Metabolic function of glycogen phosphorylase and trehalose phosphorylase in fruit-body formation ofFlammulina velutipes

Glycogen phosphorylase in the vegetative mycelium ofFlammulina velutipes converts glycogen to α-glucose 1-phosphate (G1P) in the colony during fruit-body development. Glycogen may contribute to the synthesis of trehalose as the starting material in the vegetative mycelium during the fruiting process of the colony, and the trehalose produced is translocated into the fruit-bodies as the main carbohydrate substrate for their development. Trehalose phosphorylase activity in the vegetative mycelium was at a relatively high level until fruit-body initiation, suggesting the turnover of this disaccharide during the vegetative stage of the colony development. Trehalose phosphorylase activity in the stipes showed a peak level at the early phase of fruit-body development, suggesting the continuing phosphorolysis of trehalose by this enzyme. The stipes also showed a high specific activity of phosphoglucomutase at a sufficient level to facilitate the conversion of G1P to α-glucose 6-phosphate (G6P). In the pilei a large amount of G1P remained until the growth of the fruit-bodies ceased. Trehalase activities in the stipes and pilei were at a very low level, and this enzyme may not contribute to the catabolism of trehalose in the fruit-body development.     

The stereochemical course of the reaction mechanism of trehalose phosphorylase from Schizophyllum commune

Phosphorolysis of α,α-trehalose catalyzed by trehalose phosphorylase from the basidiomycete Schizophyllum commune proceeds via net retention of anomeric configuration and yields α- -glucose 1-phosphate and α- -glucose as the products. In reverse reaction, only the α-anomers of -glucose 1-phosphate and -glucose are utilized as glucosyl donor and acceptor, respectively, and give exclusively the α,α-product. Trehalose phosphorylase converts α- -glucose 1-fluoride and phosphate into α- -glucose 1-phosphate, a reaction requiring the stereospecific protonation of the glucosyl fluoride by a Brønsted acid. The results are discussed with regard to a plausible reaction mechanism of fungal trehalose phosphorylase.     

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.     

Purification and characterization of trehalose phosphorylase from the commercial mushroom Agaricus bisporus

Trehalose phosphorylase (EC 2.4.1.64) from Agaricus bisporus was purified for the first time from a fungus. This enzyme appears to play a key role in trehalose metabolism in A. bisporus since no trehalase or trehalose synthase activities could be detected in this fungus. Trehalose phosphorylase catalyzes the reversible reaction of degradation (phosphorolysis) and synthesis of trehalose. The native enzyme has a molecular weight of 240 kDa and consists of four identical 61-kDa subunits. The isoelectric point of the enzyme was pH 4.8. The optimum temperature for both enzyme reactions was 30°C. The optimum pH ranges for trehalose degradation and synthesis were 6.0–7.5 and 6.0–7.0, respectively. Trehalose degradation was inhibited by ATP and trehalose analogs, whereas the synthetic activity was inhibited by P_i (K_i=2.0 mM). The enzyme was highly specific towards trehalose, P_i, glucose and α-glucose-1-phosphate. The stoichiometry of the reaction between trehalose, P_i, glucose and α-glucose-1-phosphate was 1:1:1:1 (molar ratio). The K_m values were 61, 4.7, 24 and 6.3 mM for trehalose, P_i, glucose and α-glucose-1-phosphate, respectively. Under physiological conditions, A. bisporus trehalose phosphorylase probably performs both synthesis and degradation of trehalose.     

Purification and Characterization of Trehalose Phosphorylase from Micrococcus varians

Trehalose phosphorylase (EC 2.4.1.64), which catalyzes the reversible reaction of phosphorolysis and synthesis of trehalose, was purified to homogeneity from a cell-free extract of Micrococcus varians strain No. 39. The enzyme was shown to have a molecular weight of 570,000 to 580,000 by gel filtration, and to have a subunit of molecular weight of 105,000 by SDS–polyacrylamide gel electrophoresis. The stoichiometry of the reaction between trehalose, Pi, glucose, and β-glucose 1-phosphate was 1: 1: 1: 1 (molar ratio). The enzyme had high specificity for trehalose, glucose, and β-glucose 1-phosphate. The K_ms for trehalose, Pi, glucose, and β-glucose 1-phosphate were 10, 3.1, 23, and 38mM, respectively. The k_cats were 200s^?1 for trehalose phosphorolysis and 660s^?1 for trehalose synthesis. The enzyme was inhibited by validamycin A, validoxylamine A, 1-deoxynojirimycin, and Cu^{2 +} during trehalose phosphorolysis, and by Cu^{2 +}, Zn^{2 +}, and Ni^{2 +} during trehalose synthesis. Inhibition competitive against trehalose was noted with validamycin A, validoxylamide A, and 1-deoxynojirimycin. Initial velocity, product inhibition, and dead-end inhibition studies suggested that both trehalose phosphorolysis and trehalose synthesis proceeded through an ordered Bi Bi mechanism.     

Survey of α-glucose 1-phosphate forming trehalose phosphorylase and trehalase in various fungi including basidiomycetous mushrooms

The distribution of α-glucose 1-phosphate forming (α-type) trehalose phosphorylase and trehalase activities in various fungi was surveyed. α-Type phosphorylase occurred in the mycelia and fruit-bodies of Agaricales and Aphyllophorales in the Holobasidiomycetidae, and at least one species of Gasteromycetes, but not in Tremellaceae or Auriculariales of the Phragmobasidiomycetidae, Heterobasidiomycetes or Hemibasidiomycetes. The test fungi in the Ascomycotina and Deuteromycotina, and the yeasts of Basidiomycotina, showed different trehalase activities, but no trehalose phosphorylase activity. The test organisms showed different levels of trehalase activity. The fruit-bodies of most mushrooms showed higher activities of α-type trehalose phosphorylase than did the mycelia.     

Trehalose phosphorylase from Pichia fermentans and its role in the metabolism of trehalose

During a screening for novel microbial trehalose phosphorylase three Pichia strains were identified as producers of this particular enzyme that have not yet been described. To our knowledge, this is the first time that this enzyme activity has been shown in yeasts. Pichia fermentans formed trehalose phosphorylase when cultivated on a growth medium containing easily metabolizable sugers such as glucose. Addition of NaCl (0.4 M) to the medium increased the synthesis of the enzyme significantly. Production of trehalose phosphorylase was found to be growth-associated with a maximum of activity formed at the transition of the exponential to the stationary phase of growth. Trehalose phosphorylase catalyzes the phosphorolytic cleavage of trehalose, yielding glucose 1-phosphate (glucose-1-P) and glucose as products. In vitro the enzyme readily catalyzes the reverse reaction, the synthesis of trehalose from glucose and glucose-1-P. For this reaction, the enzyme of P. fermentans was found to utilize -glucose-1-P preferentially. A partially purified enzyme preparation showed a pH optimum of 6.3 for the synthesis of trehalose. The enzyme was found to be rather unstable; it was easily inactivated by dilution unless Ca²⁺ or Mn²⁺ were added. This instability is presumably caused by dissociation of the enzyme. In contrast to other yeasts, P. fermentans rapidly degraded intracellularly accumulated trehalose when the carbon source in the medium was depleted. Trehalose phosphorylase seems to be a key enzyme in the degradative pathway of trehalose in P. fermentans. Additional enzymes in this catabolic pathway of trehalose include phosphoglucomutase, glucose-6-phosphate dehydrogenase, and gluconolactonase.This contribution is part of the Ph.D. thesis of Ingrid Schick     

Discovery of nigerose phosphorylase from Clostridium phytofermentans

A novel phosphorylase from Clostridium phytofermentans belonging to the glycoside hydrolase family (GH) 65 (Cphy1874) was characterized. The recombinant Cphy1874 protein produced in Escherichia coli showed phosphorolytic activity on nigerose in the presence of inorganic phosphate, resulting in the release of d-glucose and β-d-glucose 1-phosphate (β-G1P) with the inversion of the anomeric configuration. Kinetic parameters of the phosphorolytic activity on nigerose were k _cat = 67 s⁻¹ and K _m = 1.7 mM. This enzyme did not phosphorolyze substrates for the typical GH65 enzymes such as trehalose, maltose, and trehalose 6-phosphate except for a weak phosphorolytic activity on kojibiose. It showed the highest reverse phosphorolytic activity in the reverse reaction using d-glucose as the acceptor and β-G1P as the donor, and the product was mostly nigerose at the early stage of the reaction. The enzyme also showed reverse phosphorolytic activity, in a decreasing order, on d-xylose, 1,5-anhydro-d-glucitol, d-galactose, and methyl-α-d-glucoside. All major products were α-1,3-glucosyl disaccharides, although the reaction with d-xylose and methyl-α-d-glucoside produced significant amounts of α-1,2-glucosides as by-products. We propose 3-α-d-glucosyl-d-glucose:phosphate β-d-glucosyltransferase as the systematic name and nigerose phosphorylase as the short name for this Cphy1874 protein.     

Evaluation of acceptor selectivity of Lactococcus lactis ssp. lactis trehalose 6-phosphate phosphorylase in the reverse phosphorolysis and synthesis of a new sugar phosphate

Trehalose 6-phosphate phosphorylase (TrePP), a member of glycoside hydrolase family 65, catalyzes the reversible phosphorolysis of trehalose 6-phosphate (Tre6P) with inversion of the anomeric configuration to produce β-d-glucose 1-phosphate (β-Glc1P) and d-glucose 6-phosphate (Glc6P). TrePP in Lactococcus lactis ssp. lactis (LlTrePP) is, alongside the phosphotransferase system, involved in the metabolism of trehalose. In this study, recombinant LlTrePP was produced and characterized. It showed its highest reverse phosphorolytic activity at pH 4.8 and 40°C, and was stable in the pH range 5.0–8.0 and at up to 30°C. Kinetic analyses indicated that reverse phosphorolysis of Tre6P proceeded through a sequential bi bi mechanism involving the formation of a ternary complex of the enzyme, β-Glc1P, and Glc6P. Suitable acceptor substrates were Glc6P, and, at a low level, d-mannose 6-phosphate (Man6P). From β-Glc1P and Man6P, a novel sugar phosphate, α-d-Glcp-(1?1)-α-d-Manp6P, was synthesized with 51% yield.     

Purification and properties of α-glucose 1-phosphate-forming trehalose phosphorylase from a basidiomycete,Pleurotus ostreatus

Pleurotus ostreatus produced a high activity of α-glucose 1-phosphate (α-Glc 1-P) forming trehalose phosphorylase in vegetative mycelia and fruit-bodies. The enzyme was purified to homogeneity from the fruit-bodies by a procedure involving ammonium sulfate fractionation, DEAE-cellulose column chromatographies and cellulose phosphate column chromatographies. The enzyme catalyzes both the phosphorolysis of trehalose to produce α-Glc 1-P and glucose, and the synthesis of trehalose. It was not active toward other α- or β-glucosyl disaccharides and polysaccharides. The optimum pH was 7.0 for phosphorolysis and 6.4 for synthesis of trehalose. The Km values for trehalose and Pi in phospholytic reaction were 75 mM and 4.2 mM, respectively. Those for glucose and α-Glc 1-P in synthetic reaction were 505 mM and 38 mM, respectively. The estimated molecular mass by the sedimentation equilibrium method using an ultracentrifuge was 120 kDa. The molecular mass of the subunit (61 kDa) by SDS-polyacrylamide gel electrophoresis suggested that the enzyme was a dimer of two identical subunits. The addition of glycerol higher than 25% into the enzyme solution stabilized its activity. The removal of phosphorus ions from the enzyme solution, by means of dialysis or electrophoresis, caused inactivation of the enzyme, probably by dissociation of the holoenzyme into the subunit proteins.     

Trehalose Metabolism by Bacillus popilliae

Trehalose was found to be utilized more readily than glucose for the growth of Bacillus popilliae NRRL B-2309MC. The pathway of degradation of trehalose was elucidated and found to differ from that reported for other organisms. Trehalase and trehalose phosphorylase activities could not be detected. Rather, trehalose was found to undergo phosphoenolpyruvate (PEP)-dependent phosphorylation, and the resulting trehalose 6-phosphate was cleaved by a phosphotrehalase to equimolar amounts of glucose and glucose 6-phosphate. The phosphotrehalase was purified 34-fold and shown to have a pH optimum of 6.5 to 7.0 and a K(m) for trehalose 6-phosphate of 1.8 mM. A mutant missing the phosphotrehalase failed to grow on trehalose but grew normally on other sugars. The mutant accumulated [(14)C]trehalose as [(14)C]trehalose 6-phosphate. Phosphorylation of trehalose by dialyzed extracts was at least 25 times faster with PEP than with adenosine 5'-triphosphate, and the phosphorylation activity was associated primarily with the particulate fraction. These data and the results of studies of [(14)C]trehalose uptake suggest that trehalose is transported into the cell as trehalose 6-phosphate by a PEP:sugar phosphotransferase system. Cell extracts of other strains of B. popilliae were also found to produce [(14)C]sugar phosphate from [(14)C]trehalose and to have phosphotrehalase activity.     

Enzymes of alpha,alpha-Trehalose Metabolism in Soybean Nodules

Metabolism of trehalose, α,d-glucopyranosyl-α,d-glucopyranoside, was studied in nodules of Bradyrhizobium japonicum-Glycine max [L.] Merr. cv Beeson 80 symbiosis. The nodule extract was divided into three fractions: bacteroid soluble protein, bacteroid fragments, and cytosol. The bacteroid soluble protein and cytosol fractions were gel-filtered. The key biosynthetic enzyme, trehalose-6-phosphate synthetase, was consistently found only in the bacteroids. Trehalose-6-phosphate phosphatase activity was present both in the bacteroid soluble protein and cytosol fractions. Trehalase, the most abundant catabolic enzyme was present in all three fractions and showed two pH optima: pH 3.8 and 6.6. Two other degradative enzymes, phosphotrehalase, acting on trehalose-6-phosphate forming glucose and glucose-6-phosphate, and trehalose phosphorylase, forming glucose and β-glucose-1-phosphate, were also detected in the bacteroid soluble protein and cytosol fractions. Trehalase was present in large excess over trehalose-6-phosphate synthetase. Trehalose accumulation in the nodules would appear to be predicated on spatial separation of trehalose and trehalase.     

Potassium ion-dependent trehalose phosphorylase from halophilic Bacillus selenitireducens MLS10

We discovered a potassium ion-dependent trehalose phosphorylase (Bsel_1207) belonging to glycoside hydrolase family 65 from halophilic Bacillus selenitireducens MLS10. Under high potassium ion concentrations, the recombinant Bsel_1207 produced in Escherichia coli existed as an active dimeric form that catalyzed the reversible phosphorolysis of trehalose in a typical sequential bi bi mechanism releasing β-d-glucose 1-phosphate and d-glucose. Decreasing potassium ion concentrations significantly reduced thermal and pH stabilities, leading to formation of inactive monomeric Bsel_1207.     

Beta-glucose 1-phosphate-interconverting enzymes in maltose- and trehalose-fermenting lactic acid bacteria

Maltose and trehalose catabolic pathways are linked through their common enzyme, beta-phosphoglucomutase, and metabolite, beta-glucose 1-phosphate, in Lactococcus lactis. Maltose is degraded by the concerted action of maltose phosphorylase and beta-phosphoglucomutase, whereas trehalose is assimilated by a novel pathway, including the recently discovered enzyme, trehalose 6-phosphate phosphorylase, and beta-phosphoglucomutase. In the present study, 40 strains of lactic acid bacteria were investigated for utilization of metabolic reactions involving beta-glucose 1-phosphate. All genera of the low G+C content lactic acid bacteria belonging to the clostridial subbranch of Gram-positive bacteria were represented in the study. The strains, which fermented maltose or trehalose, were investigated for beta-phosphoglucomutase, maltose phosphorylase and trehalose 6-phosphate phosphorylase activity, as indications of maltose and trehalose catabolic pathways involving beta-glucose 1-phosphate interconversions. Eighty per cent of all strains fermented maltose and, of these strains, 63% were shown to use a maltose phosphorylase/beta- phosphoglucomutase pathway. One-third of the strains fermenting trehalose were found to harbour trehalose 6-phosphate phosphorylase activity, and these were also shown to possess beta-phosphoglucomutase activity. Mainly L. lactis and Enterococcus faecalis strains were found to harbour the novel trehalose 6-phosphate phosphorylase/beta-phosphoglucomutase pathway. As lower beta-glucose 1-phosphate interconverting enzyme activities were observed in the majority of glucose-cultivated lactic acid bacteria, glucose was suggested to repress the synthesis of these enzymes in most strains. Thus, metabolic reactions involving the beta-anomer of glucose 1-phosphate are frequently found in both maltose- and trehalose-utilizing lactic acid bacteria.     

Cloning and characterization of a gene encoding trehalose phosphorylase (TP) from Pleurotus sajor-caju

Complementary DNA for a gene encoding trehalose phosphorylase (TP) that reversibly catalyzes trehalose synthesis and degradation from alpha-glucose-1-phosphate (alpha-Glc-1-P) and glucose was cloned from Pleurotus sajor-caju. The cDNA of P. sajor-caju TP (designated PsTP, GenBank Accession No. AF149777) encodes a polypeptide of 751 amino acids with a deduced molecular mass of 83.7 kDa. The PsTP gene is expressed in mycelia, pilei, and stipes of fruiting bodies. Trehalose phosphorylase PsTP was purified from PsTP-transformed Escherichia coli. The enzyme catalyzes both the phosphorolysis of trehalose to produce alpha-Glc-1-P and glucose, and the synthesis of trehalose. The apparent K(m) values for trehalose and Pi in phosphorolytic reaction at pH 7.0 were 74.8 and 5.4 mM, respectively. The PsTP gene complemented Saccharomyces cerevisiae Deltatps1, Deltatps2 double-mutant cells, allowing their growth on glucose medium. Furthermore, yeast transformed with PsTP produced 2-2.5-fold more trehalose than non-transformants or cells transformed with empty vector only.     

Extensive expression regulation and lack of heterologous enzymatic activity of the Class II trehalose metabolism proteins from Arabidopsis thaliana

Trehalose metabolism has profound effects on plant growth and metabolism, but the mechanisms involved are unclear. In Arabidopsis , 21 putative trehalose biosynthesis genes are classified in three subfamilies based on their similarity with yeast TPS1 (encoding a trehalose-6-phosphate synthase, TPS) or TPS2 (encoding a trehalose-6-phosphate phosphatase, TPP). Although TPS1 (Class I) and TPPA and TPPB (Class III) proteins have established TPS and TPP activity, respectively, the function of the Class II proteins (AtTPS5-AtTPS11) remains elusive. A complete set of promoter- β -glucurinidase/green fluorescent protein reporters demonstrates their remarkably differential tissue-specific expression and responsiveness to carbon availability and hormones. Heterologous expression in yeast furthermore suggests that none of the encoded enzymes displays significant TPS or TPP activity, consistent with a regulatory rather than metabolic function for this remarkable class of proteins.     

Trehalose phosphorylase from Pleurotus ostreatus: characterization and stabilization by covalent modification, and application for the synthesis of alpha,alpha-trehalose

Trehalose phosphorylase from the basidiomycete Pleurotus ostreatus (PoTPase) was isolated from fungal fruit bodies through approximately 500-fold purification with a yield of 44%. Combined analyses by SDS-PAGE and gelfiltration show that PoTPase is a functional monomer of approximately 55 kDa molecular mass. PoTPase catalyzes the phosphorolysis of alpha,alpha-trehalose, yielding alpha-d-glucose 1-phosphate (alphaGlc 1-P) and alpha-d-glucose as the products. The optimum pH of PoTPase for alpha,alpha-trehalose phosphorolysis and synthesis is 6.8 and 6.2, respectively. Apparent substrate binding affinities (K(m)) were determined at pH 6.8 and 30 degrees C: alpha,alpha-trehalose (79 mM); phosphate (3.5 mM); d-glucose (40 mM); alphaGlc 1-P (4.1mM). A series of structural analogues of d-glucose were tested as glucosyl acceptors for the enzymatic reaction with alphaGlc 1-P, and robust activity with d-mannose (3%), 2-deoxy d-glucose (8%), 2-fluoro d-glucose (15%) and 2-keto-d-glucose (50%) was detected. Arsenate replaces, with 30% relative activity, phosphate in the conversion of alpha,alpha-trehalose, and vanadate strongly inhibits the enzyme activity (K(i) approximately 4 microM). PoTPase has a half-life (t(0.5)) of approximately 1 h at 30 degrees C in the absence of stabilizing compounds such as alpha,alpha-trehalose (300 mM; t(0.5)=11.5 h), glycerol (20%, w/v; t(0.5)=6.5h) or polyethylenglycol (PEG) 4000 (26%, w/v; t(0.5)=70 h). Covalent modification of PoTPase with activated derivatives of PEG 5000 increases the stability by up to 600-fold. Sucrose was converted to alpha,alpha-trehalose in approximately 60% yield using a coupled enzyme system composed of sucrose phosphorylase from Leuconostoc mesenteroides, glucose isomerase from Streptomyces murinus and the appropriately stabilized PoTPase.     

Involvement of the compatible solutes trehalose and sucrose in the response to salt stress of a cyanobacterial Scytonema species isolated from desert soils

The response to moderate salt stress of a Scytonema species isolated from a soil crust in the arid region of central Australia was studied. An increase in intracellular trehalose and sucrose concentrations was detected by NMR and HPLC analysis following salt stress, maximal amounts being produced by exposure to 150 mM NaCl after 48 h. When the organism was subsequently returned to normal growth conditions, the cellular concentrations of these solutes decreased. The biosynthesis of trehalose and sucrose was studied and found, in both cases, to involve both sugar phosphate synthase and phosphatase enzymes. The combined synthase activities and the individual phosphatase activities in cell extracts were increased by salt stress. Trehalose phosphorylase was the only catabolic enzyme detected for trehalose; neither trehalase nor phosphotrehalase activities could be detected. This is the first report of trehalose phosphorylase activity in cyanobacteria. Both trehalose and sucrose phosphorylase activities increased in salt-stressed cells, whereas the activity of invertase did not change.     

Production of trehalose synthase from a basidiomycete, Grifola frondosa, in Escherichia coli

The genomic DNA and cDNA for a gene encoding a novel trehalose synthase (TSase) catalyzing trehalose synthesis from α-d-glucose 1-phosphate and d-glucose were cloned from a basidiomycete, Grifola frondosa. Nucleotide sequencing showed that the 732-amino-acid TSase-encoding region was separated by eight introns. Consistent with the novelty of TSase, there were no homologous proteins registered in the databases. Recombinant TSase with a histidine tag at the NH₂-terminal end, produced in Escherichia coli, showed enzyme activity similar to that purified from the original G. frondosa strain. Incubation of α-d-glucose 1-phosphate and d-glucose in the presence of recombinant TSase generated trehalose, in agreement with the enzymatic property of TSase that the equilibrium lay far in the direction of trehalose synthesis. Received: 12 January 1998 / Received revision: 20 February 1998 / Accepted: 20 March 1998