Structural characterization of a β-xylosidase-like enzyme from Bacillus licheniformis

β-xylosidases belonging to GH43 family are involved in the breakdown of plant cell-wall hemicelluloses, catalyzing the release of xylosyl units from the nonreducing ends of xylooligosaccharides via an inverting mechanism. Here, we report the cloning, expression, purification and structural studies of a novel GH43 β-xylosidase-like enzyme from Bacillus licheniformis (XynBbl). Sequence analysis indicated that all catalytic residues are conserved; however, biochemical assays with a range of substrates revealed that XynBbl was not able to cleave any of those saccharides. Based on that result, we decided to investigate the molecular basis for its lack of activity against classical substrates for GH43 enzymes. Small Angle X-Ray Scattering (SAXS) data showed that XynBbl is a dimer in solution, which is in agreement with crystallographic data. The XynBbl crystals diffracted to 2.5 Å resolution and revealed an energetically stable dimeric configuration with the subunits aligned antiparallel to each other through the accessory domain. The structure of XynBbl consists of an N-terminal five-bladed β-propeller catalytic domain and an additional b-sandwich domain. Although the three catalytic residues (Asp41, Asp159 and Glu207) are conserved in XynBbl, the active site does not contain a pocket topology, commonly found in β-xylosidases. A hydrophobic cluster, which delineates the active-site pocket and contributes for the exo mode of action of β-xylosidases, is absent in XynBbl and it probably is the major structural determinant for the lack of β-xylosidase activity.