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.

Figure 1

Recombinant TP crystal grown from 10%(v/v) PEG 4000, 0.1 M Tris–HCl pH 7.5, 1% 2-propanol.

Figure 2

(a) Diffraction image from a typical TP crystal without dehydration. (b) Diffraction image from the TP_mut crystal, which was dehydrated according to the first method described in §2.5.

Figure 3

C^α-atom traces and packing of TP in the primitive orthorhombic cell. Subunits in dimers making up the asymmetric unit of each lattice are shown in blue and cyan and in pink and magenta, while symmetry-related molecules are shown in greyscale. This figure was prepared with the program PyMOL (DeLano, 2002 ▶).