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.
Keywords :
XynA,
GH10, bifunctional xylanase/cellulase, crystal structure,
molecular
mechanism
Introduction
With
the continuous consumption of oil resources and the development of the
global economy, developing cleaner and more economical energy is of
utmost importance (Victor & Leape, 2015). The sources of biomass energy
are very rich, including forestry byproducts and crop straws, which are
renewable energy sources (Isikgor & Becer, 2015; Sims, Mabee, Saddler,
& Taylor, 2010).
During
production, the plant cell walls must be depolymerized, which requires
synergistic action of cellulase and xylanase (Broeker et al., 2018;
Keegstra, 2010; Long et al., 2018; Xiaoyun Su et al., 2013).
In addition to biofuel production,
xylanase is also applied in various industries, such as the food
industry, feed industry, paper industry, and brewing industry (Dornez,
Verjans, Arnaut, Delcour, & Courtin, 2011; Ito et al., 2019; Kumar,
Marin-Navarro, & Shukla, 2016; Lisov et al., 2017).
The
thermostable glycoside hydrolase (GH) family with bifunctional activity
is more advantageous in degrading lignocellulose biomass, and thus has
great development potential for industrial application. Among the GH
family, the GH10 family and GH11 family contain a large number of
xylanases (Lombard, Golaconda Ramulu, Drula, Coutinho, & Henrissat,
2014). Compared with GH11 xylanases,
GH10 xylanase has an
extensive
substrate scope (Biely, Singh, & Puchart, 2016; Chakdar et al., 2016;
Collins, Gerday, & Feller, 2005).(13-15)
Furthermore,
some GH10 xylanases have been found to degrade other polysaccharides,
such as konjac glucomannan and tamarind xyloglucan (Fredriksen et al.,
2019).
The
GH10 domain of GH10 family xylanases is responsible for catalytic
activity. The regions outside the GH10 domain (N-terminal or C-terminal)
may affect the thermal stability of the protein (Mahanta, Bhardwaj,
Kumar, Reddy, & Ramakumar, 2015).
Because
the binding ability of some xylanase and the substrate is relatively
weak, it is difficult to detect their binding, such as the GH10A
xylanase from the Arctic mid-ocean ridge ventilation system (Fredriksen
et al., 2019). The xylanase GH10A is capable of degrading wheat
arabinoxylan (WAX) and tamarind xyloglucan and it is
difficult
to detect the binding ability (Fredriksen et al., 2019). The
bifunctional enzymes with xylanase/cellulase activity have been reported
such as CbXyn10C (5OFJ) (Chu et al., 2017), SlXyn10A (1E0X) (Ducros et
al., 2000a), CbXyn10B (4L4O) (Zhang et al., 2016) and CfCex (Liu, Zhang,
& Xu, 2012). CbXyn10C, Xyl10A, and CbXyn10B all have the classic
(α/β)8 TIM-barrel fold structure, but there is no
article revealing the ”switch” of the GH10 family of xylanases from
single function to dual function.
In a previous study, a novel GH10
xylanase termed XynA was identified from Bacillus sp. (Wang et
al., 2019). XynA could function at a wide range of pH (maintains more
than 60% activity at pH 5.0-7.5) and temperature (maintains more than
65% activity at 45 °C to 80 °C) (Wang et al., 2019). Interestingly,
XynA was found to be a bifunctional xylanase/cellulase enzyme,
exhibiting activities towards both xylan substrates and a variety of
cellulose substrates, including
cellobiose,
cellohexaose, carboxymethyl cellulose, filter paper, Avicel
(microcrystalline cellulose), p -nitrobenzene-cellobioside andp -nitrobenzene-glucopyranoside (Wang et al., 2019). In addition,
XynA hydrolyzed corn stover pretreated with cellulase, showing an
obvious synergistic
effect
(Wang et al., 2019). These unparalleled characteristics make XynA become
an attractive candidate in biotechnology applications, such as bioenergy
production and pulp processing (Dhiman et al., 2014; Dodd & Cann, 2009;
You et al., 2019). However, the xyanase activity of XynA is much
stronger than its cellulase activity, which may raise some problems
during application, for instance, the insufficient degradation of
substrates and the quality control (Dodd & Cann, 2009).
To
provide useful guidelines for design of XynA for future industrial
application, we investigated the catalytic molecular mechanism of XynA
for two substrates xyanase and cellulase based on 2.3 Å crystal
structure. Firstly, we designed a series of truncation at both ends of
the XynA, we found the bifunctional activity of truncation XynA_ΔN37 is
significantly improved. Then, we designed function assays and revealed
that the conserved residues Glu182 and Glu280 are crucial for the
bifunctional activity of XynA, and the other two residues Gln250 and
His252 are essential to bifunctional activity of XynA.
These
catalytic features providing clues for further structure modification of
XynA for industrial application.