4.7 Article

The chitin-binding domain of a GH-18 chitinase from Vibrio harveyi is crucial for chitin-chitinase interactions

Journal

INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
Volume 93, Issue -, Pages 1111-1117

Publisher

ELSEVIER SCIENCE BV
DOI: 10.1016/j.ijbiomac.2016.09.066

Keywords

Bacterial GH-18 chitinases; Chitin-binding domain; Chitin-protein interactions

Funding

  1. Suranaree University of Technology
  2. Office of the Higher Education Commission under the NRU project of Thailand
  3. Royal Golden Jubilee Ph.D. scholarship [PHD/0021/2552]
  4. Thailand Research Fund
  5. Suranaree University of Technology [BRG578001, SUT1-102-56-24-19]

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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 (alpha/beta)(8) TIM barrel catalytic domain; and iii) the alpha + beta insertion domain. In this study, we cloned the gene fragment encoding the chitin-binding domain of VhChiA, termed ChBD(VhChiA). The recombinant ChBD(VhChiA) was heterologously expressed in E. coli BL21 strain Tuner(DE3)pLacI host cells, and purified to homogeneity. CD measurements suggested that ChBD(VhChiA) contained beta-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 ChBD(VhChiA) bound to both alpha- and beta-chitins, with the greatest affinity for beta-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. (C) 2016 Elsevier B.V. All rights reserved.

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