Unveiling a catalytically promiscuous feruloyl esterase from Clostridium acetobutylicum.

Clostridium species have garnered increasing attention for their ability to convert lignocellulosic biomass into renewable fuels and platform chemicals. Among the enzymes involved in lignin degradation, feruloyl esterases (FAEs) cleave ester bonds between ferulic acid and polysaccharide side chains, thereby facilitating the disruption of lignin-carbohydrate complexes. However, the biochemical and structural properties of Clostridium FAEs remain poorly characterized, with activity studies largely limited to model substrates rather than native lignin-derived compounds. Here, we report the functional and crystallographic characterization of a novel FAE (CaFaeA) from Clostridium acetobutylicum. CaFaeA exhibits broad catalytic activity toward a range of hydroxycinnamate esters as well as bis(2-hydroxyethyl) terephthalate (BHET), distinguishing it from typical carboxyl esterases. Furthermore, the 2.45 Å crystal structure of CaFaeA reveals a canonical α/β-hydrolase fold with a unique lid domain of three α-helices and two antiparallel β-strands partially covering the active site. Mutagenesis identified two gatekeeper residues, Y151 and E168, that regulate substrate access and catalytic performance. Remarkably, CaFaeA demonstrates exceptional tolerance to organic solvents, retaining or even enhancing activity in the presence of 25% dimethyl sulfoxide and n-hexane. With insoluble wheat arabinoxylan (I-WAX) as substrate, its unique lid architecture enabled efficient cleavage of ferulic acid-arabinose ester linkages, resulting in a release of free ferulic acid by 5.39 mg·μmol