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.