Trehalase Inhibitor Research Articles

Molecular Basis for Trehalase Inhibition Revealed by the Structure of Trehalase in Complex with Potent Inhibitors

Molecular Basis for Trehalase Inhibition Revealed by the Structure of Trehalase in Complex with Potent Inhibitors

Trehalose metabolism in root nodules of the model legume Lotus japonicus in response to salt stress

The effect of NaCl stress (50 mM) and validamycin A treatment (30 μM) on growth and nitrogen fixation of Lotus japonicus was investigated in plants cultured under symbiotic and hydroponics conditions for teen weeks (flowering stage). Validamycin A was used as a potent trehalase inhibitor, and was able to produce a five‐fold increase in the level of trehalose during salt treatment, concomitant with an enhance in biomass (20%) in salinized plants. Alterations of nodule metabolism related to some carbohydrates and some enzyme activities were also examined. The shoot and total plant dry weight were severely affected by saline conditions decreasing by 40% and only 15–20% in plant treated without or with validamycin A, respectively. Nitrogenase activity (E.C. 1.7.9.92) was inhibited almost 40% by salt stress and no effect of validamycin was observed. Based on these results, L. japonicus might be considered as a salt‐sensitive legume. In addition, the saline conditions also inhibited the enzyme activities of sucrose synthase (E.C. 2.4.1.13), alkaline invertase (E.C. 3.2.1.26) and trehalose‐phosphate synthetase (E.C. 2.4.1.15). The validamycin A treatment mainly decreased enzyme activities: sucrose synthase, trehalose‐phosphate phosphatase (E.C. 3.1.3.12) and trehalase (E.C. 3.2.1.28). On the other hand, a high concentration of the carbohydrates, starch, sucrose and glucose, seems not to be the mechanism induced in L. japonicus to protect nodules exposed to NaCl because all these sugars decreased in such conditions. Results of the present study support the possible role of trehalose as an osmoprotectant under salt stress.

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

alpha,alpha-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.

Production of trehalase inhibitor validoxylamine A using acid-catalyzed hydrolysis of validamycin A

As a competitive trehalase inhibitor and lead compound with broad uses, validoxylamine A has been increasingly studied for its many possible applications. The process of acid-catalyzed hydrolysis of validamycin A was investigated for production of validoxylamine A. The effects of various acids and substrate concentrations were explored, and the mechanism of acid-catalyzed hydrolysis of validamycin A was proposed. Results demonstrated that the optimal condition of acid hydrolysis of validamycin A included 1 M H 2SO 4 and a 30% (w/v) initial validamycin A concentration in a 1 h reaction time.

Pyridinium Salt Photochemistry in a Concise Route for Synthesis of the Trehazolin Aminocyclitol, Trehazolamine

[reaction: see text] A strategy for the concise synthesis of trehazolamine, the aminocyclitol core of the potent trehalase inhibitor trehazolin, has been developed. The methodology takes advantage of photocyclization reaction of 1-methoxyethoxymethyl-3-pivaloxymethylpyridinium perchlorate to generate a bicyclic-aziridine intermediate, which is transformed under aziridine ring opening conditions to the key intermediate, 3,5-diacetoxy-3-pivaloxymethyl-4-(N-acetylamino)cyclopentene. In addition, the strategy is used in an enantio-divergent sequence for preparation of the natural (+)-trehazolamine and its unnatural (-)-enantiomer. In this route, the chiral auxiliary containing 1-(tetracetyl-alpha-D-glucosyl)-3-pivaloxymethylpyridinium perchlorate undergoes photocyclization to generate separable, diastereomeric bicyclic-aziridines, which are then independently transformed to enantiomeric 3,5-diacetoxy-3-pivaloxymethyl-4-(N-acetylamino)cyclopentenes.

Syntheses, Structures and Bioactivities of Fluorine‐Containing Phenylimino‐thia(oxa)zolidine Derivatives as Agricultural Bioregulators.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Physiological implications of trehalase from Phaseolus vulgaris root nodules: partial purification and characterization

The purification and characterization of trehalase from common bean nodules as well as the role of this enzyme on growth, nodulation nitrogen fixation by examining the effects of the trehalase inhibitor validamycin A, was studied. Validamycin A did not affect plant and nodule mass, neither root trehalase and nitrogenase activity; however this treatment applied at the time of sowing increased nodule number about 16% and decreased nodule trehalase activity (16-fold) and the size of nodules. These results suggest that nodule trehalase activity of Phaseolus vulgaris could be involved in nodule formation and development. In addition, acid trehalase (EC 3.2.1.28) was purified from root nodules by fractionating ammonium sulfate, column chromatography on DEAE-sepharose and sephacryl S-300, and finally on native polyacrylamide gel electrophoresis. The purified homogeneous preparation of native acid trehalase exhibited a molecular mass of 42 and 45 kDa on SDS-PAGE. The enzyme has the optimum pH 3.9, K m of 0.109 mM, V max of 3630 nkat mg –1 protein and is relatively heat stable. Besides trehalose, it shows maximal activity with sucrose and maltose and, to a lesser degree melibiose, cellobiose and raffinose, and it does not hydrolyze on lactose and turanose. Acid trehalase was activated by Na +, Mn 2+, Mg 2+, Li +, Co 2+, K + and inhibited by Fe 3+, Hg + and EDTA.

Synthesis, Inhibition Properties, and Theoretical Study of the New Nanomolar Trehalase Inhibitor 1‐Thiatrehazolin: Towards a Structural Understanding of Trehazolin Inhibition

A new trehazolin analogue, 1-thiatrehazolin, has been synthesized from carbohydrate precursors by a highly efficient route based on our previously developed ketone/oxime ether reductive carbocyclization reaction for the construction of the cyclitol ring and an intramolecular nucleophilic displacement reaction for the construction of the thiazoline ring. 1-Thiatrehazolin is a very potent, slow, tight-binding trehalase inhibitor. A structural model for trehalase inhibition by trehazolin and its analogues, based on the experimental results and supported by theoretical calculations, is proposed.

Preparation of Trehalase Inhibitor Validoxylamine A by Biocatalyzed Hydrolysis of Validamycin A With Honeybee (<I>Apis cerana</I> Fabr.) β-Glucosidase

Validoxylamine A is structurally similar to trehalose and acts a potent competitive inhibitor of trehalase. It has recently been receiving increased attention as a potential material for the development of new insecticides or drugs. In this study, beta-glucosidase extracted from honeybees (Apis cerana Fabr.) was used as a catalyst to produce validoxylamine A through enzymatic hydrolysis of validamycin A. Beta-glucosidase was separated and purified from honeybees, and its characteristics were examined. The results showed that beta-glucosidase was stable across a range of temperatures from 30 to 40 degrees C and across a relatively wide range of pH values from 5.0 to 7.5. Investigation of the biocatalyzed hydrolysis process from validamycin A to validoxylamine A with beta-glucosidase revealed that both the substrate (validamycin A) and the product (validoxylamine A) inhibited beta-glucosidase activity. The inhibition constant of the substrate Kis value was 5.01 mM, and that of the product Kip value was 1.32 mM. This product inhibition was competitive.

Facile, Efficient, and Enantiospecific Syntheses of 1,1‘-N-Linked Pseudodisaccharides as a New Class of Glycosidase Inhibitors

This article describes an efficient synthesis of a potent trehalase inhibitor, 1,1'-N-linked pseudodisaccharide 1 (consisting of two valienamines), in 14 steps with an overall yield of 12% and a first synthesis of 2 (consisting of two 2-epi-valienamines) in 15 steps with an overall yield of 24% from (-)-quinic acid. The synthesis involves a stereospecific palladium-catalyzed coupling reaction between an allylic amine and an allylic chloride as the crucial step. The acetonide blocking groups were shown to be the best hydroxyl protecting groups, compatible with the palladium-catalyzed allylic amination reaction that afforded high yields of the 1,1'-N-linked pseudodisaccharides with a minimum amount of an elimination diene side product.

Syntheses, structures and bioactivities of fluorine-containing phenylimino-thia(oxa)zolidine derivatives as agricultural bioregulators

From insight into the structure of trehazolin as trehalase inhibitor, six series of fluorine-containing phenylimino-thiazolidines (oxazolidines) derivatives were designed and prepared through a convenient synthesis of fluoroaryl isothiocyanate and a one-pot facile synthesis in high yield of fluorophenyl aminobenzoxazoles by cyclodesulfurization. The structures of the target compounds were confirmed with using IR, NMR, MS and elemental analysis. Their X-ray crystal analysis suggested that there were novel intermolecular (sp 2CF ⋯ H 3C ) and intramolecular (sp 2CF ⋯ HN) hydrogen bonds between the fluorine atom on benzene ring and hydrogen atom of methyl group or amino group on five-membered heterocycle. Their fungicidal activities against Rhizoctonia solani and Pyricuraria oryzae at 100 ppm were determined.

Stereocontrolled synthesis of the aminocyclitol moiety of (+)-trehazolin via C–H insertion reaction of alkylidenecarbene

The aminocyclitol moiety of (+)-trehazolin, a powerful trehalase inhibitor, was synthesized in a stereocontrolled manner from cis-2-butene-1,4-diol via C–H insertion reaction of the alkylidenecarbene, followed by regioselective opening of the epoxide ring. It was obtained in an enantiomerically pure form by twice using Sharpless asymmetric epoxidation.

Nitrate is a negative signal for fructan synthesis, and the fructosyltransferase-inducing trehalose inhibits nitrogen and carbon assimilation in excised barley leaves.

• Fructan biosynthesis in barley (Hordeum vulgare) has been shown to be upregulated by sugar signalling and downregulated by nitrogen. The relationship between these two regulations is investigated. • Excised third-leaves of barley were fed nitrate or glutamine under two light intensities. Other leaf blades were supplied in the dark for 24h with nitrate and trehalose in the presence of validamycin A, a trehalase inhibitor. • In the light, nitrate, but not glutamine, decreased fructan contents and sucrose:fructan 6-fructosyltransferase protein without affecting the levels of sucrose and other carbohydrates. In darkened leaves, trehalose increased and nitrate decreased the fructan contents and total sucrose:fructosyltransferase activity without altering the concentration of sucrose. The effect on fructan contents of trehalose disappeared, whereas that of nitrate remained in subsequent incubations in water under light. Trehalose decreased and nitrate increased the light- and CO2 -saturated rate of photosynthesis without significantly affecting the initial Rubisco (ribulose-1,5-bisphosphate carboxylase oxygenase) activity. Trehalose feeding decreased the activation of nitrate reductase and amino acid levels, and blocked the positive effect of nitrate on the maximal activity of this enzyme. • The results indicate that nitrate, and not a downstream metabolite, is a negative signal for fructan synthesis, independent from the positive sugar signalling and overriding it. Trehalose signalling inhibits nitrogen and carbon assimilation, at the same time, inducing fructosyltransferase activity.

The toxic and lethal effects of the trehalase inhibitor trehazolin in locusts are caused by hypoglycaemia.

The main blood sugar of locusts is trehalose, which is hydrolysed to two glucose units by trehalase. Homogenates of locust flight muscles are rich in trehalase activity, which is bound to membranes. A minor fraction of trehalase is in an overt form while the remainder is latent, i.e. active only after impairing membrane integrity. Trehazolin, an antibiotic pseudosaccharide, inhibits locust flight muscle trehalase with apparent K(i)- and EC(50) values of 10(-8) mol l(-1) and 10(-7) mol l(-1), respectively. Trehazolin is insecticidal: 50 micro g injected into locusts completely and selectively blocked the overt form of muscle trehalase (with little effect on latent activity) and killed 50% of the insects within 24 h. Here, it is demonstrated for the first time that trehazolin causes dramatic hypoglycaemia. Injection of 10 micro g trehazolin caused glucose levels to fall by over 90% in 24 h, from 2.8 mmol l(-1) to 0.23 mmol l(-1), while trehalose increased from 61 mmol l(-1) to 111 mmol l(-1). Feeding glucose to the locusts fully neutralized the effects of a potentially lethal dose of trehazolin. This indicates that hypertrehalosaemia is not acutely toxic, whereas lack of glucose causes organ failure (presumably of the nervous system), and that sufficient haemolymph glucose can only be generated from trehalose by trehalase. The results also suggest that overt flight muscle trehalase is located in the plasma membrane with the active site accessible to the haemolymph. Trehalase inhibitors are valuable tools for studying the molecular physiology of trehalase function and sugar metabolism in insects.

High concentrations of trehalose in aphid hemolymph

The major components of hemolymph were examined by 1H-NMR in seven species of aphids, Acyrthosiphon pisum, Aphis gossypii, Aphis sambuci, Lachnus tropicalis, Megoura crassicauda, Myzus persicae and Uroleucon nigrotuberculatum. Trehalose was detected as the main component from these aphids, and its concentrations in these insects were much higher (196 mM in L. tropicalis to 926 mM in A. gossypii) than those of two other homopterous insects, Cryptotympana facialis (91 mM) and Nephotettix cincticeps (53 mM). Concentrations in L. tropicalis and A. gossypii were equivalent to 1.6% to 2.0% of the fresh weight. Glucose appeared to be an artifact in the hemolymph, because it was not found in the hemolymph when validoxylamine A, a trehalase inhibitor, was added.

Trehalose-hydrolysing enzymes of Metarhizium anisopliae and their role in pathogenesis of the tobacco hornworm, Manduca sexta.

Trehalose is the main haemolymph sugar in most insects including the tobacco hornworm, Manduca sexta, and is potentially a prime target for an invading pathogenic fungus. There was considerably more trehalose-hydrolysing activity in the haemolymph of caterpillars infected with Metarhizium anisopliae than in controls. This appeared to be due primarily to additional isoforms; one of which could also hydrolyse maltose and was designated an alpha-glucosidase. A comparable isoform was identified in in vitro culture of the fungus, supporting a fungal origin for the in vivo enzyme. The in vitro fungal enzyme, alpha-glucosidase-1 (alpha-gluc-1), was purified to homogeneity and partially characterised. A study with the trehalase inhibitor trehazolin and C14 trehalose suggested that extracellular hydrolysis is important for fungal mobilisation of trehalose. Haemolymph glucose increases significantly during mycosis of tobacco hornworm larvae by M. anisopliae, consistent with the hydrolysis of trehalose by extracellular fungal enzymes. The implications for the host insect are discussed.

ChemInform Abstract: Syntheses of 2‐Aryliminooxazolidine Derivatives as Trehalase Inhibitors.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform Abstract: Syntheses and Activities as Trehalase Inhibitors of N‐Arylglycosylamines Derived from Fluorinated Anilines.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Biosynthesis of the Trehalase Inhibitor Trehazolin.

Trehazolin (1) is a trehalase inhibitor produced by Micromonospora coriacea. Biosynthesis of 1 was studied by feeding experiments with a variety of labeled precursors. Feeding experiments with [1-13C]- and [6-13C]-D-glucose revealed that the carbon skeletons of both a glucose residue and a cyclopentane ring moiety in 1 were each derived from glucose, and that C-C bond formation between C-1 and C-5 of glucose occurred during the cyclopentane ring formation. Furthermore, an experiment with [guanidino-13C, 15N2]-L-arginine revealed that two nitrogen atoms and a quaternary carbon atom involved in the aminooxazoline moiety of 1 originated from an amidino group of arginine. Further feeding experiments with [1-2H]-, [2-2H]-, [4-2H]-, [6,6-2H2]- and [1,2,3,4,5,6,6-2H7]-D-glucose as well as [1-13C]-D-fructose showed that deuteriums on C-1, C-3, C-4 and C-6 of glucose were retained during the formation of the cyclopentane ring moiety of 1.

Syntheses and activities as trehalase inhibitors of N-arylglycosylamines derived from fluorinated anilines

Twelve N-arylglycosylamines were prepared in a one-pot reaction by treatment of d-glucose, d-galactose, d-mannose or d-xylose with fluorinated anilines in the presence of small amount of hydrochloric acid. The inhibitory activity against porcine trehalase and the fungicidal activity of the title compounds toward Rhizoctonia solani have been tested.