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

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 fungus Orpinomyces . 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 FAX 3 (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 FAX 3 . The recombinant feruloyl esterase domain of XynZ alone (FAE XynZ ) and with the adjacent cellulose binding domain (FAE-CBD XynZ ) were characterized. FAE-CBD XynZ had a molecular mass of 45 kDa that corresponded to the expected product of the 1,203-bp gene. K m and V max values for FAX 3 were 5 mM and 12.5 U/mg, respectively, at pH 6.0 and 60°C. PAX 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 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 of p -coumaric acid. FAE XynZ had similar properties. Removal of the 40 C-terminal amino acids, residues 247 to 286, of FAE XynZ 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. thermocellum cellulosome together with its other hydrolytic activities demonstrates a powerful enzymatic potential of this organelle in plant cell wall decomposition.