Crystallization and preliminary X-ray diffraction analysis of Xyn30D fromPaenibacillus barcinonensis

Abstract

Xyn30D, a new member of a recently identified group of xylanases, has been purified and crystallized. Xyn30D is a bimodular enzyme composed of an N-terminal catalytic domain belonging to glycoside hydrolase family 30 (GH30) and a C-terminal family 35 carbohydrate-binding domain (CBM35) able to bind xylans and glucuronic acid. Xyn30D shares the characteristic endo mode of action described for GH30 xylanases, with the hydrolysis of the β-(1,4) bonds of xylan being directed by α-1,2-linked glucuronate moieties, which have to be placed at the −2 subsite of the xylanase active site. Crystals of the complete enzyme were obtained and a full data set to 2.3 Å resolution was collected using a synchrotron X-ray source. This represents the first bimodular enzyme with the domain architecture GH30-CBM35. This study will contribute to the understanding of the role that the different xylanases play in the depolymerization of glucuronoxylan.

Structural Analysis of Glucuronoxylan-specific Xyn30D and Its Attached CBM35 Domain Gives Insights into the Role of Modularity in Specificity*

“…Is a Bimodular Enzyme-We have purified and crystallized the glucuronoxylan-specific xylanase from P. barcinonensis (Xyn30D), as previously reported (31). We present here the three-dimensional structure of the full-length bimodular enzyme solved by molecular replacement at 2.4 Å resolution.…”

“…Crystallization and Data Collection-Crystals of Xyn30D were grown as described before (31). For data collection, native crystals were transferred to cryoprotectant solutions consisting of mother liquor plus 30% (v/v) glycerol before being cooled to 100 K in liquid nitrogen.…”

“…On the contrary, the attached xylose unit does not make any direct interaction with residues from the Xyn30D-CBM35 domain, and, in agreement with this observation, the electron density map shows weaker density at this position precluding building additional xylose units. Finally, the only significant difference observed in the ligand-bound complexes with respect to the unbound structure is a conformational change in the Glu 31 side chain that rearranges to make a hydrogen bond to GlcA O3 (Fig. 5b).…”