Title
The Chitin-Binding Domain of a GH-18 Chitinase from Vibrio harveyi is Crucial for Chitin-Chitinase Interactions
Author
Wipa Suginta, Paknisa Sirimontree, Natchanok Sritho, Takayuki Ohnuma, Tamo Fukamizo
Year
2016
Journal
International Journal of Biological Macromolecules
Abstract
Vibrio harveyi chitinase A (VhChiA) is a GH-18 glycosyl hydrolase with a structure containing three distinct domains: i) the N-terminal chitin-binding domain; ii) the (α/β)8TIM barrel catalytic domain; and iii) the α + β insertion domain. In this study, we cloned the gene fragment encoding the chitin-binding domain of VhChiA, termed ChBDVhChiA. The recombinant ChBDVhChiA was heterologously expressed in E. coli BL21 strain Tuner(DE3)pLacI host cells, and purified to homogeneity. CD measurements suggested that ChBDVhChiA contained β-sheets as major structural components and fluorescence spectroscopy showed that the protein domain was folded correctly, and suitable for functional characterization. Chitin binding assays showed that ChBDVhChiA bound to both α- and β-chitins, with the greatest affinity for β-colloidal chitin, but barely bound to polymeric chitosan. These results identified the tandem N-acetamido functionality on chitin chains as the specific sites of enzyme-substrate interactions. The binding affinity of the isolated domain was significantly lower than that of intact VhChiA, suggesting that the catalytic domain works synergistically with the chitin-binding domain to guide the polymeric substrate into the substrate binding cleft. These data confirm the physiological role of the chitin-binding domain of the marine bacterial GH-18 chitinase A in chitin-chitinase interactions.
Instrument
J-720
Keywords
Circular dichroism, Secondary structure, Biochemistry