Engineering the Active Site Pocket to Enhance the Catalytic Efficiency of a Novel Feruloyl Esterase Derived From Human Intestinal Bacteria Dorea formicigenerans

文献类型： 外文期刊

第一作者： Shen, Yang

作者： Shen, Yang;Shao, Shuli;Shen, Yang;Wang, Yulu;Wei, Xue;Wen, Boting;Liu, Shujun;Xin, Fengjiao;Tan, Huishuang;Zhang, Jingjian

作者机构：

关键词： feruloyl esterase; gut microbiota; Dorea formicigenerans; AlphaFold2; rational design; substrate access tunnel

期刊名称：FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY （ 影响因子：6.064； 五年影响因子：6.303 ）

ISSN： 2296-4185

年卷期： 2022 年 10 卷

页码：

收录情况： SCI

摘要： The human gut microbiota play essential roles in metabolism and human health, especially by enzymatically utilizing dietary fiber that the host cannot directly digest and releasing functional components including short-chain fatty acids (SCFAs) and hydroxycinnamic acids (e.g., ferulic acid). In our previous study, seven potential feruloyl esterase (FAE) genes were identified from the gut microbiota. In the current work, one of the genes encoding a novel FAE (DfFAE) from Dorea formicigenerans of Firmicutes was bacterially expressed, purified and characterized. The 30.5 kDa type-A DfFAE has an optimum pH and temperature of 8.4 and 40 degrees C, respectively, exhibiting a higher substrate specificity toward short-chain acyl-ester substrate (pNPA). The AlphaFold2 based ab initio structural modeling revealed a five alpha-helices cap domain that shaped an unusually narrow and deep active site pocket containing a specific substrate access tunnel in DfFAE. Furthermore, rational design strategy was subjected to the active site pocket in an aim of improving its enzymatic activities. The mutants V252A, N156A, W255A, P149A, and P186A showed 1.8 to 5.7-fold increase in catalytic efficiency toward pNPA, while W255A also exhibited altered substrate preference toward long-chain substrate pNPO (45.5-fold). This study highlighted an unusual active site architecture in DfFAE that influenced its substrate selectivity and illustrated the applicability of rational design for enhanced enzymatic properties.

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