Disturbed processing of the carbohydrate-binding module of family 54 significantly impairs its binding to polysaccharides.

Carbohydrate-binding modules of the family 54 (CBM54) are characterized by spontaneous rupture of the peptide bond Asn266-Ser267 (numbering corresponds to that of laminarinase Lic16A of Ruminiclostridium thermocellum). As a result of processing, two parts are formed noncovalently connected to each other. Here, to gain insights into the functional significance of the internal cleavage, we made modifications of the family-conserved processing site in CBM54 of Lic16A. We demonstrate that the introduced mutations of residues G264 or S267 to alanine block the hydrolysis. Unprocessed, modified proteins bind insoluble polysaccharides pustulan, chitin, xylan, Avicel, phosphoric acid-swollen cellulose, and β-d-glucan of the yeast cell wall 2-20 times worse than the wild-type module. The data obtained are the first to demonstrate that processing is important for the functioning of CBM54s.