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Role of Tyr-435 of Vibrio harveyi Chitinase A in Chitin Utilization

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Abstract

Vibrio harveyi chitinase A or VhChiA (EC.3.2.1.14) is a member of GH-18 chitinases that catalyzes chitin degradation from marine biomaterials. Our earlier structural data of VhChiA suggested that Tyr-435 marks the ending of subsite +2 and may influence binding of the interacting substrate at the aglycone binding sites. This study reports the effects of Tyr-435 using site-directed mutagenesis technique. Mutation of Tyr-435 to Ala (mutant Y435A) enhanced both binding and catalytic efficiency of VhChiA, whereas substitution of Tyr-435 to Trp (mutant Y435W) lessened the ability of the enzyme to bind and hydrolyze chitin substrates. The increased activity of Y435A can be explained by partial removal of a steric clash around subsite (+2), thereby allowing a chitin chain to move beyond or to access the enzyme’s active site from the aglycone side more straightforwardly.

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Abbreviations

ChBD:

chitin binding domain

DNS:

3,5-dinitrosalicylic acid

GlcNAcn :

β-1-4 linked oligomers of N-acetyl-d-glucosamine residues, where n = 1–6

IPTG:

isopropyl thio-β-d-galactoside

PMSF:

phenylmethylsulphonylfluoride

pNP:

p-nitrophenol

VhChiA:

Vibrio harveyi chitinase A

WT:

wild-type chitinase

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Acknowledgments

This work was supported financially by The Thailand Research Fund through TRF-CHE Research Grant for Mid-Career University (Grant no RMU4980028) and Suranaree University of Technology, Thailand.

Conflict of interest

None declared.

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Authors and Affiliations

  1. Biochemistry-Electrochemistry Research Unit, Schools of Chemistry and Biochemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, 30000, Thailand

    Natchanok Sritho & Wipa Suginta

Authors
  1. Natchanok Sritho
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  2. Wipa Suginta
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Corresponding author

Correspondence to Wipa Suginta.

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Supplementary Fig. S1

Michaeli–Menten plots of VhChiA variants. For pNP-GlcNAc2, the reaction was set up with concentrations of the substrate varied from 0 to 500 μM. For chitooligosaccharides, the reaction mixture (200 μL), containing 0–500 μM GlcNAc3–6 and 0.8 nM enzyme in 0.1 M sodium acetate buffer, pH 5.0, was incubated at 37 °C for 15 min. After heating at 100 °C for 10 min, the entire reaction mixture was used in the DNS assay. For colloidal and glycol chitins, the reaction was carried out the same way as for chitooligosaccharides, but concentrations of the chitins were varied from 0% to 5% (w/v). The kinetic values were evaluated the nonlinear regression function obtained from the GraphPad Prism v5.0. (JPEG 99 kb)

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Sritho, N., Suginta, W. Role of Tyr-435 of Vibrio harveyi Chitinase A in Chitin Utilization. Appl Biochem Biotechnol 166, 1192–1202 (2012). https://doi.org/10.1007/s12010-011-9504-8

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  • DOI: https://doi.org/10.1007/s12010-011-9504-8

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