Two new major subunits in the cellulosome of Clostridium thermocellum: xyloglucanase Xgh74A and endoxylanase Xyn10D

1 Research Group Microbial Biotechnology, Technische Universität München, Am Hochanger 4, D-85350 Freising-Weihenstephan, Germany 2 Institute of Molecular Genetics, Russian Academy of Science, Kurchatov Sq., 123182 Moscow, Russia 3 GSF - National Research Center for Environment and Health, Institute for Ecological Chemistry, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany Correspondence Wolfgang H. Schwarz schwarz{at}mikro.biologie.tu-muenchen.de The structure and enzymic activity of xyloglucanase Xgh74A and endoxylanase Xyn10D, components in the cellulosomes of cellulose-grown Clostridium thermocellum , were determined. Xyn10D is a thermostable endo-1,4- -xylanase with a module composition identical to Xyn10C (CBM22-GH10-Doc). It hydrolyses xylan and mixed-linkage 1,3-1,4- -glucan with a temperature optimum of 80 °C. Xyloglucanase Xgh74A contains a catalytic module of GHF74 in addition to a C-terminal dockerin module. It hydrolyses every fourth -1,4-glucan bond in the xyloglucan backbone, thus producing decorated cellotetraose units. Its low activity on CMC and lack of activity on amorphous cellulose indicates recognition of the xylosidic side chains present in xyloglucan, which is readily hydrolysed (295 U mg –1 ). The pattern of the hydrolysis products from tamarind xyloglucan resembles that of other GHF74 xyloglucan endoglucanases. The data indicate that Xgh74A and Xyn10D contribute to the in vivo degradation of the hemicelluloses xyloglucan and xylan by the cellulosome of C. thermocellum . Xgh74A is the first xyloglucanase identified in C. thermocellum and the only enzyme in the cellulosome that hydrolyses tamarind xyloglucan. Abbreviations: CBM, carbohydrate-binding module; CMC, carboxymethylcellulose; ESI/MS, electrospray ionization/mass spectrometry; GH/GHF, glycosyl hydrolase/glycosyl hydrolase family; PASC, phosphoric-acid-swollen cellulose The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AJ585344 ( xghA ) and AJ585345 ( xynD ).