Characteristics of trehalase inCandida tropicalis

Summary The trehalase content of different yeasts varies widely. A strain ofCandida tropicalis was found to be the best source of this enzyme among the yeasts tested. The trehalase activity in this yeast could be increased 8.5 times by growing it on trehalose rather than glucose. Thus trehalase is an adaptive enzyme inC. tropicalis. It was found that the amount of trehalase which could be solubilized increased with increasing pH during autolysis of the cells, none being released from the cell debris at pH 4.5 and most at pH 6.3. Some evidence was obtained to show that the solubilization was caused by an enzyme. The stability of trehalase under various conditions was studied. A partial purification was achieved by precipitation with 40% ethanol at a temperature of −18°C. The maximum temperature of the enzyme was 48°C., and the optimum pH ranged from 4.1 to 5.3     

Functional characterization of <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> neutral trehalase altered in phosphorylatable serine residues

The activation of neutral trehalase (Ntp1) by metabolic and physical stresses in Schizosaccharomyces pombe is dependent on protein kinases Pka1 or Sck1. Mutant ntp1 alleles altered for potentially phosphorylatable serine residues within the regulatory domain of the enzyme were integrated under the control of the native promoter in an ntp1-deleted background. The trehalase variants were expressed to a level similar to that of wild type trehalase from control cells. Wild type trehalase protein accumulated and became activated upon stress while a single change in the evolutionary conserved perfect consensus site for Pka1-dependent phosphorylation (Ser71), as well as point mutations in two other putative phosphorylation sites (Ser6, Ser51), produced inactive trehalases unresponsive to stress. Trehalose content in the trehalase mutated strains increased upon salt stress to a level comparable to that shown by an ntp1-deleted mutant. When exposed to heat shock, trehalose hyperaccumulated in the ntp1-null strain lacking trehalase protein and this phenotype was shown by some (Ser71), but not all, strains with serine mutated trehalases. The mutant trehalases retained the ability to form complexes with trehalose-6-phosphate synthase. These data support a role of potentially phosphorylated specific sites for the activation of S. pombe neutral trehalase and for the heat shock-induced accumulation of trehalose.     

A highly thermostable trehalase from the thermophilic bacterium <Emphasis Type="Italic">Rhodothermus marinus</Emphasis>

Trehalases play a central role in the metabolism of trehalose and can be found in a wide variety of organisms. A periplasmic trehalase (α,α-trehalose glucohydrolase, EC 3.2.1.28) from the thermophilic bacterium Rhodothermus marinus was purified and the respective encoding gene was identified, cloned and overexpressed in Escherichia coli. The recombinant trehalase is a monomeric protein with a molecular mass of 59 kDa. Maximum activity was observed at 88°C and pH 6.5. The recombinant trehalase exhibited a K _m of 0.16 mM and a V _max of 81 μmol of trehalose (min)⁻¹ (mg of protein)⁻¹ at the optimal temperature for growth of R. marinus (65°C) and pH 6.5. The enzyme was highly specific for trehalose and was inhibited by glucose with a K _i of 7 mM. This is the most thermostable trehalase ever characterized. Moreover, this is the first report on the identification and characterization of a trehalase from a thermophilic bacterium.     

Biochemical properties of an extracellular trehalase from <Emphasis Type="Italic">Malbranchea pulchella</Emphasis> var. Sulfurea

The thermophilic fungus Malbranchea pulchella var. sulfurea produced good amounts of extracellular trehalase activity when grown for long periods on starch, maltose or glucose as the main carbon source. Studies with young cultures suggested that the main role of the extracellular acid trehalase is utilizing trehalose as a carbon source. The specific activity of the purified enzyme in the presence of manganese (680 U/mg protein) was comparable to that of other thermophilic fungi enzymes, but many times higher than the values reported for trehalases from other microbial sources. The apparent molecular mass of the native enzyme was estimated to be 104 kDa by gel filtration and 52 kDa by SDS-PAGE, suggesting that the enzyme was composed by two subunits. The carbohydrate content of the purified enzyme was estimated to be 19 % and the pi was 3.5. The optimum pH and temperature were 5.0–5.5 and 55° C, respectively. The purified enzyme was stimulated by manganese and inhibited by calcium ions, and insensitive to ATP and ADP, and 1 mM silver ions. The apparent K_M values for trehalose hydrolysis by the purified enzyme in the absence and presence of manganese chloride were 2.70±0.29 and 2.58±0.13 mM, respectively. Manganese ions affected only the apparent V_max, increasing the catalytic efficiency value by 9.2-fold. The results reported herein indicate that Malbranchea pulchella produces a trehalase with mixed biochemical properties, different from the conventional acid and neutral enzymes and also from trehalases from other thermophilic fungi.     

Effects of validamycin A,a potent trehalase inhibitor,and phytohormones on trehalose metabolism in roots and root nodules of soybean and cowpea

Trehalose, a common microbial disaccharide, has been reported to be toxic to plants, and plant trehalase has therefore been hypothesized to function as a detoxifying enzyme. To test this, aseptically grown soybean (Glycine max L. Merr.) plantlets were supplied with trehalose. The plants accumulated trehalose only when validamycin A, a potent trehalase inhibitor, was added as well. Under these conditions, they accumulated trehalose to up to 8% of the dry weight in their primary leaves without any detectable impairment of growth or health. We have previously shown that in soybean nodules, trehalose is generated by the symbiotic bacteria, and trehalase is strongly induced. However, direct exposure of plants to trehalose did not affect their trehalase activity, whereas a treatment with auxin strongly increased it, indicating that the enzyme level is regulated by hormones rather than by its substrate. Addition of validamycin A to nodules caused an increase in the amount of trehalose and a decrease in the sucrose and starch pools, but nitrogen fixation was not affected. Similar results were obtained with cowpea (Vigna unguiculata L.) plantlets and nodules. These results indicate that plant trehalase is functional in metabolizing trehalose from exogenous and endogenous sources, even though the disaccharide has no obvious toxic effects.Abbreviations ABA abscisic acid - ARA acetylene-reduction activity (assay for nitrogenase) - 2,4-D 2,4-dichlorophenoxyacetic acid - DW dry weight - FW fresh weight - GA₃ gibberellic acid - -NAA, -NAA -,-naphthaleneacetic acid We are indebted to Prof. Dr. W. Broughton (University of Geneva, Switzerland) for kindly providing us cowpea seeds and the symbiont strain Rhizobium sp. NGR 234. Validamycin A was a gift of Dr. J.-P. Métraux, Ciba, Basel. This work was supported by the Swiss National Foundation.     

Inducible trehalase enzyme activity of Morchella conica Persoon mycelium

Morchella conica Pers. strains of the study were isolated from fruit bodies collected in ash-mixed forests. At first, the strains were cultured on potato dextrose agar (PDA), then on modified Murashige and Skoog (MS) solid agar media. A normal-growing strain was chosen for the trehalase induction experiments. During the trehalase induction treatment, mycelia were grown in liquid culture containing different concentrations of trehalose. After the induction period of trehalase enzymes, physiological state of the mycelium and the oxidative stress were monitored in the vegetative mycelia by measuring the change of the malondialdehyde content, superoxide dismutase enzyme activity, the fresh and dry weight. The examined Morchella conica strain utilized the trehalose properly. The rising amount of the trehalose triggered the increase of the mycelial trehalase enzyme activity. Our results clearly proved that both neutral and acidic trehalase isoenzyme activity of the Morchella conica mycelium are inducible and are playing important role in the utilization of external trehalose.     

Thermotolerance and trehalose accumulation induced by heat shock in yeast cells of Candida albicans

Candida albicans yeast cells growing exponentially on glucose are extremely sensitive to severe heat shock treatments (52.5°C for 5 min). When these cultures were subjected to a mild temperature preincubation (42°C), they became thermotolerant and displayed higher resistance to further heat stress. The intracellular content of trehalose was very low in exponential cells, but underwent a marked increase upon non-lethal heat exposure. The accumulation of trehalose is likely due to heat-induced activation of the trehalose-6-phosphate synthase complex, whereas the external trehalase remained practically unmodified. After a temperature reversion shift (from 42°C to 28°C), the pool of trehalose was rapidly mobilized without any concomitant change in trehalase activity. These results support an important role of trehalose in the mechanism of acquired thermotolerance in C. albicans and seem to exclude the external trehalase as a key enzyme in this process.     

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.     

The Arabidopsis thaliana trehalase is a plasma membrane-bound enzyme with extracellular activity

The lack of trehalose accumulation in most plant species has been partly attributed to the presence of an active trehalase. Although trehalose synthesis enzymes are thought to be cytosolic, and previous studies have indicated that trehalase activity is extracellular, the exact location of the enzyme has not yet been established in plant cell. We present evidence that the yet uncharacterised full-length Arabidopsis trehalase is a plasma membrane-bound protein, probably anchored to the membrane through a predicted N-terminal membrane spanning domain. The full-length AtTRE1, when expressed in yeast can functionally substitute for the extracellularly active trehalase Ath1p, by sustaining the growth of an ath1 null mutant strain on trehalose and at pH 4.8. We further demonstrate that AtTRE1 expressed in yeast is plasma membrane-bound as in plant cell. In light of these findings, the regulation of plant cell endogenous trehalose by trehalase is discussed.     

Biochemical characterization of neutral trehalase activity in Saccharomyces boulardii

Lyophilized cells of the non-pathogenic yeast Saccharomyces boulardii are used in many countries for the treatment of several types of diarrhoea and other gastrointestinal diseases. Although the cells must be viable, their mechanism of action is unknown. The disaccharide trehalose is a protectant against several forms of environmental stress in yeast and is involved in maintaining cell viability. There is no information on the enzymes involved in degradation of trehalose in S. boulardii. The aim of the present study was to characterize trehalase activity in this yeast. Cells of S. boulardii grown in glucose exhibited neutral trehalase activity only in the exponential phase. Acidic trehalase was not detected in glucose medium. Cells grown in trehalose exhibited acid and neutral trehalase activities at all growth stages, particularly in the exponential phase. The optimum pH and temperature values for neutral trehalase activity were determined as 6.5 and 30 °C respectively, the half-life being approximately 3 min at 45 °C. The relative molecular mass of neutral trehalase is 80 kDa and the K _m 6.4 mM (±0.6). Neutral trehalase activity at pH 6.5 was weakly inhibited by 5 mM EDTA and strongly inhibited by ATP, as well as the divalent ions Cu⁺⁺, Fe⁺⁺ and Zn⁺⁺. Enzyme activity was stimulated by Mg⁺⁺ and Ca⁺⁺ only in the absence of cAMP. The presence of cAMP with no ion additions increased activity by 40%. This revised version was published online in July 2006 with corrections to the Cover Date.     

α-Glucose-1-phosphate formation by a novel trehalose phosphorylase from Flammulina velutipes

A novel type of trehalose phosphorylase was found in a basidiomycete. Flammulina velutipes . The enzyme catalyzes both the reversible phosphorolysis of trehalose to form α-glucose 1-phosphate and glucose and also the synthesis of trehalose. Comparison of the specific activity of trehalose phosphorylase with that of trehalase suggested that the function of the former enzyme was more important in the fruit-bodies of this fungus.     

Three Enzymes for Trehalose Synthesis in Bradyrhizobium Cultured Bacteria and in Bacteroids from Soybean Nodules

α,α-Trehalose is a disaccharide accumulated by many microorganisms, including rhizobia, and a common role for trehalose is protection of membrane and protein structure during periods of stress, such as desiccation. Cultured Bradyrhizobium japonicum and B. elkanii were found to have three enzymes for trehalose synthesis: trehalose synthase (TS), maltooligosyltrehalose synthase (MOTS), and trehalose-6-phosphate synthetase. The activity level of the latter enzyme was much higher than those of the other two in cultured bacteria, but the reverse was true in bacteroids from nodules. Although TS was the dominant enzyme in bacteroids, the source of maltose, the substrate for TS, is not clear; i.e., the maltose concentration in nodules was very low and no maltose was formed by bacteroid protein preparations from maltooligosaccharides. Because bacteroid protein preparations contained high trehalase activity, it was imperative to inhibit this enzyme in studies of TS and MOTS in bacteroids. Validamycin A, a commonly used trehalase inhibitor, was found to also inhibit TS and MOTS, and other trehalase inhibitors, such as trehazolin, must be used in studies of these enzymes in nodules. The results of a survey of five other species of rhizobia indicated that most species sampled had only one major mechanism for trehalose synthesis. The presence of three totally independent mechanisms for the synthesis of trehalose by Bradyrhizobium species suggests that this disaccharide is important in the function of this organism both in the free-living state and in symbiosis.     

Evidence for nonregulatory trehalase activity inDictyostelium discoideum

An in vitro activation treatment, stimulatory to the regulatory cytoplasmic trehalase ofSaccharomyces cerevisiae, had no effect on the lysosomal trehalase ofDictyostelium discoideum. Concentrations of cAMP that produced a 19 to 22-fold increase in trehalase activity inS. cerevisiae extracts did not stimulate trehalase activity inD. discoideum extracts.. cGMP and 5-AMP were also not effective in activating the enzyme.Dictyostelium discoideum trehalase exhibits characteristics typical of nonregulatory trehalases, in agreement with its lysosomal localization. The data are consistent with the hypothesis that changes in compartmentation regulate trehalose mobilization inD. discoideum.     

Metabolism of endogenous trehalose by Streptomyces griseus spores and by spores or cells of other actinomycetes.

The disaccharide trehalose is accumulated as a storage product by spores of Streptomyces griseus. Nongerminating spores used their trehalose reserves slowly when incubated in buffer for several months. In contrast, spores rapidly depleted their trehalose pools during the first hours of germination. Extracts of dormant spores contained a high specific activity of the enzyme trehalase. The level of trehalase remained relatively constant during germination or incubation in buffer. Nongerminating spores of Streptomyces viridochromogenes, Streptomyces antibioticus, and Micromonospora echinospora and nongrowing spherical cells of Arthrobacter crystallopoietes and Nocardia corallina also maintained large amounts of trehalose and active trehalase. These trehalose reserves were depleted during spore germination or outgrowth of spherical Arthrobacter and Nocardia cells into rods.     

Trehalose synthase and trehalase behaviour in yeast cells in anhydrobiosis and hydrobiosis

• 1.I. Trehalose synthase and trehalase behaviour has been analysed in cultured yeast cells isolated from baker's yeast to increase the understanding of the mechanisms involved in trehalose content modifications observed in anyhydrobiois and hydrobiosis.
• 2.2. After desiccating yeast cells to a constant weight, trehalose levels sharply increased, whereas the glycogen content decreased, trehalose synthase was stimulated and trehalase was significantly inhibited.
• 3.3. In desiccated cells after a rehydration for 15 min, trehalose levels dropped, the glycogen content further decreased, the activity of trehalose synthase declined while that of trehalase was dramatically stimulated.
• 4.4. After rehydration for 12hr, while the trehalose and glycogen content decreased even more, the behaviour of the two enzymes was completely reversed, trehalose synthase being activated and trehalase inhibited.
• 5.5. The reasons for such impressive enzyme activity alterations in desiccated and rehydrated cells for the moment remain unknown.

     

Neurospora Trehalase and Its Structural Gene

We have isolated Neurospora trehalaseless mutants and mapped the trehalase structural gene to linkage group I. The structural gene mutations not only affect thermostability and other characteristics of the enzyme but also affect the production of an inhibitor of the wild-type trehalase. The inhibitor appears to be the mutant trehalase. We suggest that the mutant subunits act as inhibitors by entering into the multimeric forms of the enzyme and altering the ability of the normal wild-type subunits to catalyze the cleavage of trehalose.—Wild type trehalase has been purified to near homogeneity, and its characteristics have been studied. It was purified as a tetramer, with each subunit having a molecular weight of 88,000.—We have studied the regulation of trehalase and found the production of trehalase to be glucose repressible. Cells begin to produce trehalase 60 min after being transferred to glucose-free medium.     

Trehalose catabolism enzymes in L3 and L4 larvae of Anisakis simplex

The presence of trehalase and trehalose phosphorylase in L3 and L4 larvae of Anisakis simplex was demonstrated. The activity of trehalase and trehalose phosphorylase in L3 larvae was 6 and 10 times higher, respectively, than in L4 larvae. This suggests that trehalose metabolism is more important for L3 than LA larvae. Trehalases of L3 and L4 differ in their characteristics. The enzyme of L3 was present mainly in the lysosomes and cytosol, whereas in L4 the highest enzyme activity was measured in the lysosomal fraction. Trehalase activity was increased by 29% in L3 and 55% in L4 with the addition of Mg2+ (0.1 mmol). Tris inhibited trehalase in L3 larvae by 42% and in L4 by 25%. The enzymes differed in their reaction to EDTA, CaCl2, ZnCl2, and CH2ICOOH (all 0.1 mmol). High activity of trehalase from L3 larvae was measured within the pH range of 5.0 to 6.5, with an optimum pH of 6.1. The trehalase was a thermally tolerant enzyme from 25 C to 60 C. The enzyme lost half of its activity after preincubation without substrate above 75 C. The paper also discusses the similarities and differences in characteristics of trehalase from A. simplex larvae and presents the comparison to enzymes from other nematodes.     

Acid trehalase deficiency and extracellular trehalose utilization in Candida utilis

Biochemical characterization of a trehalase, detected in the mid-exponential growth phase of Candida utilis NCIM Y500, has indicated that it was a neutral trehalase and possibly the only trehalase present in this strain. Unlike Saccharomyces cerevisiae and other C. utilis strains, this strain without acid trehalase grew quite well in minimal or complete medium containing trehalose as the sole source of carbon. Both these observations were contradictory to the findings reported for acid trehalase mutants of S. cerevisiae and C. utilis. The trehalase system of the strain is suggested to be similar to that of fungi.     

Methods for the Rapid Purification of Trehalase from Dictyostelium Discoideum

A rapid and reliable method for the preparation of homogeneous trehalase from the cellular slime mold, Dictyostelium discoideum for usage in enzyme characterization studies and trehalose assays was developed. This procedure takes advantage of the fact that trehalase activity is secreted by Dictyostelium during the course of development, the major fraction being released late in fruiting body formation. Purification of trehalase to electrophoretic homogeneity was accomplished utilizing the techniques of ultrafiltration, streptomycin sulfate precipitation, ammonium sulfate fractionation, DEAE-Sephacel chromatography and preparative disc gel electrophoresis. Analysis of the purified enzyme by analytical polyacrylamide disc gel electrophoresis demonstrated the presence of a single protein band which was stainable with Coomassie blue. Assay of trehalase activity in eluates from segments of a companion gel indicated that all of the recovered trehalase activity was associated with this band of protein. Examination of the substrate specificity of the purified enzyme indicated absolute specificity for trehalose.     

A novel trehalase from Mycobacterium smegmatis - purification, properties, requirements

Trehalose is a nonreducing disaccharide of glucose (alpha,alpha-1,1-glucosyl-glucose) that is essential for growth and survival of mycobacteria. These organisms have three different biosynthetic pathways to produce trehalose, and mutants devoid of all three pathways require exogenous trehalose in the medium in order to grow. Mycobacterium smegmatis and Mycobacterium tuberculosis also have a trehalase that may be important in controlling the levels of intracellular trehalose. In this study, we report on the purification and characterization of the trehalase from M. smegmatis, and its comparison to the trehalase from M. tuberculosis. Although these two enzymes have over 85% identity throughout their amino acid sequences, and both show an absolute requirement for inorganic phosphate for activity, the enzyme from M. smegmatis also requires Mg(2+) for activity, whereas the M. tuberculosis trehalase does not require Mg(2+). The requirement for phosphate is unusual among glycosyl hydrolases, but we could find no evidence for a phosphorolytic cleavage, or for any phosphorylated intermediates in the reaction. However, as inorganic phosphate appears to bind to, and also to greatly increase the heat stability of, the trehalase, the function of the phosphate may involve stabilizing the protein conformation and/or initiating protein aggregation. Sodium arsenate was able to substitute to some extent for the sodium phosphate requirement, whereas inorganic pyrophosphate and polyphosphates were inhibitory. The purified trehalase showed a single 71 kDa band on SDS gels, but active enzyme eluted in the void volume of a Sephracryl S-300 column, suggesting a molecular mass of about 1500 kDa or a multimer of 20 or more subunits. The trehalase is highly specific for alpha,alpha-trehalose and did not hydrolyze alpha,beta-trelalose or beta,beta-trehalose, trehalose dimycolate, or any other alpha-glucoside or beta-glucoside. Attempts to obtain a trehalase-negative mutant of M. smegmatis have been unsuccessful, although deletions of other trehalose metabolic enzymes have yielded viable mutants. This suggests that trehalase is an essential enzyme for these organisms. The enzyme has a pH optimum of 7.1, and is active in various buffers, as long as inorganic phosphate and Mg(2+) are present. Glucose was the only product produced by the trehalase in the presence of either phosphate or arsenate.