Involvement of Gln679, in addition to Trp687, in chitin-binding activity of the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12

Abstract

Chitinase A1 (ChiA1) from Bacillus circulans WL-12 comprises an N-terminal catalytic domain, two fibronectin type III domains, and a C-terminal chitin-binding domain (ChBD). The ChBD of ChiA1 (ChBDChiA1) belongs to carbohydrate-binding module (CBM) family 12 and specifically binds to insoluble or crystalline chitin. It has been suggested that tryptophan-687 (Trp687) is involved in the chitin-binding activity of this ChBD. Site-directed mutagenesis was used to identify additional amino acid residues required for chitin-binding activity of this domain. Furthermore, a total of 14 amino acid residues in ChBDChiA1 were carefully selected, and it was found that mutation of Gln679, which is not well-conserved in CBM family 12, significantly decreased the binding activity to colloidal chitin. A nuclear magnetic resonance study demonstrated that neither the Q679A nor the W687A mutation altered the overall structure of ChBDChiA1. Therefore, Gln679 was identified as a new residue that is involved in the chitin-binding activity of ChBDChiA1 in addition to Trp687. However, the mechanism of chitin binding by ChBD is still unknown.