The chitin-binding domain of a GH-18 chitinase from Vibrio harveyi is crucial for chitin-chitinase interactions.
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 ChBDVhChiAwas heterologously expressed in E. coli BL21 strain Tuner(DE3)pLacI host cells, and purified to homogeneity. CD measurements suggested that ChBDVhChiAcontained 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 ChBDVhChiAbound 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.
Biochemistry-Electrochemistry Research Unit and School of Chemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, Thailand; Center of Excellence in Advanced Functional Materials, Suranaree University of Technology, Nakhon Ratchasima, Thailand. Electronic address: wipa@sut.ac.th.
Wipa
W
Suginta
Biochemistry-Electrochemistry Research Unit and School of Chemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, Thailand.
Paknisa
P
Sirimontree
Biochemistry-Electrochemistry Research Unit and School of Chemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, Thailand.
Natchanok
N
Sritho
Department of Advanced Biosciences, Kinki University, Nara, Japan.
Takayuki
T
Ohnuma
Department of Advanced Biosciences, Kinki University, Nara, Japan.
Tamo
T
Fukamizo
D020134:Catalytic Domain; D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D014733:Vibrio / Q000201:enzymology*
D002686:Chitin; D002688:Chitinases
Bacterial GH-18 chitinases; Chitin-binding domain; Chitin-protein interactions