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\begin{document}

\title{Structure-function relationship of bifunctional XynA}



\author[1]{Wei Xie}%
\author[1]{Qi Yu}%
\author[1]{Ruiqin Zhang}%
\author[1]{Yun Liu}%
\author[1]{Ruoting Cao}%
\author[1]{Sidi Wang}%
\author[1]{Ruoting Zhan}%
\author[1]{Zhongqiu Liu}%
\author[2]{kui wang}%
\author[2]{Caiyan Wang}%
\affil[1]{Guangzhou University of Traditional Chinese Medicine}%
\affil[2]{Guangzhou University of Chinese Medicine}%


\vspace{-1em}



  
  \date{May 15, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{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 \selectlanguage{ngerman}Å. XynA is
characterized with a classic (\selectlanguage{greek}α/β\selectlanguage{english})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\_\selectlanguage{greek}Δ\selectlanguage{english}N37 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.%
\end{abstract}%



\sloppy


\textbf{Structure-function relationship of b} \textbf{ifunctional XynA}

Wei Xie\textsuperscript{1}, Qi Yu\textsuperscript{1}, Ruiqing
Zhang\textsuperscript{2}, Yun Liu\textsuperscript{2}, Ruoting
Cao\textsuperscript{2}, Sidi Wang\textsuperscript{3}, Ruoting
Zhan\textsuperscript{2}, Zhongqiu Liu\textsuperscript{1}, Kui
Wang\textsuperscript{2}*, Caiyan Wang\textsuperscript{1}*

\textsuperscript{1}Guangdong Key Laboratory for Translational Cancer
Research of Chinese Medicine, Joint Laboratory for Translational Cancer
Research of Chinese Medicine of the Ministry of Education of the
People's Republic of China, International Institute for Translational
Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou,
Guangdong, 510006, China.

\textsuperscript{2}Research Center of Chinese Herbal Resource Science
and Engineering, Key Laboratory of Chinese Medicinal Resource
from\emph{Lingnan} (Guangzhou University of Chinese Medicine), Ministry
of Education, Joint Laboratory of National Engineering Research Center
for the Pharmaceutics of Traditional Chinese Medicines, Guangzhou
University of Chinese Medicine, Guangzhou, Guangdong, 510006,
China.\textsuperscript{3}College of Fundamental Medical Sciences,
Guangzhou University of Chinese Medicine, Guangzhou 510006, China.

\textsuperscript{\textbf{*}}\textbf{Corresponding authors:}

Kui Wang,

Mailing address: Research Center of Chinese Herbal Resource Science and
Engineering, Guangzhou University of Chinese Medicine, Guangzhou Higher
Education Mega Center, No.232 Outer Ring East Rd., Panyu District,
Guangzhou 510006, Guangdong Province, China.

Phone: 86-020-39358066; Fax: 86-020-39358066;

E-mail: kuiwang@gzucm.edu.cn.

Caiyan Wang,

Guangdong Key Laboratory for Translational Cancer Research of Chinese
Medicine, Joint Laboratory for Translational Cancer Research of Chinese
Medicine of the Ministry of Education of the People's Republic of China,
International Institute for Translational Chinese Medicine, Guangzhou
University of Chinese Medicine, Guangzhou, Guangdong, 510006, China.
Tel.: 86-020-39358875; Fax: 86-020-39358071;

E-mail: wangcaiyan@gzucm.edu.cn.

\textbf{Running title:} Structural basis of bifunctional GH10 xylanase
XynA

\textbf{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 \emph{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 \selectlanguage{ngerman}Å.
XynA is characterized with a classic (\selectlanguage{greek}α/β\selectlanguage{english})\textsubscript{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\_\selectlanguage{greek}Δ\selectlanguage{english}N37 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.

\textbf{Keywords} : XynA, GH10, bifunctional xylanase/cellulase, crystal
structure, molecular mechanism

\textbf{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 (\selectlanguage{greek}α/β\selectlanguage{english})\textsubscript{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 \emph{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 \selectlanguage{ngerman}°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), \emph{p} -nitrobenzene-cellobioside
and\emph{p} -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\_\selectlanguage{greek}Δ\selectlanguage{english}N37 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.

\textbf{Materials and Methods}

\textbf{Site-directed mutagenesis}

The GenBank of XynA is QCO69162. The target protein was preceded by a
6xHis tag. We designed primer pairs with mutation points. The primers of
E182A were used to cause the plasmid pET28a-XynA to undergo PCR. Then,
the PCR product was digested with \emph{Dpn} I (Thermo Scientific,
Waltham, United States) for 1.5 hours at 37 \selectlanguage{ngerman}°C, and the product was
heated at 80 °C for 5 minutes to denature. Finally, the product was
transformed into competent DH5\selectlanguage{greek}α \selectlanguage{english}cells. The successfully sequenced
mutants were transformed into BL21-CodonPlus (DE3) cells to express the
protein. The mutant E280A needed to be mutated based on pET28a-E182A.
XynA E182Q/E280Q had the same construction method as XynA E182A/E280A.
The purification methods of various mutants and truncations of XynA are
the same as XynA.

\textbf{Expression and purification of the wild-type and mutant XynA
proteins}

The recombinant plasmid pET28a-XynA was transformed into BL21-Codonplus
(DE3) competent cells, and then a single colony was selected and
inoculated into 100 mL of LB medium containing 30 \selectlanguage{greek}μ\selectlanguage{english}g/mL kanamycin
(Sangon Biotech, Shanghai, China) and 32 \selectlanguage{greek}μ\selectlanguage{english}g/mL chloramphenicol (Sangon
Biotech, Shanghai, China) and cultured in a 37 \selectlanguage{ngerman}°C constant temperature
shaking table at 180 rpm for 10 hours. When the
OD\textsubscript{600}reached 0.6-0.8, the \emph{E. coli} had reached the
logarithmic growth stage. Protein expression was then induced with 0.2
mM isopropyl-\selectlanguage{greek}β-\selectlanguage{english}D-thiogalactopyranoside (IPTG, Sangon Biotech, Shanghai,
China) at 25 \selectlanguage{ngerman}°C on a constant temperature shaker for 12 hours. Finally,
the samples were collected by centrifugation at 4000 rpm for 20 minutes
at 4 °C and stored at -80 °C until use.

The collected \emph{E. coli} was suspended in buffer A containing 40 mM
Tris-HCl at pH 8.0 with 250 mM NaCl and 10 mM imidazole at 4 °C, and
then 1 mM \selectlanguage{greek}β-\selectlanguage{english}mercaptoethanol (Amresco, Guangzhou, China) and 1 mM PMSF
(Sigma, Beijing, China) were added. The resuspended \emph{E. coli} cells
were passed through a low-temperature ultrahigh-pressure cell disrupter
at 1200 psi, and the obtained lysate was centrifuged at 14000 rpm for 60
minutes (4 \selectlanguage{ngerman}°C). After centrifugation, portions of the supernatant and
the precipitate were retained (Coomassie brilliant blue: running glue
samples) (Laemmli, 1970). The supernatant was divided into two parts:
one part was directly combined with nickel-nitrilotriacetic acid
(Ni-NTA) affinity resin (Qiagen, China); the other part was heated in a
65 °C water bath for 60 minutes and then centrifuged at 14000 rpm for 60
minutes. After the second centrifugation, a part of the supernatant and
the precipitate were retained, and the supernatant was combined with
Ni-NTA. The two supernatants above were combined with Ni-NTA for 45-60
minutes. The hybrid protein was removed by using buffer A (40 mM
Tris-HCl at pH 8.0 with 250 mM NaCl, 10 mM imidazole at 4 °C), and the
target proteins were eluted by using buffer B (40 mM Tris-HCl at pH 8.0
with 250 mM NaCl, 250 mM imidazole at 4 °C). XynA was purified in two
ways, one of which was heating in a 65 °C water bath for 60 minutes and
the second was using a Hi Trap QHP column (1 mL GE Healthcare, United
States). The protein is bound to the column at loading. The conditions
are then changed, and the bound components elute separately.
Purification was carried out on a 1 mL QHP column with a NaCl gradient,
and the protein solution was injected into the equilibrium column at a
flow rate of 0.5 mL/ minute. A linear NaCl gradient elution of 50 to 250
mM was used, and 2 mL fractions of the eluate were collected over the
entire gradient length.

The state of XynA in solution was analyzed by a Superdex 200 (10/300)
increase column (GE Healthcare, United States). First, the
chromatographic column was balanced with a buffer containing 150 mM
NaCl, 40 mM Tris-HCl at pH 8.0 and 1 mM dithiothreitol (DTT, Sangon
Biotech, Shanghai, China). Second, XynA loading. Finally, the column was
eluted with buffer solution, and 2 mL fractions of eluate was collected.

\textbf{Protein crystallization, data collection and structure
determination}

The home-grown crystals were initially screened manually using a
laboratory-made kit at room temperature using a 96-well plate with
diffused vapor diffusion. A 0.8 \selectlanguage{greek}μ\selectlanguage{english}L aliquot of XynA protein solution (8.0
mg/mL, 20 mM Tris-HCl pH 8.0, 287 mM NaCl) was mixed with a 0.8 \selectlanguage{greek}μ\selectlanguage{english}L
reservoir solution and equilibrated with a 100 \selectlanguage{greek}μ\selectlanguage{english}L reservoir solution.
Twenty-four hours later, the initial XynA crystals were obtained in 28\%
PEG600 (w/v), 0.1 M CaCl\textsubscript{2} and 0.1 M MES pH 6.0. The
optimized crystallization conditions were 24\% PEG600 (w/v), 0.2 M
CaCl\textsubscript{2}, 0.1 M MES pH 6.0, crystal length reaches 2 mm.
29\% PEG600 (w/v), 0.1 M CaCl\textsubscript{2}, 0.1 M MES pH 6.0 and
25\% glycerol were used as refrigerating protectors. Individual crystals
were installed on a nylon loop and immediately cooled with liquid
nitrogen prior to data collection. The individual crystals are installed
on a nylon loop and immediately cooled with liquid nitrogen. In Shanghai
Synchrotron Radiation Facility (SSRF-BL19U1) diffraction data was
collected through X-ray diffraction and finally processed using HKL2000
(Otwinowski \& Minor, 1997). We use PDB 1V0L as a model to obtain the
initial model. Based on the initial model, we use COOT to accurately
modify the structure, and then use CCP4 for refinement. Finally, repeat
the above steps until the structural requirements are met (Emsley,
Lohkamp, Scott, \& Cowtan, 2010; McCoy et al., 2007). XynA finally
obtained a 2.3\selectlanguage{ngerman}Å resolution crystal structure. The crystallization
conditions of the XynA E182A/E280A were the same as those of XynA, and
the structure of the XynA E182A/E280A was obtained with a resolution of
2.9 Å. Information about the protein crystal structure is provided
in\textbf{Table 1} . Conservation analysis of protein structure was
carried out through the ConSurf Server website and \emph{PyMOL} software
was used to prepare images.

\textbf{Multiple residue sequence alignment}

We downloaded the residue sequences of the GH10 family of xylanases such
as CbXyn10C and SlXyn10A from the GenBank protein database. Then, we
compared the residue sequences of XynA, the residue sequence of the GH10
family of monofunctional xylanases, and the residue sequence of the GH10
family of bifunctional xylanases using ClustalW software and the
ESPript3 website.

\textbf{Biolayer interferometry binding studies}

The ForteBio Octet® RED96 interaction analyzer(39-41) (ForteBio, United
States) was used to detect the binding of XynA to the oligosaccharide
substrates (cellobiose, xylohexaose and cellohexaose) (Megazyme,
Ireland) and xylan (BWX) (Shanghai, China) (Li, Tian, Wang, Zhu, \& Sun,
2017; Petersen, 2017). First, XynA was dialyzed against PBS. Second, the
equivalent biotinylation kit (Biotinylation Kit, Jiangsu Bomeida Life
Science Co., Ltd.) was added to bind XynA protein for 60 minutes for
biotinylating. Finally, the unbound biotin was removed with a
desalination column. Before the experiment, we wetted the biosensor
(SSA, Shenzhen Baokeda Biotechnology Co., Ltd.) with PBS for 10 minutes.
In the first step, we equilibrated the sensor with PBS for 2 minutes. In
the second step, XynA was loaded onto the SSA sensor until it was
saturated. Third, we equilibrated with PBS for 3 minutes. Fourth,
diluted substrate solutions (12.5, 25, 50, 100, 200, and 250 \selectlanguage{greek}μ\selectlanguage{english}M) were
sequentially added and dissociated from low concentration to high
concentration, and each combination and dissociation time was 300
seconds. A sensor not bound to the biotinylated XynA was used as a
baseline control for calibration. The whole process was carried out at
room temperature, and all tests were repeated three times.

\textbf{Protein thermal shift assay}

SYPRO Orange Protein Gel Stain 5000\selectlanguage{ngerman}× (Thermo Scientific, United States)
was diluted with protein buffer solution (20 mM Tris-HCl at pH 8.0 with
287 mM NaCl) to 200×. A 30 \selectlanguage{greek}μ\selectlanguage{english}g portion of protein (5.0 mg/mL) was mixed
with BWX and incubated on ice for 60 minutes, then centrifuged at 14000
rpm/ minutes for 10 minutes. After centrifugation, the protein was
evenly mixed with 2.5 \selectlanguage{greek}μ\selectlanguage{english}L 200\selectlanguage{ngerman}× SYPRO Orange, and the total volume was 20
\selectlanguage{greek}μ\selectlanguage{english}L. The mixture was heated from 25 \selectlanguage{ngerman}°C to 99 °C at a rate of 0.2
°C/minute, and each sample was prepared in triplicate. An Applied
Biosystems real-time PCR instrument (7500 Fast, Thermo Scientific,
United States) was used to determine the protein denaturation
temperature (exposure temperature of hydrophobic amino acid residues),
and the results were generated using \emph{Origin} software (Niesen,
Berglund, \& Vedadi, 2007).

\textbf{Molecular docking}

First, the PDB files of XynA, xylohexaose and cellohexaose were prepared
using the Protein Preparation Wizard of Maestro (Maestro11.9,
Schrödinger, LLC, New York, 2019) to prepare the model structure for
docking (Friesner et al., 2004; Sastry, 2013). Then, the appropriate
docking pocket was selected, and the molecular docking was performed.
The docking scores of each ligand were determined with the best posture
on Maestro. The criterion for hydrogen bond judgment is that the
distance between the acceptor and donor atoms should be less than 3 Å.
Output the docking file through Maestro, and finally use
\emph{PyMOL}software to make figure.

\textbf{Determination of the enzyme activity by DNS}

The DNS method was used to detect the xylanase activity of the XynA and
XynA E182A/E280A proteins. In this study, BWX (Lot\# X4252, \selectlanguage{english}[?] 90\%
purity) was used as the substrate, and xylose was used as the standard.
The reducing sugars released from BWX were measured by DNS reagent to
estimate the xylanase activity. The hydrolyzed product oligosaccharides
(reducing sugars) undergo redox reactions with 3,5-dinitrosalicylic acid
(DNS) to produce 3-amino-5-nitrosalicylic acid (brown red). First, we
took the appropriate concentration (25, 250, 500, 1000 nM) of the
protein and 0.5\% (w/v) of BWX and mixed them evenly in 100 mM sodium
citrate at pH 6.5, and the total volume was 50 \selectlanguage{greek}μ\selectlanguage{english}L. Second, the reaction
system was incubated at the optimal temperature of 65 \selectlanguage{ngerman}°C for 10 minutes.
Third, the DNS reagent was added to the reaction system, and the mixture
was incubated at 95 °C for 5 minutes. Finally, the reaction system was
cooled in an ice bath to room temperature and diluted with 100 mM sodium
citrate buffer solution at pH 6.5, and the absorbance was determined at
540 nm with an enzyme label analyzer.

\textbf{Results}

\textbf{The overall structure of XynA}

The full-length XynA protein was expressed and purified with removal of
nucleic acids by anion exchange column QHP. XynA contains 408 residues
and the molecular weight of the tagged full-length protein was 49.8 kDa
with an isoelectric point of 6.64. It was identified as one monomer in
the solution by gel filtration \textbf{(Figure 1A)} . We found that
xylanase 10A (SlXyn10A, PDB code 1V0L) from\emph{S. lividans} has the
highest sequence similarity (30\%) with XynA by using Phyre2 server
(Gloster et al., 2004; Kelley, Mezulis, Yates, Wass, \& Sternberg,
2015). We used this structure as a search model to solve the crystal
structure of XynA by molecular replacement method.

The determined 2.3 Å crystal structure of XynA (residues 22-393) with
a\emph{R} \textsubscript{free} of 0.245 (\textbf{Table 1} ). The space
group is \emph{P4122} , and one molecule was present in the asymmetric
unit. In the refined model, residues Leu52, Ala53, Asp359, Ala360,
Asn361, Gly378, and Glu379 are missing due to disordered configuration.
The XynA structure is composed of 12 \selectlanguage{greek}α-\selectlanguage{english}helices and 14 \selectlanguage{greek}β-\selectlanguage{english}sheets, among
which \selectlanguage{greek}α\selectlanguage{english}2-\selectlanguage{greek}α\selectlanguage{english}11 helices and \selectlanguage{greek}β\selectlanguage{english}3-\selectlanguage{greek}β\selectlanguage{english}10 sheets form a classical
(\selectlanguage{greek}α/β\selectlanguage{english})\textsubscript{8} TIM-barrel fold\textbf{(Figure 1B)} . Overall,
the mug-shaped XynA is comprised of an N-terminal domain (residues
1-67), GH10 domain (residues 68-326) and C-terminal domain (residues
327-408) \textbf{(Figure 1C)} . The body of ``mug'' is GH10 domain,
which has 3 \selectlanguage{greek}α-\selectlanguage{english}helices separated by loop regions, named \selectlanguage{greek}α\selectlanguage{english}2, \selectlanguage{greek}α\selectlanguage{english}4, and \selectlanguage{greek}α\selectlanguage{english}7
\textbf{(Figure 1B)} . These three \selectlanguage{greek}α-\selectlanguage{english}helices are not affecting the
overall conformation of classical (\selectlanguage{greek}α/β\selectlanguage{english})\textsubscript{8} TIM-barrel
fold.

\textbf{The thermostability and activity of N-terminal and C-terminal to
XynA}

We compare the crystal structures of XynA with other GH10 family
enzymes. Of note, only the GH10 domain is in the other protein
structures mentioned above, the N-terminal and C-terminal domains are
unique for XynA (\textbf{Figure 2A} ). The typical GH10 domain is well
conserved in GH10 family, as evidenced by the low RMSD (1.599 \selectlanguage{ngerman}Å) of
structural superimposition of XynA, TsXy1A and SlXyn10A. In order to
explore the effects of N-terminal and C-terminal on XynA, we constructed
deleted residues 1-67 named XynA\_\selectlanguage{greek}Δ\selectlanguage{english}N and deleted of residues 327-408
named XynA\_\selectlanguage{greek}Δ\selectlanguage{english}C. Only XynA\_\selectlanguage{greek}Δ\selectlanguage{english}N37 (delete residues 1-36) expresses soluble
protein, XynA\_\selectlanguage{greek}Δ\selectlanguage{english}C has been unable to express soluble protein. We tested
the activity and thermal stability of XynA\_\selectlanguage{greek}Δ\selectlanguage{english}N37 protein. We found that
the melting temperature of XynA\_\selectlanguage{greek}Δ\selectlanguage{english}N37 is 71.32 \selectlanguage{ngerman}°C, which is 2.21 °C
lower than that of XynA (\textbf{Figure 2B} ). XynA\_\selectlanguage{greek}Δ\selectlanguage{english}N37 has a
significant increase in the hydrolytic activity of the three substrates
BWX, WAX and barley \selectlanguage{greek}β-\selectlanguage{english}glucan (\textbf{Figure 2C} ). Since 71.32 \selectlanguage{ngerman}°C is a
relatively high melting temperature in xylanase, the difference in
melting temperature between XynA\_\selectlanguage{greek}Δ\selectlanguage{english}N37 and XynA can be ignored. We
believe that XynA\_\selectlanguage{greek}Δ\selectlanguage{english}N37 increases the activity while maintaining the
original thermal stability. The N-terminal and C-terminal form the
handle of this ''mug'' together. We find that the N-terminal residues
Arg25, Asn29, Arg31, and Arg36 establish abundant hydrogen bond
interactions with the residues Glu296, Arg300, Phe303, Ser304, His305,
and Val308 at the body GH10 domain (\textbf{Figure 2D} ). Similarly, the
C-terminal residues Val330, His332, Arg342, Tyr343 and Glu344 form
extensive hydrogen bond interactions with residues His89, Glu296,
Phe316, Trp317, and Ala318 of the GH10 domain (\textbf{Figure 2E} ). The
residues participated in hydrogen bond interactions are all located in
\selectlanguage{greek}α-\selectlanguage{english}helices and loops. The difference between XynA and other GH10 families
exists in the N-terminal and C-terminal domain. The C-terminal residues
(Glu338-Glu349) form \selectlanguage{greek}α-\selectlanguage{english}helix\textsubscript{12}, which makes the
classical (\selectlanguage{greek}α/β\selectlanguage{english})\textsubscript{8} TIM-barrel fold more complete. The
N-terminal (\selectlanguage{greek}α\selectlanguage{english}1, \selectlanguage{greek}β\selectlanguage{english}1 and \selectlanguage{greek}β\selectlanguage{english}2) and C-terminal domain (\selectlanguage{greek}α\selectlanguage{english}12, \selectlanguage{greek}β\selectlanguage{english}11-\selectlanguage{greek}β\selectlanguage{english}14) is
partially crossover, and there are two \selectlanguage{greek}α-\selectlanguage{english}helices and six \selectlanguage{greek}β-\selectlanguage{english}sheets that
are adjacent to the TIM barrel.

\textbf{Sequence and structure analyses of XynA with other GH10
xylanase}

To find the catalytic residues of XynA, we performed multiple sequence
alignments of XynA with five GH10 family enzymes, namely CbXyn10C
(\emph{C. bescii} ), SlXyn10A (\emph{S. lividans} ), BsXynA
(\emph{Bacillus sp.} ), TmXynB (\emph{T. maritima} ) and TsXy1A
(\emph{T. saccharolyticum} ). Among them, XynA, CbXyn10C and SlXyn10A
are xylanases with xylanase and cellulase activity. BsXynA, TmXynB, and
TsXy1A are strict xylanase enzyme. We find that the residues in the GH10
domain, such as Phe90, Glu96, Lys100, Gly133 and Trp137 are much
conserved\textbf{(Figure 3)} . Though XynA and BsXynA both come
from\emph{Bacillus} sp., BsXynA is a traditional GH10 family xylanase
that only catalyzes the degradation of xylan (Zhou et al., 2014a). The
architecture of GH10 domain is similar in XynA and BsXynA, but the
subtle sequence difference in fifteen residues indicates that these
sites may determine the monofunction or bifunction (\textbf{Figure 3} ).
By structural superposition and sequence comparison among CbXyn10C,
SlXyn10A, TmXynB, TsXy1A and XynA, we infer that Glu182 and Glu280 are
putative catalytic residues of XynA (Chu et al., 2017; Han et al., 2013;
Ihsanawati et al., 2005; V Ducros et al., 2000). Further analysis
indicates that most residues related to monofunctional and bifunctional
activity are conserved and overlapped, containing Glu96, Asn97, Lys100,
His133 and Trp137. The residues specific to bifunctional activity
include Tyr216, Asn217, Ile223, Trp257 and Tyr288.

\textbf{The xylanase activity of XynA}

To explore the catalytic properties of XynA, we first investigated the
optimal enzyme concentration and the xylanase activity of XynA. Using
0.5\% (w/v) beechwood xylan (BWX) as substrate in 100 mM sodium citrate
buffer, pH6.5 at 65\selectlanguage{ngerman}°C for10 minutes, as the concentration of XynA
protein increased, the percentage of degraded sugar increased until it
reached a plateau \textbf{(Figure 4A)} . Based on this reaction curve,
500 nM is regarded as the optimal concentration for XynA.

The activity of GH10 family enzymes requires two conserved catalytic
residues glutamate, and the mutation of glutamate will completely
inactivate the protein (Derewenda et al., 1994). To verify the effect of
these two glutamic acids on XynA activity, we double mutated Glu182 and
Glu280 to alanine and produced the mutant XynA E182A/E280A protein. The
xylanase activity of the mutants (XynA E182A/E280A) was determined using
the DNS method. The results displayed that WT had the highest activity
at a concentration of 500 nM, while the E182A/E280A mutant is inactive,
even at an extremely high concentration (1 mM) \textbf{(Figure 4B)} .

To analyze the binding mode and interaction patterns of XynA and its
substrates, we have tried hard to prepare the crystals of XynA WT,
E182A/E280A or E182Q/E280Q with cellobiose, cellohexaose, or xylohexaose
complexes, respectively. However, all trials failed, only the apo
crystals of WT and mutant XynA were obtained. This may be explained by
the weak and difficult detection features for the GH10 family proteins
and their substrates according to previous studies (Baumann et al.,
2011). However, through Surface Plasmon Resonance (SPR) assay, we
detected the binding affinity of BWX to both the WT and E182A/E280A
mutant XynA\textbf{(Figure 4C)} . The binding constants indicated that
the binding ability of the WT to BWX was stronger than that of the
E182A/E280A mutant. We found that WT does not bind to oligosaccharides
by SPR assay, which is consistent with the fact that it is difficult for
oligosaccharides to enter the crystal structure \textbf{(Figure 4C)} .

The protein thermal shift assay indicates that XynA is much
thermostable, as evidenced by the Tm value 73.53 °C. Moreover, the Tm
value 75.5 °C of XynA bound with BWX is higher than that of the apo
protein, indicating that substrate binding could stabilize
XynA\textbf{(Figure 4D)} . Similarly, the Tm value of the mutant before
and after the BWX binding also increased, indicating that the substrate
binding also affects the stability of the mutant.

Crystal structure can provide direct hints to study the protein features
and properties. To elucidate how E182/E280 affect the enzyme activity of
XynA, we determined the crystal structure of XynA E182A/E280A at 2.9 Å.
The overall architecture of XynA E182A/E280A is highly similar to the WT
XynA, yielding a C\selectlanguage{greek}α \selectlanguage{english}RMSD of 0.218 \selectlanguage{ngerman}Å. A major difference observed between
the WT and mutant is an angle swing in the N- and the C-terminus. We
found that the previously established hydrogen bonds between Glu182 and
Trp137, Asn181 and Gln250 as well as Glu280 and with Asn217 and His252
disappeared in the E182A/E280A mutant \textbf{(Figure 4E, 4F)} .

\textbf{Molecular docking of XynA with xylohexaose and cellohexaose and
the mutant activity confirmation}

Since the cocrystal structure of XynA complexed with substrates is
unsuccessful, to investigate the catalytic mechanism of XynA, we
performed molecular docking studies. The sugar chains of xylan and
cellulose are too long to suitable for molecular docking, therefore,
xylohexaose and cellohexaose are substrates for docking. Viewing the
putative binding mode, these two substrates situated in the center of
the mouth of the XynA ''mug'' structure \textbf{(Figure 5A, 5B)} .
Xylohexaose formed ten hydrogen bonds with surrounding residues in the
active pocket (mug mouth), including Asp141, Asn143, Glu96, Lys100,
Trp314, Asn220 and Ser223. Cellohexaose formed eleven hydrogen bond
interactions with surrounding residues, such as Asp74, Glu96, Glu98,
Try102, Glu182, Gln250, His252 and Asn220. Among the residues in
putative substrate binding pocket, only five residues are varied between
bifunctional enzymes XynA and CbXyn10C, namely 140, 216, 223, 257 and
288, indicating conservation in sequence and structure (\textbf{Figure
5F} ). The centric Glu182 and Glu280 are surrounded by Glu96, Asn97,
Lys100, Tyr216 and Asn217, forming a pocket embracing the sugar chains
of substrate. Based on molecular docking results, residues Gln250 and
His252 are speculated with relation to the bifunctionality of XynA.
According to the results, Asp74, Glu96, Gln250, His252 and Asn220 formed
hydrogen bond with cellohexaose. Based on the classification of
residues, we designed mutants D74A, E96A, Q250A, Q250E, H252A and N220E.
In light of the residues of CbXyn10C interacting with the substrate,
Val216 and Ala254 are potential residues that could interact with the
substrate in the XynA sequence (Chu et al., 2017). We designed mutants
N97E, V216E and A254E. We used WAX, BWX and barley \selectlanguage{greek}β-\selectlanguage{english}glucan as
substrates, and the activities of these mutants relative to that of the
XynA (abbreviated as WT in \textbf{Figure 5} ) was determined by the
3,5-dinitrosalicylic acid (DNS) method \textbf{(Figure 5C, 5D, 5E)} . We
found that the four mutants (E182A/E280A, E182Q/E280Q, Q250A, and H252A)
were inactive towards three substrates. The mutants E96A and N97E lost
their activity towards BWX and were unable to degrade WAX and barley
\selectlanguage{greek}β-\selectlanguage{english}glucan by more than 50\%. The mutants V216E, N220E, and A254E showed
significantly reduced degradation activity on WAX, BWX, and barley
\selectlanguage{greek}β-\selectlanguage{english}glucan. The mutant Q250E showed reduced degradation activity on WAX
and BWX and no activity against barley \selectlanguage{greek}β-\selectlanguage{english}glucan. The mutant D74A showed
significantly weaker degradation activities against WAX and barley
\selectlanguage{greek}β-\selectlanguage{english}glucan. E182A/E280A and E182Q/E280Q were completely inactive against
these three substrates, further confirming that Glu182 and Glu280 were
the key catalytic residue residues.

\textbf{Discussion}

The most important features of XynA are its thermal stability,
bifunctionality and low homology. It has been reported that its
half-lives at 65 \selectlanguage{ngerman}°C and 70 °C are 12 hours and 1.5 hours, respectively
(Wang et al., 2019). We can take advantage of the thermal stability of
this protein. We tried to purify the protein by heating it in a water
bath at 65 °C for 1 hour, which may affect the structure of the protein,
so we also used an anion-exchange column QHP for purification. During
the crystallization process, we found that the protein purified by the
QHP column crystallized quickly, and the crystal morphology was regular,
while the protein purified by heating crystallized slowly, and the
crystal morphology was irregular. These two kinds of crystallization
indicate that the high temperature influences the conformation and
stability of the protein, resulting in differences in the
crystallization.

In addition to the typical (\selectlanguage{greek}α/β\selectlanguage{english})\textsubscript{8} TIM-barrel fold GH10
domain structure, XynA has N-terminal and C-terminal. By homology
analysis of the overall structure of XynA, the homology of the ''mug
handle'' formed by the N-terminal and C-terminal parts of XynA is low,
the \selectlanguage{greek}β-\selectlanguage{english}sheet conservatism inside the ''mug'' is high, and the outer part
of the \selectlanguage{greek}α-\selectlanguage{english}helix outside the ``mug'' is not conserved, while the inner
part of the \selectlanguage{greek}α-\selectlanguage{english}helix is very conserved \textbf{(Figure 5F).}It has been
reported that the N-terminus and C-terminus might affect the thermal
stability, activity and substrate specificity of the protein (Mahanta et
al., 2015; Song, Tsang, \& Sylvestre, 2015; Wang et al., 2019; Zheng et
al., 2016). By truncating the N-terminal \selectlanguage{greek}α \selectlanguage{english}helix (1-36), we proved that
the N-terminal did not affect the thermal stability of XynA, and
truncating the N-terminal alpha helix enhanced the activity of XynA
\textbf{(Figure 2B, 2C)} . In the structure, we see that the N-terminal
alpha helix (1-36) interacts with the GH10 domain by hydrogen bonding
\textbf{(Figure 2D)} . This hydrogen bond restricts the (\selectlanguage{greek}α/β\selectlanguage{english})8
TIM-barrel fold and cannot swing better to accommodate the substrate and
promote its hydrolysis. We tried many C-terminal truncations, but none
of them were soluble. This proves that the presence of the C-terminus is
essential for protein expression. Our analysis showed that the inside of
the ''mug'' is bound to the substrate and degrades the substrate, so the
residues are more conserved.

We found and verified two key residues Glu182 and Glu280 by structural
analysis of XynA. In the catalytic process, Glu182 and Glu280 are
catalytic nucleophiles/bases and catalytic proton donors, respectively
(Withers et al., 1986). Comparing the properties of WT with those of the
double mutant showed the activities, structures and stabilities were all
different, proving the importance of Glu182 and Glu280. Glu182 and
Glu280 formed hydrogen bonds with surrounding residues could ``lock''
the substrate to be catalyzed after entering the active pocket. The
hydrogen bonds formed by the side chains of Glu182 and Glu280 with
surrounding residues account for the majority interaction. Since the
residues 182 and 280 are located in the loop area, the reduced
interaction also makes this part more flexible, thereby reducing the
binding affinity to substrate. This is in consistent with the SPR
result. At the same time, WT has the ability to hydrolyze the substrate,
while the E182A/E280A has no hydrolysis ability, which also shows that
the mutation affects the hydrolysis activity by affecting the binding of
the substrate. We attempted to determine the cocrystal structure of the
proteins (WT, E182A/E280A, and E182Q/E280Q) with substrates xylohexaose
and cellohexaose to reveal the specific catalytic mechanism. However,
neither the incubation of the protein with the substrate nor the
immersion of the crystal in the substrate successfully afforded
cocrystals. From the SPR results, we also observed that XynA does not
bind to xylohexaose and cellohexaose. The substrate cannot be
immobilized in the protein, so it is difficult to obtain a cocrystal
structure.

We obtained the simulated structures of XynA and xylohexaose and of XynA
and cellohexaose by molecular docking. The glucose unit has one more
-CH\textsubscript{2}OH group than the xylose unit in its molecular
structure. Therefore, cellohexaose requires more space than xylohexaose
to accommodate the sugar chains. It is found that the XynA active pocket
can hold xylohexaose and cellohexaose. This shows that XynA can
accommodate sugar chains of different substituents and thus exhibit a
broad substrate scope. Compared with the residues that formed hydrogen
bonds with xylohexaose and cellohexaose, Asp74, Glu182, Gln250 and
His252 are unique residues that form hydrogen bonds between XynA and
cellohexaose. According to the molecular docking, Glu96, Lys100, Gln250
and His252 are particularly conserved in the residue sequences of the
GH10 family and interact with substrates in CbXyn10C and SlXyn10A.

We find that four mutants, E182A/E280A, E182Q/E280Q, Q250A, and H252A
are all inactive towards three substrates. We are not surprised that
Glu182 and Glu280 are required for activity, as our results show that
they are key catalytic residues. After being replaced with alanine, the
activities for the three substrates completely disappeared, which proves
that the two residues His252 and Gln250 also related to catalysis. Our
analysis reveals the conservation of residue His252 and Gln250 in the
GH10 family and the uniformity of its interactions with substrates
(\textbf{Figure 3} ). The mutant H252A and Q250A only reduce the
activity of the CbXyn10C protein (Chu et al., 2017). However, H252A and
Q250A of XynA lose its activity towards the three substrates, proving
that His252 and Gln250 are important residues in XynA. For most of the
xylanases in the GH10 family, the catalytic residues are Glu residue
(Chu et al., 2017). But we find that in addition to Glu182 and Glu280 as
catalytic residues, XynA also has the two residues His252 and Gln250.
From the crystal structure, it is found that Glu280 is located in the
loop region (more flexible). The side chain residues of His252 form
hydrogen bonds with Glu280 side chains, and the main chain residues of
Gln250 form hydrogen bonds with Glu280 main chain residues. The hydrogen
bonding interaction makes the structure of Glu280 more stable, so that
the side chain is closer to the substrate, thereby playing the role of
electron transfer. The mutation of His252 prevents Glu280 from getting
close to the substrate, making it difficult to carry out electron
transfer and making the protein inactive \textbf{(Figure 6)} . Glu182 is
also located in the loop area, which is easier to swing and difficult to
bind to the substrate. The side chain residues of Gln250 and Glu182 side
chain residues form hydrogen bonds, which make the structure of Glu182
more stable and play the role of electron transfer \textbf{(Figure 6)} .
The mutation of Gln250 prevents Glu182 from getting close to the
substrate, making it difficult to perform electron transfer. Therefore,
we believe that a hydrogen bond network is formed around Gln250 to
mediate the effects of Glu182 and Glu280.

The mutants D74A, E96A, N97E, N220E, V216E and A254E all have reduced
activity to various extents, proving that these sites all contribute to
the activity of the substrate. We indicate that the residues that
interact with Glu182 including Trp137, Asn181, Asn217 and Gln250 and the
residues that interact with Glu280 including His133, Asn217, Gln250 and
His252 are very conserved in CbXyn10C, and these residues interact with
the substrate, fully reflecting the unity of the GH10 family. Finally,
we propose the catalytic mechanism of XynA. In the first glycosylation
step, E280 acts as a nucleophile (since Glu280 has less interaction with
surrounding residues than Glu182, Glu280 is more flexible to generate
electron transfer first), attacking the anomeric center to displace
glycosides and form glycosylase intermediates. At the same time, Glu182
acts as an acid catalyst and protonates the glycoside oxygen when the
bond is broken. In the second deglycosylation step, the glycosylase is
hydrolyzed by water, and Glu182 acts as a base catalyst, deprotonating
the water molecules when it attacks. As the catalysis progresses, Gln250
and His252 interact with the catalytic residue to bring the catalytic
residue close to the substrate. At the same time, Gln250 and His252
interact with the substrate to affect the catalysis \textbf{(Figure 6)}
.

XynA is a heat-stable and acid-stable xylanase in the GH10 family, and
Celluclast 1.5L can synergistically hydrolyze pretreated corn stover.
These properties indicate that it may be effective in food, animal feed
and biofuel production. Analysis of the XynA crystal structure can
provide a basis for its transformation, making it more suitable for
commercial applications such as biofuel production.

\textbf{Data availability}

The atomic coordinates and structure factors of xylanase XynA structure
has been uploaded to the Protein Data Bank (XynA PDB code: 7D88 and XynA
E182A/E280A PDB code: 7D89). All remaining data are included in the
article.

\textbf{Acknowledgements}

This work was supported by the following grants: the Youth Scientific
Research Training Project of GZUCM (No. 2019QNPY07), the National
Natural Science Foundation of China (No. 31700625), Guangdong Key
Laboratory for Translational Cancer Research of Chinese Medicine (No.
2018B030322011), the National Natural Science Foundation of China
(31802099), the Pearl River Talent Recruitment Program of Guangdong
Province (2017GC010372), the Natural Science of Foundation of Guangdong
Province, China (2018A030310497), the Research Program for Young
Innovative Talents in Higher Education Institutions of Guangdong
Province (2017KQNCX037) and Shanghai Synchrotron Radiation Facility.

\textbf{Conflict of Interest}

The authors declare no conflict of interest.

\textbf{Author contributions}

Caiyan Wang, Kui Wang and Zhongqiu Liu: Conceptualization, Methodology.

Wei Xie: Data curation, Writing- Original draft preparation.

Wei Xie, Qi Yu, Ruiqing Zhang, Yun Liu, Ruoting Cao, Sidi Wang and
Ruoting Zhan: Software.

Wei Xie, Qi Yu, Caiyan Wang and Kui Wang: Visualization, Investigation.

Caiyan Wang, Kui Wang and Zhongqiu Liu: Writing-Reviewing and Editing.

\textbf{Table 1} . Data collection and refinement statistics.\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
& XynA (7D88) & XynA E182A/E280A (7D89)\tabularnewline
\midrule
\endhead
Data collection & &\tabularnewline
Wavelength (\selectlanguage{ngerman}Å) & SSRF-BL19U1 & SSRF-BL19U1\tabularnewline
Space group & \emph{P}4122 & \emph{P}4122\tabularnewline
a, b, c (\selectlanguage{ngerman}Å) & 109.842, 109.842, 85.57 & 110.193, 110.193,
84.983\tabularnewline
\selectlanguage{greek}α, β, γ \selectlanguage{english}(\selectlanguage{ngerman}°) & 90.00, 90.00, 90.00 & 90.00, 90.00, 90.00\tabularnewline
Resolution (\selectlanguage{ngerman}Å) & 15.29-2.345 (2.429-2.345) \textsuperscript{a} &
49.28-2.894 (2.997- 2.894) \textsuperscript{a}\tabularnewline
R\textsubscript{merge}\textsuperscript{b} & 0.158 (1.074) &
0.108(0.550)\tabularnewline
I/\selectlanguage{greek}σ\selectlanguage{english}\textsubscript{(I)} & 30(2.6) & 34(5.83)\tabularnewline
Completeness (\%) & 99.35 (98.73) & 99.56 (96.27)\tabularnewline
Redundancy & 25.3(23.6) & 25.5(27.1)\tabularnewline
Refinement & &\tabularnewline
Resolution (\selectlanguage{ngerman}Å) & 15.29-2.345 & 49.28-2.894\tabularnewline
No. reflections & 22400 (2182) & 12123 (1135)\tabularnewline
R\textsubscript{work}\textsuperscript{c}/R\textsubscript{free}\textsuperscript{d}
& 0.2213/0.2446 & 0.2378/0.2690\tabularnewline
No. atoms & 3041 & 2884\tabularnewline
Protein & 2972 & 2856\tabularnewline
Ligand & 2 & 2\tabularnewline
Water molecules & 67 & 26\tabularnewline
B-factors (\selectlanguage{ngerman}Å \textsuperscript{2}) & &\tabularnewline
Protein & 55.49 & 63.42\tabularnewline
Ligand & 51.66 & 49.77\tabularnewline
Water molecules & 56.34 & 47.08\tabularnewline
Bond lengths (\selectlanguage{ngerman}Å) & 0.009 & 0.014\tabularnewline
Bond angles (\selectlanguage{ngerman}º) & 1.08 & 1.53\tabularnewline
Ramachandran favored (\%) & 95.51 & 96.21\tabularnewline
allowed & 3.93 & 3.21\tabularnewline
Outliers (\%) & 0.56 & 0.58\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{a}Values in parentheses are for the highest-resolution
shell. \textsuperscript{b}R\textsubscript{merge} =\selectlanguage{greek}Σ\selectlanguage{english}\textbar{}(I
-\textless{} I \textgreater{} )\textbar{}/\selectlanguage{greek}σ\selectlanguage{english}(I), where I is the observed
intensity. \textsuperscript{c}R\textsubscript{work} =
\selectlanguage{greek}Σ\selectlanguage{english}\textsubscript{hkl}\textbar{}\textbar{}Fo\textbar{} -
\textbar{}Fc\textbar{}\textbar{}/
\selectlanguage{greek}Σ\selectlanguage{english}\textsubscript{hkl}\textbar{}Fo\textbar{}, calculated from working data
set. \textsuperscript{d}R\textsubscript{free} is calculated from 5.0\%
of data randomly chosen and not included in refinement.

\textbf{Figures and Figure legends}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1.} XynA protein and its structure. (A) Gel-filtration
analysis showing XynA as monomer in solution. The red and black lines
represent the UV absorption of XynA and the standard proteins at 280 nM
respectively. (B) The topological organization of XynA. The GH10
catalytic domain, the N-terminal domain and C-terminal domain are
colored in cyan, pink and purple, respectively. (C) Overall structure of
XynA is like a shape of ``mug''. The color is assigned as shown in
front.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2.} The analysis of N-terminal and C-terminal to
XynA\textbf{.} (A) Structural superposition of XynA, SlXyn10A (PDB code
1V0L), and TsXy1A (PDB code 3W25). (B) Protein thermal shift assay of
XynA and XynA\_\selectlanguage{greek}Δ\selectlanguage{english}N37. (C) Relative activities of the XynA\_\selectlanguage{greek}Δ\selectlanguage{english}N37 in the
hydrolysis of BWX, WAX and barley \selectlanguage{greek}β-\selectlanguage{english}glucan respectively. * indicates P
\textless{}0.05, **** indicates P \textless{}0.0001. (D) The hydrogen
bonding interaction between the N-terminal and GH10 domains. The red
dashed line indicates hydrogen bonding interaction. (E) The hydrogen
bonding interaction between the C-terminal and GH10 domains. The red
dotted line represents hydrogen bonds. The criterion for hydrogen bond
judgment is that the distance between the acceptor and donor atoms
should be less than 4 \selectlanguage{ngerman}Å.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\par\null

\textbf{Figure 3.} Multiple sequence alignment and structure analyses of
XynA. Multiple sequence alignment of XynA (QCO69162) with CbXyn10C
(ACM60945) from \emph{C. bescii} (Chu et al., 2017); SlXyn10A (AAC26525)
from \emph{S. lividans} (Ducros et al., 2000b); BsXynA (AHH02587)
from\emph{Bacillus} sp.(Zhou et al., 2014b); TmXynB (AAD35164) from
\emph{T. maritima} (Ihsanawati et al., 2005); and TsXy1A (AFK86466) from
\emph{T. saccharolyticum} (Han et al., 2013)\emph{.} The red background
color represents strictly conserved residues, and the red font
represents the substitution of conserved residues. The pink five-pointed
star indicates the two key catalytic residues of XynA, and the original
secondary structure of XynA is shown above the sequence alignment. The
black squares represent the residues that interact with the substrate in
CbXyn10C, and the white squares represent the residues that interact
with the substrate in TsXy1A. The triangles indicate the residues of
XynA and BsXynA that differ in the GH10 domain.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4.} Comparison of XynA with XynA E182A/E280A in terms of
binding, catalysis activity and structures. (A) Effect of XynA
concentration on its xylanase activity. (B) XynA and XynA E182A/E280A
activities for degrading substrates at different concentrations. Each
experiment was repeated three times. XynA and XynA E182A/E280A were
analyzed for significance, ns means no significant difference, * means P
\textless{}0.05, ** means P \textless{}0.01, *** means P
\textless{}0.0002, and **** means P \textless{}0.0001. (C) XynA binding
experiment with BWX; XynA E182A/E280A binding experiment with BWX.
Different colored lines in the figure represent different concentrations
of BWX. The binding constants are shown in the table. (D) Protein
thermal shift assay of XynA and XynA E182A/E280A. RFU of the Y-axis
represents relative fluorescence units. The Tm value represents the
denaturation or exposure temperature of the hydrophobic residues of the
protein. \selectlanguage{greek}Δ\selectlanguage{english}Tm indicates the temperature change after adding BWX. (E)
Interaction between two residues Glu182 and Glu280 and surrounding
residues of XynA. (F) The interaction between the Ala182 and Ala280
residues and the surrounding residues of XynA E182A/E280A. Color cyan
and pink indicate the crystal structure of WT and double mutant
respectively. The red dotted line represents hydrogen bonds. The
criterion for hydrogen bond judgment is that the distance between the
acceptor and donor atoms should be less than 4 \selectlanguage{ngerman}Å.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5.}The molecular docking with xylohexaose and
cellohexaose and activity analysis of the XynA. (A-B) The docking of
XynA with xylohexaose and cellohexaose displayed in a cartoon mode,
respectively, and the hydrogen bonds between the XynA with xylohexaose
or cellohexaose are partially enlarged. The red dotted line indicates
hydrogen bonding interaction. The criterion for hydrogen bond judgment
is that the distance between the acceptor and donor atoms should be less
than 3 \selectlanguage{ngerman}Å. (C-E) Relative activities of the XynA mutants in the
hydrolysis of BWX, WAX and barley \selectlanguage{greek}β-\selectlanguage{english}glucan respectively. Each experiment
was repeated three times. WT enzyme activity was set to 100\%. WT group
was used as a control for a significant analysis, * means P
\textless{}0.05, ** means P \textless{}0.01, *** means P
\textless{}0.0002, and **** means P \textless{}0.0001. (F) The spatial
position and conservation of residues in space fill model of XynA that
may interact with the substrate. The green to purple gradient indicates
the degree of conservation. The annotated residues are the mutation
sites found by molecular docking.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Figure 6.}Proposed endo-1,4-\selectlanguage{greek}β-\selectlanguage{english}xylanase catalytic mechanism for
XynA. E280 acts as a nucleophile, and E182 acts as an acid/base
catalyst. Q250 and H252 will interact with catalytic residues. The blue
sugar represents the sugar that has been hydrolyzed. The pink dotted
line represents hydrogen bond interaction.

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