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°C for10 minutes, as the concentration of XynA protein increased, the percentage of degraded sugar increased until it reached a plateau (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) (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(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 (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(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 Cα RMSD of 0.218 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 (Figure 4E, 4F) .
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 (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 (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 β-glucan as substrates, and the activities of these mutants relative to that of the XynA (abbreviated as WT in Figure 5 ) was determined by the 3,5-dinitrosalicylic acid (DNS) method (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 β-glucan by more than 50%. The mutants V216E, N220E, and A254E showed significantly reduced degradation activity on WAX, BWX, and barley β-glucan. The mutant Q250E showed reduced degradation activity on WAX and BWX and no activity against barley β-glucan. The mutant D74A showed significantly weaker degradation activities against WAX and barley β-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.
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 °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 (α/β)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 β-sheet conservatism inside the ”mug” is high, and the outer part of the α-helix outside the “mug” is not conserved, while the inner part of the α-helix is very conserved (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 α 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 (Figure 2B, 2C) . In the structure, we see that the N-terminal alpha helix (1-36) interacts with the GH10 domain by hydrogen bonding (Figure 2D) . This hydrogen bond restricts the (α/β)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 -CH2OH 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 (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 (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 (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 (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.