The structure of the feruloyl esterase module of xylanase 10B from Clostridium thermocellum provides insights into substrate recognition

Background: Degradation of the plant cell wall requires the synergistic action of a consortium of predominantly modular enzymes. In Clostridiae, these biocatalysts are organized into a supramolecular assembly termed a cellulosome. This multienzyme complex possesses, in addition to its well-described cellulolytic activity, an apparatus specific for xylan degradation. Cinnamic acid esterases hydrolyze the ferulate groups involved in the crosslinking of arabinoxylans to lignin and thus play a key role in the degradation of the plant cell wall in addition to having promising industrial and medical applications. Results: We have cloned and overexpressed the feruloyl esterase module from a 5 domain xylanase, Xyn1 OB from Clostridium thermocellum. The native structure at 1.6 Angstrom resolution has been solved with selenomethionine multiple wavelength anomalous dispersion and refined to a final R-free of 17.8%. The structure of a hydrolytically inactive mutant, S954A, in complex with the reaction product ferulic acid has been refined at a resolution of 1.4 Angstrom with an R-free of 16.0%. Conclusions: The C. thermocellum Xyn1 OB ferulic acid esterase displays the alpha/beta -hydrolase fold and possesses a classical Ser-His-Asp catalytic triad. Ferulate esterases are characterized by their specificity, and the active center reveals the binding site for ferulic acid and related compounds. Ferulate binds in a small surface depression that possesses specificity determinants for both the methoxy and hydroxyl ring substituents of the substrate. There appears to be a lack of specificity for the xylan backbone, which may reflect the intrinsic chemical heterogeneity of the natural substrate.