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

doi:10.1128/JB.182.5.1346-1351.2000

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Journal of Bacteriology, March 2000, p. 1346-1351, Vol. 182, No. 5
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

David L. Blum, Irina A. Kataeva, Xin-Liang Li, and Lars G. Ljungdahl^*

Department of Biochemistry and Molecular Biology and the Center for Biological Resource Recovery, The University of Georgia, Athens, Georgia 30602

Received 3 September 1999/Accepted 9 December 1999

The cellulosome of Clostridium thermocellum is a multiprotein complex with endo- and exocellulase, xylanase, $beta$ -glucanase,and acetyl xylan esterase activities. XynY and XynZ, componentsof the cellulosome, are composed of several domains includingxylanase domains and domains of unknown function (UDs). Databasesearches revealed that the C- and N-terminal UDs of XynY and XynZ,respectively, have sequence homology with the sequence of a feruloylesterase of strain PC-2 of the anaerobic fungus Orpinomyces. Purifiedcellulosomes from C. thermocellum were found to hydrolyze FAXX(O-{5-O-[(E)-feruloyl]- $alpha$ -L-arabinofuranosyl}-(1 $right-arrow$ 3)-O- $beta$ -D-xylopyranosyl-(1 $right-arrow$ 4)-D-xylopyranose)and FAX₃ (5-O-[(E)-feruloyl]-[O- $beta$ -D-xylopyranosyl-(1 $right-arrow$ 2)]-O- $alpha$ -L-arabinofuranosyl-[1 $right-arrow$ 3]}-O- $beta$ -D-xylopyranosyl-(1 $right-arrow$ 4)-D-xylopyranose),yielding ferulic acid as a product, indicating that they haveferuloyl esterase activity. Nucleotide sequences correspondingto the UDs of XynY and XynZ were cloned into Escherichia coli,and the expressed proteins hydrolyzed FAXX and FAX₃. The recombinantferuloyl esterase domain of XynZ alone (FAE_XynZ) and with theadjacent cellulose binding domain (FAE-CBD_XynZ) were characterized.FAE-CBD_XynZ had a molecular mass of 45 kDa that corresponded tothe expected product of the 1,203-bp gene. K_m and V_max valuesfor FAX₃ were 5 mM and 12.5 U/mg, respectively, at pH 6.0 and60°C. PAX₃, a substrate similar to FAX₃ but with a p-coumaroylgroup instead of a feruloyl moiety was hydrolyzed at a rate 10times slower. The recombinant enzyme was active between pH 3 to10 with an optimum between pH 4 to 7 and at temperatures up to70°C. Treatment of Coastal Bermuda grass with the enzyme releasedmainly ferulic acid and a lower amount of p-coumaric acid. FAE_XynZhad 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 ligninfrom hemicellulose and may be involved in lignin solubilization.The presence of feruloyl esterase in the C. thermocellum cellulosometogether with its other hydrolytic activities demonstrates a powerfulenzymatic potential of this organelle in plant cell walldecomposition.

^* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, A214 Life Sciences Building, TheUniversity of Georgia, Athens, GA 30602-7229. Phone: (706) 542-7640.Fax: (706) 542-2222. E-mail: larsljd{at}arches.uga.edu.

Present address: University of California $---$ San Diego, Department of Medicine, La Jolla, CA 92093-0822.

Journal of Bacteriology, March 2000, p. 1346-1351, Vol. 182, No. 5
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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