Structural Analysis of a Glycoside Hydrolase Family 11 Xylanase from Neocallimastix patriciarum
Structural Analysis of a Glycoside Hydrolase Family 11 Xylanase from Neocallimastix patriciarum
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DOI：
10.1074/jbc.m114.550905
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发表时间：
2014-04-18
影响因子：
4.8
通讯作者：
Guo, Rey-Ting
中科院分区：
文献类型：
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作者：
Cheng, Ya-Shan;Chen, Chun-Chi;Guo, Rey-Ting
背景：嗜热木聚糖酶在许多工业应用中是有价值的。结果如下：测定了木聚糖酶XynCDBFV及其与低聚木糖复合物的结构，其N-末端区域（NTR）有助于热稳定性。结论：NTR可以稳定XynCDBFV的蛋白折叠。意义：XynCDBFV是一种来源于瘤胃真菌新美鞭菌（Neocallimastix patriciarum）的糖苷水解酶家族11（GH 11）木聚糖酶，具有较高的比活和较宽的pH适应性，其催化结构域具有巨大的商业应用潜力。在这里，XynCDBFV及其与底物的复合物的晶体结构被确定为1.27-1.43分辨率。这些结构揭示了一个典型的GH 11-卷曲折叠和详细的酶和配体之间的相互作用网络。值得注意的是，在XynCDBFV结构中鉴定了由11个氨基酸组成的延伸的N-末端区域（NTR），其在GH 11木聚糖酶中是独特的。NTR通过氢键和堆积力沿着与Cys-4和Cys-172之间的二硫键连接到催化核心。有趣的是，与野生型酶相比，NTR缺失突变体在55 ℃和75 ℃分别保留了61.5%和19.5%的酶活性，而C4 A/C172 A突变体显示出86.8%和23.3%的活性。这些结果表明NTR在XynCDBFV热稳定性中起作用，并且Cys-4/Cys-172二硫键对NTR介导的相互作用至关重要。此外，我们还表明，毕赤酵母生产的XynCDBFV具有更高的催化活性，在较高的温度比大肠杆菌，其中可能发生不正确的NTR折叠和低效的二硫键形成。总之，这些结构和功能分析的工业青睐XynCDBFV提供了NTR的贡献，其热稳定性的分子基础。
Background: Thermophilic xylanases are valuable in many industrial applications. Results: The structures of a xylanase XynCDBFV and its complex with xylooligosaccharides were determined, and its N-terminal region (NTR) contributes to thermostability. Conclusion: NTR may stabilize the overall protein folding of XynCDBFV. Significance: The structural and functional investigation of unprecedented NTR of XynCDBFV provides a new insight into the molecular basis of thermophilic xylanases.The catalytic domain of XynCDBFV, a glycoside hydrolase family 11 (GH11) xylanase from ruminal fungus Neocallimastix patriciarum previously engineered to exhibit higher specific activity and broader pH adaptability, holds great potential in commercial applications. Here, the crystal structures of XynCDBFV and its complex with substrate were determined to 1.27-1.43 resolution. These structures revealed a typical GH11 -jelly-roll fold and detailed interaction networks between the enzyme and ligands. Notably, an extended N-terminal region (NTR) consisting of 11 amino acids was identified in the XynCDBFV structure, which is found unique among GH11 xylanases. The NTR is attached to the catalytic core by hydrogen bonds and stacking forces along with a disulfide bond between Cys-4 and Cys-172. Interestingly, the NTR deletion mutant retained 61.5% and 19.5% enzymatic activity at 55 degrees C and 75 degrees C, respectively, compared with the wild-type enzyme, whereas the C4A/C172A mutant showed 86.8% and 23.3% activity. These results suggest that NTR plays a role in XynCDBFV thermostability, and the Cys-4/Cys-172 disulfide bond is critical to the NTR-mediated interactions. Furthermore, we also demonstrated that Pichia pastoris produces XynCDBFV with higher catalytic activity at higher temperature than Escherichia coli, in which incorrect NTR folding and inefficient disulfide bond formation might have occurred. In conclusion, these structural and functional analyses of the industrially favored XynCDBFV provide a molecular basis of NTR contribution to its thermostability.