For enzymes with buried active sites, transporting substrates/products ligands between active sites and bulk solvent via access tunnels is a key step in the catalytic cycle of these enzymes. Thus, tunnel engineering is becoming a powerful strategy to refine the catalytic properties of these enzymes. The tunnel-like structures have been described in enzymes catalyzing bulky substrates like glycosyl hydrolases, while it is still uncertain whether these structures involved in ligands exchange. Till so far, no studies have been reported on the application of tunnel engineering strategy for optimizing properties of enzymes catalyzing biopolymers. In this study, xylanase S7-xyl (PDB: 2UWF) with a deep active cleft was chosen as a study model to evaluate the functionalities of tunnel-like structures on the properties of biopolymer-degrading enzymes. Three tunnel-like structures in S7-xyl were identified and simultaneously reshaped through multi-sites saturated mutagenesis; the most advantageous mutant 254RL1 (V207N/Q238S/W241R) exhibited 340% increase in specific activity compared to S7-xyl. Deconvolution analysis revealed that all three mutations contributed synergistically to the improved activity of 254RL1. Enzymatic characterization showed that larger end products were released in 254RL1, while substrate binding and structural stability were not changed. Dissection of the structural alterations revealed that both the tun_1 and tun_2 in 254RL1 have larger bottleneck radius and shorter length than those of S7-xyl, suggesting that these tunnel-like structures may function as products transportation pathways. Attributed to the improved catalytic efficiency, 254RL1 represents a superior accessory enzyme to enhance the hydrolysis efficiency of cellulase towards different pretreated lignocellulose materials. In addition, tunnel engineering strategy was also successfully applied to improve the catalytic activities of three other xylanases including xylanase NG27-xyl from Bacillus sp. strain NG-27, TSAA1-xyl from Geobacillus sp. TSAA1 and N165-xyl from Bacillus sp. N16-5, with 80%, 20% and 170% increase in specific activity, respectively. This study represents a pilot study of engineering and functional verification of tunnel-like structures in enzymes catalyzing biopolymer. The specific activities of four xylanases with buried active sites were successfully improved by tunnel engineering. It is highly likely that tunnel reshaping can be used to engineer better biomass-degrading abilities in other lignocellulolytic enzymes with buried active sites.

中文翻译：

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隧道工程加速产品释放，以提高木质纤维素分解酶的生物质降解能力

对于具有埋藏活性位点的酶，通过通道在活性位点和本体溶剂之间运输底物/产物配体是这些酶催化循环中的关键步骤。因此，隧道工程正在成为改进这些酶催化特性的有力策略。隧道状结构已在催化大体积底物（如糖基水解酶）的酶中得到描述，但仍不确定这些结构是否参与配体交换。到目前为止，还没有关于应用隧道工程策略优化酶催化生物聚合物性能的研究报道。在本研究中，选择具有深活性裂隙的木聚糖酶 S7-xyl (PDB: 2UWF) 作为研究模型，以评估隧道状结构对生物聚合物降解酶特性的功能性。通过多位点饱和诱变，鉴定出S7-xyl中的三个隧道状结构并同时重塑；与 S7-xyl 相比，最有利的突变体 254RL1 (V207N/Q238S/W241R) 比活性增加了 340%。反卷积分析显示，所有三种突变都对 254RL1 的活性提高有协同作用。酶学表征表明，254RL1 释放了较大的最终产物，而底物结合和结构稳定性没有改变。结构变化的解剖表明，254RL1中的tun_1和tun_2都比S7-xyl具有更大的瓶颈半径和更短的长度，这表明这些隧道状结构可能作为产品运输途径。由于提高了催化效率，254RL1 代表了一种优越的辅助酶，可提高纤维素酶对不同预处理木质纤维素材料的水解效率。此外，隧道工程策略还成功应用于提高其他三种木聚糖酶的催化活性，包括来自芽孢杆菌属的木聚糖酶 NG27-xyl。来自 Geobacillus sp. 的菌株 NG-27、TSAA1-xyl。来自芽孢杆菌属的 TSAA1 和 N165-xyl。N16-5，比活性分别增加 80%、20% 和 170%。这项研究代表了在酶催化生物聚合物中隧道状结构的工程和功能验证的初步研究。隧道工程成功地提高了四种活性位点埋藏的木聚糖酶的比活性。