Xylanases (1,4-β-D-xylan xylanhydrolases; EC 18.104.22.168) hydrolyze the 1,4-β-D-xylopyranosyl linkage of xylans. The detailed structural characterization of these enzymes is of interest for the elucidation of their catalytic mechanism and for their rational modification toward improved stability and specificity. An extracellular xylanase from Geobacillus stearothermophilus T-6 (XT6) has recently been cloned, overexpressed, purified and biochemically characterized. Previous crystallographic efforts resulted in a hexagonal crystal form, which subsequently proved to be of limited use for structural analysis, mainly because of its relatively poor diffraction quality and resolution. A systematic search for more suitable crystals of XT6 recently resulted in a new crystal form of this enzyme with significantly improved diffraction characteristics. The new crystals belong to a C-centred monoclinic crystal system (space group C2), with unit-cell parameters a = 121.5, b = 61.7, c = 89.1 Å, β = 119.7°. These crystals diffract X-rays to better than 1.5 Å resolution, showing a very clear diffraction pattern of relatively high quality. The crystals are mechanically strong and exhibit excellent radiation-stability when frozen under cold nitrogen gas. A full diffraction data set to 1.45 Å resolution (94.1% completeness, R merge = 7.0%) has been collected from flash-frozen crystals of the native enzyme at 95 K using synchrotron radiation. Crystals of the E159A/E265A catalytic double mutant of XT6 were found to be isomorphous to those of native XT6. They were used for a full measurement of 1.8 Å resolution diffraction data at 100 K (90.9% completeness; Rmerge = 5.0%). These data are currently being used for the high-resolution structure determination of XT6 and its mutant for mechanistic interpretations and rational introduction of thermostability.
|Original language||American English|
|Number of pages||5|
|Journal||Acta Crystallographica Section D: Biological Crystallography|
|State||Published - Mar 2004|