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 (Figure 2A ). The typical GH10 domain is well conserved in GH10 family, as evidenced by the low RMSD (1.599 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_ΔN and deleted of residues 327-408 named XynA_ΔC. Only XynA_ΔN37 (delete residues 1-36) expresses soluble protein, XynA_ΔC has been unable to express soluble protein. We tested the activity and thermal stability of XynA_ΔN37 protein. We found that the melting temperature of XynA_ΔN37 is 71.32 °C, which is 2.21 °C lower than that of XynA (Figure 2B ). XynA_ΔN37 has a significant increase in the hydrolytic activity of the three substrates BWX, WAX and barley β-glucan (Figure 2C ). Since 71.32 °C is a relatively high melting temperature in xylanase, the difference in melting temperature between XynA_ΔN37 and XynA can be ignored. We believe that XynA_ΔN37 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 (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 (Figure 2E ). The residues participated in hydrogen bond interactions are all located in α-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 α-helix12, which makes the classical (α/β)8 TIM-barrel fold more complete. The N-terminal (α1, β1 and β2) and C-terminal domain (α12, β11-β14) is partially crossover, and there are two α-helices and six β-sheets that are adjacent to the TIM barrel.
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 (C. bescii ), SlXyn10A (S. lividans ), BsXynA (Bacillus sp. ), TmXynB (T. maritima ) and TsXy1A (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(Figure 3) . Though XynA and BsXynA both come fromBacillus 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 (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.