Structural perspectives of an engineered β-1,4-xylanase with enhanced thermostability.

The glycoside hydrolase 10 (GH10) xylanase from Streptomyces sp. 9 (XynAS9) can operate in a broad range of pH and temperature, and thus is a potential candidate for commercial applications. Recently, we engineered XynAS9 via mutating several residues in accordance with the consensus sequences of GH10 thermophilic xylanases in an attempt to improve the enzyme thermostability and thermotolerance. The most promising effects were observed in the double mutant V81P/G82E. In order to investigate the molecular mechanism of the improved thermal profile of XynAS9, complex crystal structures of the wild type (WT) and mutant (MT) enzyme were solved at 1.88-2.05Å resolution. The structures reveal a classical GH10 (β/α)8 TIM-barrel fold. In MT XynAS9, E82 forms several interactions to its neighboring residues, which might aid in stabilizing the local structure. Furthermore, the MT structure showed lower B factors for individual residues compared to the WT structure, reflecting the increased MT protein rigidity. Analyses of the XynAS9 structures also delineate the detailed enzyme-substrate interaction network. More importantly, possible explanations for the enhanced thermal profiles of MT XynAS9 are proposed, which may be a useful strategy for enzyme engineering in the future.