Purification and crystallographic studies of a putative carbohydrate-binding module from the Ruminococcus flavefaciens FD-1 endoglucanase Cel5A.

Ruminant herbivores meet their carbon and energy requirements from a symbiotic relationship with cellulosome-producing anaerobic bacteria that efficiently degrade plant cell-wall polysaccharides. The assembly of carbohydrate-active enzymes (CAZymes) into cellulosomes enhances protein stability and enzyme synergistic interactions. Cellulosomes comprise diverse CAZymes displaying a modular architecture in which a catalytic domain is connected, via linker sequences, to one or more noncatalytic carbohydrate-binding modules (CBMs). CBMs direct the appended catalytic modules to their target substrates, thus facilitating catalysis. The genome of the ruminal cellulolytic bacterium Ruminococcus flavefaciens strain FD-1 contains over 200 modular proteins containing the cellulosomal signature dockerin module. One of these is an endoglucanase Cel5A comprising two family 5 glycoside hydrolase catalytic modules (GH5) flanking an unclassified CBM (termed CBM-Rf2) and a C-terminal dockerin. This novel CBM-Rf2 has been purified and crystallized, and data from cacodylate-derivative crystals were processed to 1.02 and 1.29 Å resolution. The crystals belonged to the orthorhombic space group P212121. The CBM-Rf2 structure was solved by a single-wavelength anomalous dispersion experiment at the As edge.