-d-Glucuronidases cleave the -1,2-glycosidic bond of the 4-Omethyl--d-glucuronic acid side chain in xylan. Of the xylandebranching hydrolases, these enzymes are the least studied and characterized. The -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 78 480 and a pI of 5.42. -Glucuronidase T-6 shows high homology to the -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 -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-speci®c mutagenesis. In this report, the crystallization and preliminary crystallographic characterization of the native -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 A Ê . These crystals are mechanically strong, are stable in the X-ray beam and diffract X-rays to better than 2.4 A Ê resolution. A full 3.0 A Ê 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 imagingplate 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 P2 1 ) with unit-cell dimensions a = 65. 8, b = 127.4, c = 96.6 A Ê and = 97.9 . These crystals are also quite strong and stable, and diffract to better than 2.8 A Ê resolution. A full 2.8 A Ê 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.

Xylanases (1,4-beta-D-xylan xylanhydrolases; E.C. 3.2.1.8) hydrolyze the 1,4-beta-D-xylopyranosyl linkage of xylans. The structural characterization of xylanase active sites is of great interest, since it can lead to a better understanding of their catalytic mechanism and contribute significant knowledge to the rational design of specific oligosaccharide-binding sites via protein engineering. An intracellular xylanase gene (xynA2) from Bacillus stearothermophilus T-6 has recently been cloned and sequenced. The xynA2 gene encodes for an intracellular enzyme (IXT6) of 331 amino acids, with a calculated molecular weight of 38 639 Da and a pI of 5.72. Based on sequence homology, the enzyme belongs to family 10 of the glycosyl hydrolases. The xynA2 gene product (IXT6) was overproduced in Escherichia coli and purified to homogeneity. Crystallographic studies of IXT6 were initiated in order to study the specificity and mechanism of catalysis of this unique xylanase, as well as to provide a structural basis for rational introduction of enhanced thermostability by site-specific mutagenesis. The M1 crystal form was found to be the most suitable for detailed crystal structure analysis. These crystals belong to a C--centered monoclinic crystal system (space group C2) with unit-cell parameters a = 170.6, b = 82.5, c = 80.0 A, beta = 91.43 degrees. They are mechanically strong, are fairly stable in the X-ray beam and diffract X--rays to better than 2.5 A resolution. A full 2.9 A resolution diffraction data set (97.9% completeness, R(merge) = 8.4%) has recently been collected from one crystal at room temperature using X-ray synchrotron radiation (lambda = 1.125 A) and a MAR300 imaging-plate area detector. A comparable 2.5 A data set was measured at 90 K using a rotating-anode X-ray source and an R-AXIS IIc imaging-plate area detector (97.2% completeness, R(merge) = 6.9%). Molecular-replacement studies and multiple anomalous dispersion (MAD) experiments are currently in progress in order to determine the detailed three-dimensional structure of IXT6.

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