Crystallization and preliminary X-ray analysis of α-D-glucuronidase from Bacillus stearothermophilus T-6

alpha-D-Glucuronidases cleave the alpha-1,2-glycosidic bond of the 4-O-methyl-alpha-D-glucuronic acid side chain in xylan. Of the xylan-debranching hydrolases, these enzymes are the feast studied and characterized. The alpha-glucuronidase gene (aguA) from Bacillus stearothermophilus T-6 has been cloned, sequenced and overproduced in Escherichia coli. The gene encodes for a protein of 679 amino acids with a calculated molecular weight of 78480 and a pr of 5.42. alpha-Glucuronidase T-6 shows high homology to the alpha-glucuronidases of Thermotoga maritima (60% identity) and of Trichoderma reesei (44% identity). Based on the amino-acid sequence similarity, it is likely that these enzymes represent a new class of glycosyl hydrolases. Crystallographic studies of alpha-glucuronidase T-6 were initiated to study the mechanism of catalysis, as well as to provide a structural basis for rational introduction of enhanced thermostability by site-specific mutagenesis. In this report, the crystallization and preliminary crystallographic characterization of the native alpha-glucuronidase T-6 enzyme is described. Two crystal forms were found suitable for detailed crystal structure analysis. The T1 form was obtained by the vapour-diffusion method using PEG 4000 as a precipitant and 2-propanol as an organic additive. The crystals belong to a primitive tetragonal crystal system (space group P4(1)2(1)2 or P4(3)2(1)2) with unit-cell dimensions a = b = 76.1 and c = 331.2 Angstrom. These crystals are mechanically strong, are stable in the X-ray beam and diffract X-rays to better than 2.4 Angstrom resolution. A full 3.0 Angstrom resolution diffraction data set (97.3% completeness, R-merge 9.8%) has recently been collected on one crystal at room temperature using a rotating-anode X-ray source and an R-AXIS IIc imaging-plate detector. The M1 form was obtained and characterized by similar techniques. The best crystallization occurred at a slightly lower pH and a lower concentration of 2-propanol. The crystals belong to a primitive monoclinic crystal system (space group PZ,) with unit-cell dimensions a = 65.8, b = 127.4, c = 96.6 Angstrom and beta = 97.9 degrees. These crystals are also quite strong and stable, and diffract to better than 2.8 Angstrom resolution. A full 2.8 A resolution diffraction data set (96.2% completeness, R-merge 7.6%) has recently been collected on one crystal at room temperature using the same R-AXIS IIc setup. Both forms are currently being used to obtain crystallographic phasing via isomorphous heavy-atom derivatives and selenomethionine MAD experiments.