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Volume 69, Issue 1


Identification of critical amino acid residues for chloride binding of Bacillus licheniformis trehalose-6-phosphate hydrolase

Ping-Lin Ong
  • Department of Biochemical Science and Technology, National Chiayi University, 300 Syuefu Road, Chiayi County, 60004, Taiwan
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/ Tzu-Ting Chuang
  • Department of Biochemical Science and Technology, National Chiayi University, 300 Syuefu Road, Chiayi County, 60004, Taiwan
  • Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi County, 60004, Taiwan
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/ Tzu-Fan Wang / Long-Liu Lin
Published Online: 2013-11-15 | DOI: https://doi.org/10.2478/s11756-013-0290-3


Based on sequence alignment of selected Cl− dependent and independent glycoside hydrolase family 13 enzymes, two invariant residues (Arg201 and Asn347) and one tyrosine (Tyr365) that might be responsible for the binding of Bacillus licheniformis trehalose-6-phosphate hydrolase (BlTreA) to chloride ion were identified. The role of these three residues was further explored by mutational and biophysical analyses. The mutant enzymes (R201Q/E/K, N327Q/D/K, and Y365A/R) and BlTreA were individually overexpressed in Escherichia coli M15 host cells and purified by one-step nickel affinity chromatography on Ni-NTA resin. The purified BlTreA and Y365A had a specific activity of 236.9 and 47.6 U/mg protein, respectively. The remaining enzymes lost their hydrolase activity completely even in the presence of high salt. With the exception of Y365A, all mutant enzymes did not have the ability to bind fluoride, chloride and nitrate anions. Structural analyses showed that the circular dichroism spectra of the mutant proteins were consistent with those of BlTreA. However, wild-type and mutant enzymes displayed a slight difference in the profiles of intrinsic tryptophan fluorescence. Collectively, these results clearly indicate that Arg201 and Agr327 residues might play an essential role in chloride binding of BlTreA.

Keywords: Bacillus licheniformis; trehalose-6-phosphate hydrolase; site-directed mutagenesis; chloride binding; arginine

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About the article

Published Online: 2013-11-15

Published in Print: 2014-01-01

Citation Information: Biologia, Volume 69, Issue 1, Pages 1–9, ISSN (Online) 1336-9563, DOI: https://doi.org/10.2478/s11756-013-0290-3.

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© 2013 Slovak Academy of Sciences. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. BY-NC-ND 3.0

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