Feruloyl esterase activity of the Clostridium thermocellum cellulosome canbe attributed to previously unknown domains of XynY and XynZ

The cellulosome of Clostridium thermocellum is a multiprotein complex with endo- and exocellulase, xylanase, beta-glucanase, and acetyl xylan esterase activities. XynY and XynZ, components of the cellulosome, are composed of several domains including xylanase domains and domains of unknown function (UDs), Database searches revealed that the C- and N-terminal UDs of XynY an d XynZ, respectively, have sequence homology with the sequence of a feruloy l esterase of strain PC-2 of the anaerobic fungus Orpinomyces. Purified cel lulosomes from C. thermocellum were found to hydrolyze FAXX (O-{5-O-[(E)-fe ruloyl]-alpha-L-arabinofuranosyl}-(1-->3)-O-beta-D-xylopyranosyl-(1-->4)-D- xylopyranose) and FAX(3) (5-O-[(E)-feruloyl]-[O-beta-D-xylopyranosyl-(1-->2 )]-O-alpha-L-arabinofuranosyl-[1-->3]}-O-beta-D-xylopyranosyl-(1-->4)-D-xyl opraynose), yielding ferulic acid as a product, indicating that they have f eruloyl esterase activity, Nucleotide sequences corresponding to the UDs of XynY and XynZ were cloned into Escherichia call, and the expressed protein s hydrolyzed FAXX and FAX(3). The recombinant feruloyl esterase domain of X ynZ alone (FAE(XynZ)) and with the adjacent cellulose binding domain (FAE-C BDXynZ) were characterized. FAE-CBDXynZ had a molecular mass of 45 kDa that corresponded to the expected product of the 1,203-bp gene. K-m and V-max v alues for FAX(3) were 5 mM and 12.5 U/mg, respectively, at pH 6.0 and 60 de grees C, FAX(3), a substrate similar to FAX(3) but with a p-coumaroyl group instead of a feruloyl moiety was hydrolyzed at a rate 10 times slower, The recombinant enzyme was active between pH 3 to 10 with an optimum between p H 4 to 7 and at temperatures up to 70 degrees C, Treatment of Coastal Bermu da grass with the enzyme released mainly ferulic acid and a lower amount of p-coumaric acid, FAE(XynZ) had similar properties. Removal of the 40 C-ter minal amino acids, residues 247 to 286, of FAE(XynZ) resulted in protein wi thout activity. Feruloyl esterases are believed to aid in a release of lign in from hemicellulose and may be involved in lignin solubilization. The pre sence of feruloyl esterase in the C. thermocellum cellulosome together with its other hydrolytic activities demonstrates a powerful enzymatic potentia l of this organelle in plant cell wall decomposition.