title: Crystallization and X-ray diffraction studies of inverting trehalose phosphorylase from Thermoanaerobacter sp.
abstract: 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-alpha(1,1)alpha-D-glucopyranose] to beta-D-glucose 1-phosphate and D-glucose. Recombinant purified protein was produced in Escherichia coli and crystallized in space group P2(1)2(1)2(1). Crystals of recombinant TP were obtained in their native form and were soaked with glucose, with n-octyl-beta-D-glucoside and with trehalose. The crystals presented a number of challenges including an unusually large unit cell, with a c axis measuring 420 A, and variable diffraction quality. Crystal-dehydration protocols led to improvements in diffraction quality that were often dramatic, typically from 7-8 to 3-4 A resolution. The structure of recombinant TP was determined by molecular replacement to 2.8 A 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.
authors.affiliation: Unit for Structural Biology and Biophysics, Laboratory for Protein Biochemistry and Biomolecular Engineering, K. L. Ledeganckstraat 35, Ghent University, B-9000 Ghent, Belgium.
authors.forename: Annelies
authors.initials: A
authors.lastname: Van Hoorebeke
authors.forename: Jan
authors.initials: J
authors.lastname: Stout
authors.forename: Ruben
authors.initials: R
authors.lastname: Van der Meeren
authors.forename: John
authors.initials: J
authors.lastname: Kyndt
authors.forename: Jozef
authors.initials: J
authors.lastname: Van Beeumen
authors.forename: Savvas N
authors.initials: SN
authors.lastname: Savvides
mesh_terms: D003460:Crystallization; D018360:Crystallography, X-Ray; D005964:Glucosyltransferases / Q000737:chemistry*; D008958:Models, Molecular; D020836:Protein Structure, Quaternary; D046968:Thermoanaerobacter / Q000201:enzymology*
chemical_list: D005964:Glucosyltransferases; C023181:alpha,alpha-trehalose phosphorylase
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