionine (AdoMet) level were noticed during stationary phase of growth. Acidic polyacrylamide gel electrophoresis and subsequent autoradiography revealed that substrate of IMT was a protein of molar mass around 82 kDa which could be an AT. Methylated AT was found to be more active while non-methylated AT was relatively less active in comparison to the untreated sample. Since AT existed as an equilibrium mixture of protomer and oligomer, it was suggested that IMT catalysed carboxyl methylation might have some contribution towards the regulation of AT activity.

Electrophoresis, Polyacrylamide Gel↗
PubMed1987

Rat intestinal trehalase. Studies of the active site.

Rat intestinal trehalase was solubilized, purified and reconstituted into proteoliposomes. With octyl glucoside as the solubilizing detergent, the purified protein appeared as a single band on SDS/polyacrylamide-gel electrophoresis with an apparent molecular mass of 67 kDa. Kinetic studies indicated that the active site of this enzyme can be functionally divided into two adjacent regions, namely a binding site (with pKa 4.8) and a catalytic site (with pKa 7.2). Other findings suggested that the catalytic site contains a functional thiol group, which is sensitive to inhibition by N-ethylmaleimide, Hg2+ and iodoacetate. Substrate protection and iodoacetate labelling of the thiol group demonstrated that only a protein of 67 kDa was labelled. Furthermore, sucrose and phlorizin protected the thiol group, but Tris-like inhibitors did not. Structure-inhibition analysis of Tris-like inhibitors, the pH effect of Tris inhibition and Tris protection of 1-(3-dimethylaminopropyl)-3-ethylcarbodi-imide inactivation permitted characterization and location of a separate site containing a carboxy group for Tris binding, which may also be the binding region. On the basis of these findings, a possible structure for the active site of trehalase is proposed.

Animals↗
PubMed2000

Induction of neutral trehalase Nth1 by heat and osmotic stress is controlled by STRE elements and Msn2/Msn4 transcription factors: variations of PKA effect during stress and growth.

Saccharomyces cerevisiae neutral trehalase, encoded by NTH1, controls trehalose hydrolysis in response to multiple stress conditions, including nutrient limitation. The presence of three stress responsive elements (STREs, CCCCT) in the NTH1 promoter suggested that the transcriptional activator proteins Msn2 and Msn4, as well as the cAMP-dependent protein kinase (PKA), control the stress-induced expression of Nth1. Here, we give direct evidence that Msn2/Msn4 and the STREs control the heat-, osmotic stress- and diauxic shift-dependent induction of Nth1. Disruption of MSN2 and MSN4 abolishes or significantly reduces the heat- and NaCl-induced increases in Nth1 activity and transcription. Stress-induced increases in activity of a lacZ reporter gene put under control of the NTH1 promoter is nearly absent in the double mutant. In all instances, basal expression is also reduced by about 50%. The trehalose concentration in the msn2 msn4 double mutant increases less during heat stress and drops more slowly during recovery than in wild-type cells. This shows that Msn2/Msn4-controlled expression of enzymes of trehalose synthesis and hydrolysis help to maintain trehalose concentration during stress. However, the Msn2/Msn4-independent mechanism exists for heat control of trehalose metabolism. Site-directed mutagenesis of the three STREs (CCCCT changed to CATCT) in NTH1 promoter fused to a reporter gene indicates that the relative proximity of STREs to each other is important for the function of NTH1. Elimination of the three STREs abolishes the stress-induced responses and reduces basal expression by 30%. Contrary to most STRE-regulated genes, the PKA effect on the induction of NTH1 by heat and sodium chloride is variable. During diauxic growth, NTH1 promoter-controlled reporter activity strongly increases, as opposed to the previously observed decrease in Nth1 activity, suggesting a tight but opposite control of the enzyme at the transcriptional and post-translational levels. Apparently, inactive trehalase is accumulated concomitant with the accumulation of trehalose. These results might help to elucidate the general connection between control by STREs, Msn2/Msn4 and PKA and, in particular, how these components play a role in control of trehalose metabolism.

Base Sequence↗
PubMed1997

Inhibition of trehalase activity enhances trehalose accumulation in transgenic plants.

As a first step toward the exploitation of the disaccharide trehalose as a stress-protective and preservative agent in plants, we engineered trehalose biosynthesis in tobacco (Nicotiana tabacum) and potato (Solanum tuberosum) by introducing the otsA and otsB genes from Escherichia coli, which encode trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase, respectively. In leaves of transgenic tobacco plants, very low levels of trehalose accumulation were obtained (0.11 mg g-1 fresh weight), whereas in transgenic potato tubers, no trehalose accumulated at all. Plant trehalase activity was shown to affect the accumulation of trehalose in these plants. An increase in trehalose accumulation, up to 0.41 and 4.04 mg g-1 fresh weight in tobacco leaves and potato micro-tubers, respectively, was noted when the potent trehalase inhibitor validamycin A was added to in vitro plants and to hydroponically grown greenhouse plants. Stunted growth and the formation of lancet-shaped leaves by trehalose-accumulating tobacco plants suggest a negative effect of trehalose biosynthesis on N. tabacum development. It is surprising that experiments with wild-type plants cultured in the presence of validamycin A indicate that, despite current belief, the capacity to synthesize trehalose may not be restricted to primitive phyla of vascular plants and certain "resurrection plants," but may exist throughout the angiosperms.

Cloning, Molecular↗
PubMed1991

Osmotic induction of the periplasmic trehalase in Escherichia coli K12: characterization of the treA gene promoter.

The Escherichia coli treA gene encodes an osmotically inducible periplasmic trehalase. A strain carrying a treA-lacZ transcriptional fusion was constructed. The beta-galactosidase activity produced in this strain growing exponentially in a medium of high osmotic pressure was 10-fold higher than that produced in a medium of low osmotic pressure, demonstrating that treA transcription is osmotically inducible. treA transcriptional induction depends neither on the presence of trehalase itself nor on the synthesis of cytoplasmic trehalose which occurs in response to osmotic stress in wild-type E. coli strains. The treA promoter was identified by S1 nuclease protection. Deletion analysis demonstrated that sequences sufficient for the osmotic induction lie downstream from nucleotide -40 with respect to the transcription start. Transcription initiation at treAp required the presence of a functional sigma 70 subunit of RNA polymerase. treA expression was increased in the presence of a mutation in osmZ, which was previously identified as leading to a partially constitutive expression of the osmotically inducible proU operon.

Amino Acid Sequence↗
PubMed1980

Stereochemistry of the hydrolysis of trehalose by the enzyme trehalase prepared from the flesh fly Sarcophaga barbata.

1. The enzyme trehalase was prepared from whole flesh flies, Sarcophaga barbata. 2. The rate of mutorotation of glucose released by the enzyme was compared with that of alpha-D-glucose, beta-D-glucose and an equimolar mixture of the two. 3. Derivatives of the glucose released by the enzyme were prepared and analysed by gas chromatography for anomeric composition. 4. The enzyme was incubated with trehalose in a medium enriched with H218O and the glucose was analysed by gas chromatography/mass spectrometry. 5. It was found that an equimolar mixture of alpha-D-glucose and beta-D-glucose is formed on hydrolysis of trehalose by trehalase and 18O is incorporated into beta-D-glucose. 6. These results indicate that there is an inversion of configuration at C-1 of trehalose on hydrolysis and that the hydrolysis proceeds by a bimolecular nucleophilic substitution mechanism.

Animals↗
PubMed2006

Kinetic analysis of a trehalase-overexpressing strain grown on trehalose: a new tool for respiro-fermentative transition studies in Saccharomyces cerevisiae.

AIMS: The aim was to demonstrate the use of a trehalase-overexpressing Saccharomyces cerevisiae strain grown on trehalose as a valuable tool in the studies of respiro-fermentative transition at a reduced scale. METHODS AND RESULTS: A trehalase-overexpressing strain was cultivated in synthetic medium on trehalose under aerobic conditions. This strain grew at a maximum specific growth rate of 0.16 h(-1) and showed a pure oxidative metabolism. Glucose pulse experiments were carried out in this system in order to quantify the short-term Crabtree effect. These data were then compared with glucose pulse experiments carried out in the conventional way with the wild-type strain in glucose-limited chemostats. Glucose-pulse experiments in aerobic batch cultures grown on trehalose led to a metabolic respiro-fermentative transition similar to the one observed in glucose-limited chemostats. CONCLUSIONS: This cultivation system allowed us to quantitatively mimic at the flask scale the Crabtree effect observed in conventional chemostat studies. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is of primary interest in S. cerevisiae studies in which: (i) the implementation of oxidative growth is required (as with studies of the Crabtree effect and heterologous protein production); (ii) small-scale culture systems are required (e.g. high-throughput mutant screening and isotopic labelling experiments).

Fermentation↗
PubMed2006

The cellular resistance against oxidative stress (H2O2) is independent of neutral trehalase (Ntc1p) activity in Candida albicans.

The protective role of trehalose against oxidative stress caused by hydrogen peroxide in Candida albicans has been investigated in the homozygous mutant ntc1Delta/ntc1Delta, disrupted in the NTC1 gene, which encodes the neutral (cytosolic) trehalase (Ntc1p). After a severe oxidative exposure (50 mM H(2)O(2)), both parental (CAI-4) and ntc1Delta/ntc1Delta exponential-phase cells stored large amounts of intracellular trehalose. In turn, the degree of cell survival was roughly equivalent in both strains, although slightly higher in ntc1Delta/ntc1Delta cultures. The mechanism of 'adaptive tolerance' was functional in the two strains. Thus, a gently oxidative pretreatment (5 mM H(2)O(2)) increased the recovery of cellular viability when it was followed by a severe challenge (50 mM H(2)O(2)); this phenomenon was accompanied by a significant elevation of the endogenous trehalose content. Oxidative stress also induced specific activation of the antioxidant enzymes catalase and glutathione reductase upon gentle oxidative treatment (5 mM H(2)O(2)), whereas superoxide dismutase activity was only activated upon prolonged exposure. Taken together, these results strongly suggest that in C. albicans neutral trehalase activity does not play an essential role in the protective response against oxidative stress. They also suggest that a diminished Ntc1p activity might favour the growth of C. albicans cells subjected to a strong oxidative exposure.

Candida albicans↗
PubMed1984

Activation of trehalase by membrane-depolarizing agents in yeast vegetative cells and ascospores.

The membrane-depolarizing agents 2,4-dinitrophenol, carbonylcyanide m-chlorophenylhydrazone, and nystatin are known to cause a rapid increase in the cyclic AMP level in fungal cells. Addition of these proton ionophores to yeast stationary-phase cells or ascospores causes an immediate 10-fold increase in trehalase activity. This observation is in agreement with a role for cyclic AMP-induced phosphorylation in the activation process of trehalase. It also provides an explanation for previous results on the induction of trehalose breakdown by 2,4-dinitrophenol in resting yeast cells.

2,4-Dinitrophenol↗
PubMed1991

Synthesis, accumulation, and excretion of trehalose in osmotically stressed Escherichia coli K-12 strains: influence of amber suppressors and function of the periplasmic trehalase.

It has been reported previously that Escherichia coli K-12 carries an amber mutation that prevents osmotic stress-dependent accumulation of trehalose (M. L. Rod, K. Y. Alam, P. R. Cunningham, and D. P. Clark, J. Bacteriol. 170:3601-3610, 1988). We report that E. coli K-12 and W1485 (sup0) accumulated trehalose but that they required a higher osmotic strength in the growth medium than that required by their sup+ derivatives. Furthermore, the sup+ derivatives displayed both strongly increased trehalose-6-phosphate synthase activity and expression of otsA-lacZ and otsB-lacZ operon fusions compared with their parental strains. It is suggested that the amber mutation in question may be in a gene system encoding a transcriptional activator of the ots genes which govern the synthase. The much-used sup0 strain MC4100 behaved like the sup+ derivatives of W1485 with respect to trehalose synthesis. treA mutants with a defective periplasmic trehalase accumulated trehalose extracellularly under osmotic stress. The amount of trehalose excreted correlated with their synthase activity. Strains with an intact trehalase did not display extracellular trehalose accumulation. Thus, stressed E. coli cells regulate the cytoplasmic level of trehalose by a futile cycle involving overproduction, excretion, and degradation to glucose, which is reutilized.

Escherichia coli↗
PubMed1996

Characterization of a cytoplasmic trehalase of Escherichia coli.

Escherichia coli can synthesize trehalose in response to osmotic stress and is able to utilize trehalose as a carbon source. The pathway of trehalose utilization is different at low and high osmolarity. At high osmolarity, a periplasmic trehalase (TreA) is induced that hydrolyzes trehalose in the periplasm to glucose. Glucose is then taken up by the phosphotransferase system. At low osmolarity, trehalose is taken up by a trehalose-specific enzyme II of the phosphotransferase system as trehalose-6-phosphate and then is hydrolyzed to glucose and glucose-6-phosphate. Here we report a novel cytoplasmic trehalase that hydrolyzes trehalose to glucose. treF, the gene encoding this enzyme, was cloned under ara promoter control. The enzyme (TreF) was purified from extracts of an overexpressing strain and characterized biochemically. It is specific for trehalose exhibiting a Km of 1.9 mM and a Vmax of 54 micromol of trehalose hydrolyzed per min per mg of protein. The enzyme is monomeric, exhibits a broad pH optimum at 6.0, and shows no metal dependency. TreF has a molecular weight of 63,703 (549 amino acids) and is highly homologous to TreA. The nonidentical amino acids of TreF are more polar and more acidic than those of TreA. The expression of treF as studied by the expression of a chromosomal treF-lacZ fusion is weakly induced by high osmolarity of the medium and is partially dependent on RpoS, the stationary-phase sigma factor. Mutants producing 17-fold more TreF than does the wild type were isolated.

Amino Acid Sequence↗
PubMed1978

alpha,alpha-Trehalase of Trichoderma reesei.

A simple adsorption and elution of the trehalase of Trichoderma reesei on bentonite increased the specific activity 70-80 times, with a recovery of 90%. This alpha,alpha-trehalase has an optimum pH of 4.4, a pl of 5.7, a Km of 3.1 X 10(-3) M, and a specific activity of 50 mumol/mg. min-1.

Adsorption↗
PubMed1982

Trehalase deficiency. Prevalence and relation to single-cell protein food.

Perspectives in nutrition suggest a greater use of single-cell proteins in the future. The problems of a high purine content and of potential allergens have been solved by technology. The single-cell proteins may contain trehalose, the absorption of which by the human intestine necessitates prior hydrolysis by trehalase, a specific brush-border disaccharidase. The enzymatic analysis of 100 intestinal biopsies discovered 2 cases of very low trehalase level, with an expected clinical intolerance to even small doses of trehalose. It is concluded that tolerance to high doses of trehalose should be excellent in about 98% of healthy people.

Dietary Proteins↗