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

alpha-D-Glucuronidases cleave the alpha-1,2-glycosidic bond of the 4-O-meth yl-alpha-D-glucuronic acid side chain in xylan. Of the xylan-debranching hy drolases, these enzymes are the feast studied and characterized. The alpha- glucuronidase gene (aguA) from Bacillus stearothermophilus T-6 has been clo ned, 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-glu curonidases 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. Crystallog raphic studies of alpha-glucuronidase T-6 were initiated to study the mecha nism of catalysis, as well as to provide a structural basis for rational in troduction of enhanced thermostability by site-specific mutagenesis. In thi s report, the crystallization and preliminary crystallographic characteriza tion 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 precip itant and 2-propanol as an organic additive. The crystals belong to a primi tive 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 ar e 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 diffrac tion data set (97.3% completeness, R-merge 9.8%) has recently been collecte d on one crystal at room temperature using a rotating-anode X-ray source an d an R-AXIS IIc imaging-plate detector. The M1 form was obtained and charac terized by similar techniques. The best crystallization occurred at a sligh tly lower pH and a lower concentration of 2-propanol. The crystals belong t o a primitive monoclinic crystal system (space group PZ,) with unit-cell di mensions a = 65.8, b = 127.4, c = 96.6 Angstrom and beta = 97.9 degrees. Th ese 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 d erivatives and selenomethionine MAD experiments.