Preliminary crystallographic analysis of Xyn52B2, a GH52 β-D-xylosidase from Geobacillus stearothermophilus T6

Abstract

Geobacillus stearothermophilus T6 is a thermophilic bacterium that possesses an extensive hemicellulolytic system, including over 40 specific genes that are dedicated to this purpose. For the utilization of xylan, the bacterium uses an extracellular xylanase which degrades xylan to decorated xylo-oligomers that are imported into the cell. These oligomers are hydrolyzed by side-chain-cleaving enzymes such as arabinofuranosidases, acetylesterases and a glucuronidase, and finally by an intracellular xylanase and a number of β-xylosidases. One of these β-xylosidases is Xyn52B2, a GH52 enzyme that has already proved to be useful for various glycosynthesis applications. In addition to its demonstrated glycosynthase properties, interest in the structural aspects of Xyn52B2 stems from its special glycoside hydrolase family, GH52, the structures and mechanisms of which are only starting to be resolved. Here, the cloning, overexpression, purification and crystallization of Xyn52B2 are reported. The most suitable crystal form that has been obtained belonged to the orthorhombic P212121 space group, with average unit-cell parameters a = 97.7, b = 119.1, c = 242.3 Å. Several X-ray diffraction data sets have been collected from flash-cooled crystals of this form, including the wild-type enzyme (3.70 Å resolution), the E335G catalytic mutant (2.95 Å resolution), a potential mercury derivative (2.15 Å resolution) and a selenomethionine derivative (3.90 Å resolution). These data are currently being used for detailed three-dimensional structure determination of the Xyn52B2 protein.

Keywords: GH52; Geobacillus stearothermophilus; SAD data collection; enzymatic glycosynthesis; glycoside hydrolase; glycosynthase; selenomethionine; xylan utilization; xylobiose; xylose; xylosidase.

Figure 1

Typical crystals of Xyn52B2-E335G. (a) A complete 5 µl crystallization drop from a typical hanging-drop experiment, showing fully grown crystals of the ESF1 crystal form. The length of the observed crystals is in the range 0.05–0.3 mm. (b) A close-up view of a typical ‘elongated snowflake’ cluster of the ESF1 crystals, demonstrating the unusual shapes of the crystals and their clusters. (c) The 0.30 µl crystallization micro-drop produced by the Mosquito LCP crystallization robot which resulted in the ERB1 crystal form. (d) The 0.30 µl crystallization micro-drop produced by the Mosquito LCP crystallization robot which resulted in the ERB2 crystal form.

Figure 2

X-ray diffraction pattern of Xyn52B2-E335G-Hg (ESF1 form) obtained using a synchrotron source (BM14, ESRF). The outer circle corresponds to 2.2 Å resolution. The insets represent magnified views of the sections indicated by the corresponding squares (bottom, low resolution; top, medium resolution).

Figure 3

X-ray diffraction pattern of Xyn52B2-E335G-Se (ESF1 form) obtained using a synchrotron source (BM14, ESRF). The outer circle corresponds to 3.9 Å resolution. The insets represent magnified views of the sections indicated by the corresponding squares (top, low resolution; bottom, medium resolution). It is noted that in principle the diffraction pattern exceeds 3.9 Å resolution, but the diffraction spots in this range are rather diffuse and were therefore not included in the final data set.