Feruloyl Esterase Activity of the Clostridium thermocellum Cellulosome Can Be Attributed to Previously Unknown Domains of XynY and XynZ

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Abstract
The cellulosome of Clostridium thermocellum is a multiprotein complex with endo- and exocellulase, xylanase, β-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 and XynZ, respectively, have sequence homology with the sequence of a feruloyl esterase of strain PC-2 of the anaerobic fungusOrpinomyces. Purified cellulosomes from C. thermocellum were found to hydrolyze FAXX (O-{5-O-[(E)-feruloyl]-α-l-arabinofuranosyl}-(1→3)-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose) and FAX3(5-O-[(E)-feruloyl]-[O-β-d-xylopyranosyl-(1→2)]-O-α-l-arabinofuranosyl-[1→3]}-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose), yielding ferulic acid as a product, indicating that they have feruloyl esterase activity. Nucleotide sequences corresponding to the UDs of XynY and XynZ were cloned into Escherichia coli, and the expressed proteins hydrolyzed FAXX and FAX3. The recombinant feruloyl esterase domain of XynZ alone (FAEXynZ) and with the adjacent cellulose binding domain (FAE-CBDXynZ) were characterized. FAE-CBDXynZhad a molecular mass of 45 kDa that corresponded to the expected product of the 1,203-bp gene. Km andVmax values for FAX3 were 5 mM and 12.5 U/mg, respectively, at pH 6.0 and 60°C. PAX3, a substrate similar to FAX3 but with ap-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 pH 4 to 7 and at temperatures up to 70°C. Treatment of Coastal Bermuda grass with the enzyme released mainly ferulic acid and a lower amount ofp-coumaric acid. FAEXynZ had similar properties. Removal of the 40 C-terminal amino acids, residues 247 to 286, of FAEXynZ resulted in protein without activity. Feruloyl esterases are believed to aid in a release of lignin from hemicellulose and may be involved in lignin solubilization. The presence of feruloyl esterase in the C. thermocellumcellulosome together with its other hydrolytic activities demonstrates a powerful enzymatic potential of this organelle in plant cell wall decomposition.