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

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

doi:10.3389/fbioe.2022.936914

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

Front Bioeng Biotechnol. 2022 Jun 20:10:936914. doi: 10.3389/fbioe.2022.936914. eCollection 2022.

Authors

Yang Shen^{1

2}, Yulu Wang², Xue Wei², Boting Wen², Shujun Liu², Huishuang Tan³, Jingjian Zhang⁴, Shuli Shao¹, Fengjiao Xin²

Affiliations

¹ Department of Life Science and Agroforestry, Qiqihar University, Qiqihar, China.
² Laboratory of Biomanufacturing and Food Engineering, Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing, China.
³ Key Laboratory of Ministry of Education for Protein Science, School of Life Sciences, Tsinghua University, Beijing, China.
⁴ Cangzhou Academy of Agriculture and Forestry Sciences, Cangzhou, China.

Abstract

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 °C, respectively, exhibiting a higher substrate specificity toward short-chain acyl-ester substrate (pNPA). The AlphaFold2 based ab initio structural modeling revealed a five α-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.

Keywords: AlphaFold2; Dorea formicigenerans; feruloyl esterase; gut microbiota; rational design; substrate access tunnel.