Expression, purification and crystallization of a novel carbohydrate-binding module from the Ruminococcus...

Anaerobic bacteria organize carbohydrate-active enzymes into a multi-component complex, the cellulosome, which degrades cellulose and hemicellulose highly efficiently. Genome sequencing of Ruminococcus flavefaciens FD-1 offers extensive information on the range and diversity of the enzymatic and structural components of the cellulosome. The R. flavefaciens FD-1 genome encodes over 200 dockerin-containing proteins, most of which are of unknown function. One of these modular proteins comprises a glycoside hydrolase family 5 catalytic module (GH5) linked to an unclassified carbohydrate-binding module (CBM-Rf1) and a dockerin. The novel CBM-Rf1 has been purified and crystallized. The crystals belonged to the trigonal space group R32:H. The CBM-Rf1 structure was determined by a multiple-wavelength anomalous dispersion experiment using AutoSol from the PHENIX suite using both selenomethionyl-derivative and native data to resolutions of 2.28 and 2.0 Å, respectively.