Bacteria
and fungi utilize carbohydrate-active enzymes, such as
feruloyl esterases (FAEs), to degrade lignocellulosic biomass. FAEs
in subfamily 5 (SF5) of carbohydrate esterase family 1 target larger
substrates, making them particularly interesting. However, their mechanisms
are not well understood due to limited structural information. This
study presents the first structure of the catalytic domain (CD) of
an SF5 FAE from Acremonium alcalophilum (AaFaeD), both free and in a complex with ferulic acid (FA).
FA binds within a hydrophobic cleft formed by two hydrophobic walls
facing each other. Structure-based functional mutagenesis of key residues
in these walls clarified their roles in catalysis. Notably, the F120Y
mutant of the AaFaeD catalytic domain (AaFaeD-CD) showed a 1.5-fold increase in catalytic activity toward
methyl ferulate compared with the wild type. Structural comparisons
with SF2 and SF3 FAEs revealed a more open substrate-binding site
in SF5. High-performance liquid chromatography and gas chromatography–mass
spectrometry analysis of destarched wheat bran hydrolysis by AaFaeD-CD showed that SF5 FAEs can process both monomeric
and dimeric phenolic substrates, like 5,5′-dehydrodiferulate,
unlike SF2 and SF3 FAEs, which prefer monomeric substrates.