Abstract
Xylan and cellulose are the two major constituents in numerous types of
lignocellulose. 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. was 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 Å. XynA is
characterized with a classic (α/β)8 TIM-barrel fold (GH10 domain)
flanked by the flexible N-terminal domain and C-terminal domain. XynA
has a longer N-terminal and C-terminal than most other GH10 family
enzymes. The important thing is that the activity of our N-terminal
truncated XynA_ΔN37 is significantly improved. And we found that the
C-terminus is crucial to protein expression in solution. 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 double mutant
E182A/E280A. Molecular docking studies of XynA with xylohexaose and
cellohexaose, together with site-directed mutagenesis and enzyme
activity assay, demonstrate that Gln250 and His252 are indispensable to
bifunctional activity. These results elucidate the structural and
biochemical features of XynA, providing clues for further modification
of XynA for industrial application.