Description:
<jats:p>α-D-Glucuronidases cleave the α-1,2-glycosidic bond of the 4-<jats:italic>O</jats:italic>-methyl-α-D-glucuronic acid side chain in xylan. Of the xylan-debranching hydrolases, these enzymes are the least studied and characterized. The α-glucuronidase gene (<jats:italic>aguA</jats:italic>) from <jats:italic>Bacillus stearothermophilus</jats:italic> T-6 has been cloned, sequenced and overproduced in <jats:italic>Escherichia coli</jats:italic>. The gene encodes for a protein of 679 amino acids with a calculated molecular weight of 78480 and a p<jats:italic>I</jats:italic> of 5.42. α-Glucuronidase T-6 shows high homology to the α-glucuronidases of <jats:italic>Thermotoga maritima</jats:italic> (60% identity) and of <jats:italic>Trichoderma reesei</jats:italic> (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 α-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 α-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 <jats:italic>P</jats:italic>4<jats:sub>1</jats:sub>2<jats:sub>1</jats:sub>2 or <jats:italic>P</jats:italic>4<jats:sub>3</jats:sub>2<jats:sub>1</jats:sub>2) with unit-cell dimensions <jats:italic>a</jats:italic> = <jats:italic>b</jats:italic> = 76.1 and <jats:italic>c</jats:italic> = 331.2 Å. These crystals are mechanically strong, are stable in the X-ray beam and diffract X-rays to better than 2.4 Å resolution. A full 3.0 Å resolution diffraction data set (97.3% completeness, <jats:italic>R</jats:italic>
<jats:sub>merge</jats:sub> 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 <jats:italic>P</jats:italic>2<jats:sub>1</jats:sub>) with unit-cell dimensions <jats:italic>a</jats:italic> = 65.8, <jats:italic>b</jats:italic> = 127.4, <jats:italic>c</jats:italic> = 96.6 Å and β = 97.9°. These crystals are also quite strong and stable, and diffract to better than 2.8 Å resolution. A full 2.8 Å resolution diffraction data set (96.2% completeness, <jats:italic>R</jats:italic>
<jats:sub>merge</jats:sub> 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 <jats:italic>via</jats:italic> isomorphous heavy-atom derivatives and selenomethionine MAD experiments.</jats:p>