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Trehalose Phosphorylase (trehalose + phosphorylase)

Distribution by Scientific Domains
Chemistry83%

Selected Abstracts

Crystallization and X-ray diffraction studies of inverting trehalose phosphorylase from Thermoanaerobacter sp.

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2010
Annelies Van Hoorebeke
Disaccharide phosphorylases are attractive enzymatic platforms for tailor-made sugar synthesis owing to their ability to catalyze both the synthesis and the breakdown of disaccharides. Trehalose phosphorylase from Thermoanaerobacter sp. (TP) is a glycoside hydrolase family 65 enzyme which catalyzes the reversible breakdown of trehalose [d -glucopyranosyl-,(1,1),- d -glucopyranose] to ,- d -glucose 1-phosphate and d -glucose. Recombinant purified protein was produced in Escherichia coli and crystallized in space group P212121. Crystals of recombinant TP were obtained in their native form and were soaked with glucose, with n -octyl-,- d -glucoside and with trehalose. The crystals presented a number of challenges including an unusually large unit cell, with a c axis measuring 420,Å, and variable diffraction quality. Crystal-dehydration protocols led to improvements in diffraction quality that were often dramatic, typically from 7,8 to 3,4,Å resolution. The structure of recombinant TP was determined by molecular replacement to 2.8,Å resolution, thus establishing a starting point for investigating the structural and mechanistic determinants of the disaccharide phosphorylase activity. To the best of our knowledge, this is the first crystal structure determination of an inverting trehalose phosphorylase. [source]

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

FEBS JOURNAL, Issue 5 2008
Christiane Goedl
Schizophyllum commune,,,-trehalose phosphorylase utilizes a glycosyltransferase-like catalytic mechanism to convert its disaccharide substrate into ,- d -glucose 1-phosphate and ,- d -glucose. Recruitment of phosphate by the free enzyme induces ,,,-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 phosphate-binding sub-site. Loss of wild-type catalytic efficiency for reaction with phosphate (kcat/Km = 21 000 m,1·s,1) was dramatic (,107 -fold) in purified Arg507,Ala (R507A) and Lys512,Ala (K512A) enzymes, reflecting a corresponding change of comparable magnitude in kcat (Arg507) and Km (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 H2PO4,, 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 ,,,-trehalose (Ki = 0.4 ,m) was completely disrupted in the R507A mutant but only weakened in the K512A mutant (Ki = 300 ,m). Phosphate (50 mm) enhanced the basal hydrolase activity of the K512A mutant toward ,,,-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. [source]

Crystallization and X-ray diffraction studies of inverting trehalose phosphorylase from Thermoanaerobacter sp.

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2010
Annelies Van Hoorebeke
Disaccharide phosphorylases are attractive enzymatic platforms for tailor-made sugar synthesis owing to their ability to catalyze both the synthesis and the breakdown of disaccharides. Trehalose phosphorylase from Thermoanaerobacter sp. (TP) is a glycoside hydrolase family 65 enzyme which catalyzes the reversible breakdown of trehalose [d -glucopyranosyl-,(1,1),- d -glucopyranose] to ,- d -glucose 1-phosphate and d -glucose. Recombinant purified protein was produced in Escherichia coli and crystallized in space group P212121. Crystals of recombinant TP were obtained in their native form and were soaked with glucose, with n -octyl-,- d -glucoside and with trehalose. The crystals presented a number of challenges including an unusually large unit cell, with a c axis measuring 420,Å, and variable diffraction quality. Crystal-dehydration protocols led to improvements in diffraction quality that were often dramatic, typically from 7,8 to 3,4,Å resolution. The structure of recombinant TP was determined by molecular replacement to 2.8,Å resolution, thus establishing a starting point for investigating the structural and mechanistic determinants of the disaccharide phosphorylase activity. To the best of our knowledge, this is the first crystal structure determination of an inverting trehalose phosphorylase. [source]

Enzymatic production of ,- D -glucose-1-phosphate from trehalose

BIOTECHNOLOGY JOURNAL, Issue 9 2010
Jef Van der Borght
Abstract ,- D -Glucose-1-phosphate (,Glc1P) is an efficient glucosyl donor for both enzymatic and chemical glycosylation reactions but is currently very costly and not available in large amounts. This article provides an efficient production method of ,Glc1P from trehalose and phosphate using the thermostable trehalose phosphorylase from Thermoanaerobacter brockii. At the process temperature of 60°C, Escherichia coli expression host cells are lysed and cell treatment prior to the reaction is, therefore, not required. In this way, the theoretical maximum yield of 26% could be easily achieved. Two different purification strategies have been compared, anion exchange chromatography or carbohydrate removal by treatment with trehalase and yeast, followed by chemical phosphate precipitation. In a next step, ,Glc1P was precipitated with ethanol but this did not induce crystallization, in contrast to what is observed with other glycosylphosphates. After conversion of the product to its cyclohexylammonium salt, however, crystals could be readily obtained. Although both purification methods were quantitative (>99% recovery), a large amount of product (50%) was lost during crystallization. Nevertheless, a production process for crystalline ,Glc1P is now available from the cheap substrates trehalose and inorganic phosphate. [source]