Mutational analysis of amino acid residues involved in catalytic activity of a family 18 chitinase from tulip bulbs

Keisuke Suzukawa, Takeshi Yamagami, Takayuki Ohnuma, Hideki Hirakawa, Satoru Kuhara, Yoichi Aso, Masatsune Ishiguor

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9 Citations (Scopus)

Abstract

We expressed chitinase-1 (TBC-1) from tulip bulbs (Tulipa bakeri) in E. coli cells and used site-directed mutagenesis to identify amino acid residues essential for catalytic activity. Mutations at Glu-125 and Trp-251 completely abolished enzyme activity, and activity decreased with mutations at Asp-123 and Trp-172 when glycolchitin was the substrate. Activity changed with the mutations of Trp-251 to one of several amino acids with side-chains of little hydrophobicity, suggesting that hydrophobic interaction of Trp-251 is important for the activity. Molecular dynamics (MD) simulation analysis with hevamine as the model compound showed that the distance between Asp-123 and Glu-125 was extended by mutation of Trp-251. Kinetic studies of Trp-251-mutated chitinases confirmed these various phenomena. The results suggested that Glu-125 and Trp-251 are essential for enzyme activity and that Trp-251 had a direct role in ligand binding.

Original languageEnglish
Pages (from-to)341-346
Number of pages6
JournalBioscience, Biotechnology and Biochemistry
Volume67
Issue number2
DOIs
Publication statusPublished - Jan 1 2003

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Analytical Chemistry
  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Molecular Biology
  • Organic Chemistry

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