Structural and biochemical characterization of bifunctional XynA

Abstract

Xylan and cellulose are the two major constituents in numerous types of lignocellulosic biomass, representing a promising resource for biofuels and other biobased industries. The efficient degradation of lignocellulose requires the synergistic actions of cellulase and xylanase. Thus, bifunctional enzyme incorporated xylanase/cellulase activity has attracted considerable attention since it has great cost savings potential. Recently, a novel GH10 family enzyme XynA identified from Bacillus sp. is found to degrade both cellulose and xylan. To understand its molecular catalytic mechanism, here we first solve the crystal structure of XynA at 2.3 [A]. XynA is characterized with a classic (/{beta})8 TIM-barrel fold (GH10 domain) flanked by the flexible N-terminal domain and C-terminal domain. Circular dichroism, protein thermal shift and enzyme activity assays reveal that conserved residues Glu182 and Glu280 are both important for catalytic activities of XynA, which is verified by the crystal structure of XynA with E182A/E280A double mutant. Molecular docking studies of XynA with xylohexaose and cellohexaose as well as site-directed mutagenesis and enzyme activity assay demonstrat that Gln250 and His252 are indispensible to cellulase and bifunctional activity, separately. These results elucidate the structural and biochemical features of XynA, providing clues for further modification of XynA for industrial application.