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Default NMR analysis of substrate binding to a two-domain chitinase: Comparison between soluble and insoluble chitins.

NMR analysis of substrate binding to a two-domain chitinase: Comparison between soluble and insoluble chitins.

Related Articles NMR analysis of substrate binding to a two-domain chitinase: Comparison between soluble and insoluble chitins.

Carbohydr Res. 2018 Feb 08;458-459:52-59

Authors: Takashima T, Ohnuma T, Fukamizo T

Abstract
CJP-4 is a two-domain chitinase from Japanese cedar (Cryptomeria japonica) pollen, consisting of an N-terminal CBM18 domain and a GH19 catalytic domain. The substrate binding to an inactive mutant protein of full-length CJP-4, in which the catalytic acid Glu108 was mutated to glutamine, CJP-4(E108Q), was analyzed by NMR spectroscopy. Based on the chemical shift perturbations of 1H-15N HSQC signals of Gly26 (CBM18 domain) and Trp185 (GH19 domain), the association constants for individual domains of CJP-4(E108Q) toward soluble chitin hexamer (GlcNAc)6 were determined to be 2300 and 3500 M-1, respectively. Isothermal titration calorimetry provided a similar association constant for (GlcNAc)6 (1980 M-1) with the one-site binding model. One (GlcNAc)6 molecule appeared to bind to a single binding site of CJP-4(E108Q), spanning from CBM18 to GH19 domains. When chitin nanofibers, insoluble chitinase substrate, were added to the CJP-4(E108Q) solution, strong line-broadening was observed for the majority of the backbone resonances in CBM18 domain but not in GH19 domain, indicating a binding preference of CBM18 domain to the insoluble chitin. We here demonstrated importance of CBM18 domain in insoluble chitin recognition based on the NMR binding data obtained for full-length CJP-4. Chitin nanofibers were found to be useful for spectroscopic observation of insoluble chitin binding to proteins.


PMID: 29459179 [PubMed - as supplied by publisher]



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