Characterization of trehalose phosphorylase from Schizophyllum commune

The Cori ester ␣-D-glucose 1-phosphate (␣Glc 1-P) is a high-energy intermediate of cellular carbohydrate metabolism. Its glycosidic phosphomonoester moiety primes ␣Glc 1-P for flexible exploitation in glucosyl and phosphoryl transfer reactions. Two structurally and mechanistically distinct sugar-phosphate phosphatases from Escherichia coli were characterized in this study for utilization of ␣Glc 1-P as a phosphoryl donor substrate. The agp gene encodes a periplasmic ␣Glc 1-P phosphatase (Agp) belonging to the histidine acid phosphatase family. Had13 is from the haloacid dehydrogenase-like phosphatase family. Cytoplasmic expression of Agp (in E. coli Origami B) gave a functional enzyme preparation (k cat for phosphoryl transfer from ␣Glc 1-P to water, 40 s ؊1 ) that was shown by mass spectrometry to exhibit no free cysteines and the native intramolecular disulfide bond between Cys 189 and Cys 195 . Enzymatic phosphoryl transfer from ␣Glc 1-P to water in H 2 18 O solvent proceeded with complete 18 O label incorporation into the phosphate released, consistent with catalytic reaction through O-1-P, but not C-1-O, bond cleavage. Hydrolase activity of both enzymes was not restricted to a glycosidic phosphomonoester substrate, and D-glucose 6-phosphate was converted with a k cat similar to that of ␣Glc 1-P. By examining phosphoryl transfer from ␣Glc 1-P to an acceptor substrate other than water (D-fructose or D-glucose), we discovered that Agp exhibited pronounced synthetic activity, unlike Had13, which utilized ␣Glc 1-P mainly for phosphoryl transfer to water. By applying D-fructose in 10-fold molar excess over ␣Glc 1-P (20 mM), enzymatic conversion furnished D-fructose 1-phosphate as the main product in a 55% overall yield. Agp is a promising biocatalyst for use in transphosphorylation from ␣Glc 1-P. Phosphorylation of sugar substrates is a common biochemical transformation of central importance to cellular metabolism (1-3). It usually involves phosphoryl transfer from a phosphoactivated donor substrate, such as ATP, to an acceptor group, typically a hydroxyl, on the sugar backbone (4-7). Various phosphotransferases (EC 2.7) catalyze sugar phosphorylation (8-12). In an alternative reaction catalyzed by glycoside phosphorylases (EC 2.4), where phosphorylation occurs exclusively at the sugar's anomeric position, a glycosyl residue is transferred from a sugar donor substrate to phosphate (Fig. 1A) (13, 14). The phosphomonoester moiety attached to sugars is a key element of biological recognition, across all steps of glycolysis, for example, and it serves to prime sugars for further conversion in different biochemical pathways (15-18). It is known from intracellular-metabolite-profiling studies that changes in concentrations of common sugar phosphates (e.g., D-glucose 6-phosphate [Glc 6-P] and D-fructose 6-phosphate [Fru 6-P]) are often linked to major alterations in cellular physiology (19)(20)(21)(22). Due to the requirement for authentic reference material in different biological investigations, there is considerable ...

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“…␣Glc 1-P and Glc 6-P were measured in a coupled enzymatic system with phosphoglucomutase and Glc 6-P dehydrogenase (59). Fru 6-P was …”

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confidence: 99%

Phosphoryl Transfer from α-d-Glucose 1-Phosphate Catalyzed by Escherichia coli Sugar-Phosphate Phosphatases of Two Protein Superfamily Types

Wildberger

Pfeiffer

Brecker

et al. 2015

Appl Environ Microbiol

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“…Unless otherwise noted, all materials used have been described elsewhere [17,43]. Purified preparations of wildtype ScTPase as well as R507A and K512A mutants thereof were obtained using previously reported procedures [16].…”

Section: Materials and Enzymesmentioning

confidence: 99%

“…Initial rates of phosphorolysis of a,a-trehalose were recorded using a reported discontinuous assay [17,43] where the formation of G1P was measured. The concentration of G1P was determined as NADH produced in a second coupled enzymatic reaction catalyzed by phosphoglucomutase and glucose 6-phosphate dehydrogenase.…”

Section: Steady-state Kinetic Characterizationmentioning

confidence: 99%

The phosphate site of trehalose phosphorylase from Schizophyllum commune probed by site-directed mutagenesis and chemical rescue studies

Goedl¹,

Nidetzky²

2008

FEBS Journal

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Glycosyltransferases (GTs) constitute a diverse class of enzymes that catalyze the synthesis of glycosidic bonds in oligosaccharides and glycoconjugates. A nucleotide-, phospho-or lipid-phospho-activated sugar is typically utilized as the donor substrate, and transfer of the glycosyl moiety to the acceptor molecule occurs with either inversion or retention of configuration at the reactive anomeric carbon [1]. After detailed studies of glycogen phosphorylase spanning many decades [2-6], there has been recent rekindled interest in the mechanistic characterization of retaining glycosyltransferases, particularly in relation to glycoside hydrolases, the physiological counterpart enzymes that catalyze the breakdown of glycosidic linkages [7]. The canonicalSchizophyllum commune a,a-trehalose phosphorylase utilizes a glycosyltransferase-like catalytic mechanism to convert its disaccharide substrate into a-d-glucose 1-phosphate and a-d-glucose. Recruitment of phosphate by the free enzyme induces a,a-trehalose binding recognition and promotes the catalytic steps. Like the structurally related glycogen phosphorylase and other retaining glycosyltransferases of fold family GT-B, the trehalose phosphorylase contains an Arg507-XXXX-Lys512 consensus motif (where X is any amino acid) comprising key residues of its putative phosphatebinding sub-site. Loss of wild-type catalytic efficiency for reaction with phosphate (k cat ⁄ K m = 21 000 m )1 AEs )1 ) was dramatic ( ‡10 7 -fold) in purified Arg507 fi Ala (R507A) and Lys512 fi Ala (K512A) enzymes, reflecting a corresponding change of comparable magnitude in k cat (Arg507) and K m (Lys512). External amine and guanidine derivatives selectively enhanced the activity of the K512A mutant and the R507A mutant respectively. Analysis of the pH dependence of chemical rescue of the K512A mutant by propargylamine suggested that unprotonated amine in combination with H 2 PO 4 ) , the protonic form of phosphate presumably utilized in enzymatic catalysis, caused restoration of activity. Transition state-like inhibition of the wild-type enzyme A by vanadate in combination with a,a-trehalose (K i = 0.4 lm) was completely disrupted in the R507A mutant but only weakened in the K512A mutant (K i = 300 lm). Phosphate (50 mm) enhanced the basal hydrolase activity of the K512A mutant toward a,a-trehalose by 60% but caused its total suppression in wild-type and R507A enzymes. The results portray differential roles for the side chains of Lys512 and Arg507 in trehalose phosphorylase catalysis, reactant state binding of phosphate and selective stabilization of the transition state respectively.Abbreviations G1P, a-D-glucose 1-phosphate; GTs, glycosyltransferases; K512A, Lys512 fi Ala mutant; R507A, Arg507 fi Ala mutant; ScTPase, Schizophyllum commune trehalose phosphorylase; S N i-like, internal return-like mechanism.

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“…In particular, trehalose phosphorylase (TP) produces trehalose by glucose-1-phosphate and glucose in various microorganisms including green algae (Belocopitow and Mare´chal, 1970;Mare´chal and Belocopitow, 1972), yeast, Pichia fermentans (Schiek et al, 1995), and various fungi (Kitamoto et al, 1988;Aisaka and Masuda, 1995;Saito et al, 1998;Eis and Nidetzky, 1999).…”

Section: Introductionmentioning

confidence: 99%

Genetic engineering of drought-resistant tobacco plants by introducing the trehalose phosphorylase (TP) gene from Pleurotus sajor-caju

Han

Park

Kwon

et al. 2005

Plant Cell Tiss Organ Cult

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This study generated transgenic tobacco plants expressing trehalose phosphorylase of Pleurotus sajor-caju (PsTP) constitutively under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Sixteen transgenic lines were selected by genomic Southern blot analysis for further study. Unlike yeast TPS1-transformed or Escherichia coli TPS1-transformed tobacco or potato, all of the PsTP transgenic tobacco lines showed normal growth phenotypes both in the culture tubes and soil mixture. The study measured the trehalose contents of PsTP-transformed tobacco plants as well as the wild type and empty vector-transformed control plants. Results showed that the PsTP transformant contained 6.3 lmol g )1 of plant tissues, while the wild type and the control plants had only minimal levels of trehalose. Because this study detected a significant amount of trehalose in PsTP transgenic tobacco plants, it decided to carry out a bioanalysis of the PsTP transgenic tobacco plants by drought treatment by not watering the plants for over 10 days. A significant difference in drought resistance was observed from the second nonwatering day between the transgenic and the control tobacco plants. The transgenic tobacco plants had normal growth and did not wither, while the wild type and the only empty vector-transformed control plants withered severely. Among all the transgenic lines, line 10-4 showed the strongest resistance to drought stress. It did not wither even after 10 days without watering. In addition, when the drought resistance of PsTP transgenic tobacco plants was tested using detached leaves, most transgenic plants, except one line, showed better capacity to retain water than the empty vector-transformed transgenic plant.

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Characterization of trehalose phosphorylase from Schizophyllum commune

Cited by 48 publications

References 16 publications

Phosphoryl Transfer from α-d-Glucose 1-Phosphate Catalyzed by Escherichia coli Sugar-Phosphate Phosphatases of Two Protein Superfamily Types

Phosphoryl Transfer from α-d-Glucose 1-Phosphate Catalyzed by Escherichia coli Sugar-Phosphate Phosphatases of Two Protein Superfamily Types

The phosphate site of trehalose phosphorylase from Schizophyllum commune probed by site-directed mutagenesis and chemical rescue studies

Genetic engineering of drought-resistant tobacco plants by introducing the trehalose phosphorylase (TP) gene from Pleurotus sajor-caju

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