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

Abstract

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.

The Function of CBM32 in Alginate Lyase VxAly7B on the Activity on Both Soluble Sodium Alginate and Alginate Gel

“…Although the mechanism of the spontaneous cleavage of VxAly7B is still unclear, we found that the reaction was slow and the increase in temperature and the extension of time will accelerate its progress (Figure 1D and Supplementary Figure 4). The internal cleavage of CBM54 of laminarinase Lic16A and chitinase ChiW is a common feature, and the spontaneous cleavage of CBM54 is essential for the combination of the catalytic module and insoluble polysaccharides (Itoh et al, 2014;Dvortsov et al, 2018). The N-terminal extension (CBM_4_9 module) of AlyA and aly-SJ02 helps the protein to be folded correctly and then breaks, which is a common phenomenon of PL18 family alginate lyases (Matsushima et al, 2010;Dong et al, 2014).…”