Structural Insights into the Mechanisms Underlying the Kinetic Stability of GH28 Endo-Polygalacturonase

文献类型： 外文期刊

第一作者： Tu, Tao

作者： Tu, Tao;Wang, Zhiyun;Luo, Yan;Li, Yeqing;Su, Xiaoyun;Wang, Yuan;Zhang, Jie;Yao, Bin;Luo, Huiying;Rouvinen, Juha;Hakulinen, Nina

作者机构：

关键词： glycoside hydrolase; endo-polygalacturonase; kinetic stability; carbon-oxygen hydrogen bond; stable conformation

期刊名称：JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY （ 影响因子：5.279； 五年影响因子：5.269 ）

ISSN： 0021-8561

年卷期： 2021 年 69 卷 2 期

页码：

收录情况： SCI

摘要： Thermostability is a key property of industrial enzymes. Endo-polygalacturonases of the glycoside hydrolase family 28 have many practical applications, but only few of their structures have been determined, and the reasons for their stability remain unclear. We identified and characterized the Talaromyces leycettanus JCM12802 endo-polygalacturonase TlPGA, which differs from other GH28 family members because of its high catalytic activity, with an optimum temperature of 70 degrees C. Distinctive features were revealed by comparison of thermophilic TlPGA and all known structures of fungal endo-polygalacturonases, including a relatively large exposed polar accessible surface area in thermophilic TlPGA. By mutating potentially important residues in thermophilic TlPGA, we identified Thr284 as a critical residue. Mutant T284A was comparable to thermophilic TlPGA in melting temperature but exhibited a significantly lower half-life and half-inactivation temperature, implicating residue Thr284 in the kinetic stability of thermophilic TlPGA. Structure analysis of thermophilic TlPGA and mutant T284A revealed that a carbon-oxygen hydrogen bond between the hydroxyl group of Thr284 and the C alpha atom of Gln255, and the stable conformation adopted by Gln255, contribute to its kinetic stability. Our results clarify the mechanism underlying the kinetic stability of GH28 endo-polygalacturonases and may guide the engineering of thermostable enzymes for industrial applications.

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