Structural insights into the molecular mechanisms of substrate recognition and hydrolysis by feruloyl esterase from Aspergillus sydowii.

The depolymerization of lignocellulosic biomass is facilitated by feruloyl esterases (FAEs), which hydrolyze ester bonds between lignin and polysaccharides. Fungal FAEs belonging to subfamily (SF) 6 release precursors such as ferulic acid derivatives, attractive for biochemical production. Among these, Aspergillus sydowii FAE (As FaeE), an SF6 FAE, exhibits remarkable activity across various substrates. In this study, we conducted X-ray crystallography and kinetic analysis to unravel the molecular mechanisms governing substrate recognition and catalysis by As FaeE. As FaeE exhibits a typical α/β -hydrolase fold, characterized by a catalytic triad of serine, aspartate, and histidine. Comparative analysis of substrate-free, ferulic acid-bound, and sinapic acid-bound forms of As FaeE suggests a conformational change in the loop covering the substrate-binding pocket upon binding. Notably, Pro158 and Phe159 within this loop cover the phenolic part of the substrate, forming three layers of planar rings. Our structure-based functional mutagenesis clarifies the roles of the residues involved in substrate binding and catalytic activity. Furthermore, distinct substrate-binding mechanisms between As FaeE and other studied FAEs are identified. This investigation offers the initial structural insights into substrate recognition by SF6 FAEs, equipping us with structural knowledge that might facilitate the design of FAE variants capable of efficiently processing a wider range of substrate sizes. • 3D structures of As FaeE in substrate-free and substrate-bound forms were revealed. • The loop covering the substrate-binding pocket plays a pivotal role in binding. • Functional roles of the residues in the substrate-binding pocket are unveiled. • Mutants with different substrate preferences were found. • SF6 FAE's preference towards monomeric hydroxycinnamates is rationally understood. [ABSTRACT FROM AUTHOR]