Insight into the functional roles of Glu175 in the hyperthermostable xylanase XYL10C-ΔN through structural analysis and site-saturation mutagenesis.
Insight into the functional roles of Glu175 in the hyperthermostable xylanase XYL10C-ΔN through structural analysis and site-saturation mutagenesis.
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通过结构分析和位点饱和诱变深入了解 Glu175 在超热稳定木聚糖酶 XYL10C-ΔN 中的功能作用
DOI：
10.1186/s13068-018-1150-8
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发表时间：
2018
影响因子：
6.3
通讯作者：
Yao B
中科院分区：
文献类型：
--
作者：
You S;Chen CC;Tu T;Wang X;Ma R;Cai HY;Guo RT;Luo HY;Yao B
研究背景提高半纤维素酶的水解性能以降解木质纤维素类生物质对于第二代生物炼制具有重要意义。木聚糖酶作为一种重要的半纤维素酶，必须具有较高的活性和耐热性，才能在生物乙醇工业中得到应用。为了获得优良的木聚糖酶候选物，有必要了解其结构与功能的关系，为改进酶的性质提供有意义的参考。结果通过去除N端66个氨基酸，XYL 10 C-Δ N的酶催化效率提高了1.8倍，能更有效地水解玉米秸秆，但其热稳定性与野生型XYL 10 C相似。基于XYL 10 C-ΔN及其与木二糖复合物的晶体结构，发现位于环3上的Glu 175对GH 10木聚糖酶具有特异性，并且可能通过与环2上的Lys 135和Met 137相互作用来解释该酶的优异性质。位点饱和诱变证实在175位具有谷氨酸的XYL 10 C-ΔN具有最高的催化效率、比活性和最宽的pH-活性曲线。Glu 175在GH 10木聚糖酶XylE和XynE 2突变体中的功能也得到了验证，突变体的催化效率和pH-活性曲线均得到了提高。结论XYL 10 C-ΔN具有良好的热稳定性、催化效率和木质纤维素降解能力，是一种有价值的生物燃料木聚糖酶候选菌株。通过结构分析和功能验证，对XYN 10 C-ΔN催化剂活性提高的机理进行了研究，并确定Glu 175在提高催化效率中起关键作用。该研究揭示了XYN 10 C-ΔN中一个关键残基Glu 175的重要性，为GH 10木聚糖酶的改性提供了参考。
BackgroundImproving the hydrolytic performance of hemicellulases to degrade lignocellulosic biomass is of considerable importance for second-generation biorefinery. Xylanase, as the crucial hemicellulase, must be thermostable and have high activity for its potential use in the bioethanol industry. To obtain excellent xylanase candidates, it is necessary to understand the structure–function relationships to provide a meaningful reference to improve the enzyme properties. This study aimed to investigate the catalytic mechanism of a highly active hyperthermophilic xylanase variant, XYL10C-ΔN, for hemicellulose degradation.ResultsBy removing the N-terminal 66 amino acids, the variant XYL10C-ΔN showed a 1.8-fold improvement in catalytic efficiency and could hydrolyze corn stover more efficiently in hydrolysis of corn stover; however, it retained similar thermostability to the wild-type XYL10C. Based on the crystal structures of XYL10C-ΔN and its complex with xylobiose, Glu175 located on loop 3 was found to be specific to GH10 xylanases and probably accounts for the excellent enzyme properties by interacting with Lys135 and Met137 on loop 2. Site-saturation mutagenesis confirmed that XYL10C-ΔN with glutamate acid at position 175 had the highest catalytic efficiency, specific activity, and the broadest pH-activity profile. The functional roles of Glu175 were also verified in the mutants of another two GH10 xylanases, XylE and XynE2, which showed increased catalytic efficiencies and wider pH-activity profiles.ConclusionsXYL10C-ΔN, with excellent thermostability, high catalytic efficiency, and great lignocellulose-degrading capability, is a valuable candidate xylanase for the biofuel industry. The mechanism underlying improved activity of XYN10C-ΔN was thus investigated through structural analysis and functional verification, and Glu175 was identified to play the key role in the improved catalytic efficiency. This study revealed the importance of a key residue (Glu175) in XYN10C-ΔN and provides a reference to modify GH10 xylanases for improved catalytic performance.
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影响因子：
6.3
作者：
Hu J;Arantes V;Pribowo A;Saddler JN
通讯作者：
Saddler JN
影响因子：
11.4
作者：
Banerjee, Goutami;Car, Suzana;Walton, Jonathan D.
通讯作者：
Walton, Jonathan D.
深入了解具有纤维素活性的 GH10 木聚糖酶活性位点中非催化残基的作用
影响因子：
4.8
作者：
Chu, Yindi;Tu, Tao;Su, Xiaoyun
通讯作者：
Su, Xiaoyun
影响因子：
2.1
作者：
Dias, FMV;Goyal, A;Fontes, CMGA
通讯作者：
Fontes, CMGA
影响因子：
2.8
作者：
Kim, JH;Irwin, D;Wilson, DB
通讯作者：
Wilson, DB