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

Research output: Contribution to journalArticle

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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

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Tulipa
Chitinases
Enzyme activity
Catalyst activity
Amino Acids
Mutagenesis
Mutation
Hydrophobicity
Hydrophobic and Hydrophilic Interactions
Escherichia coli
Molecular dynamics
Ligands
Kinetics
Essential Amino Acids
Computer simulation
Substrates
Enzymes
Molecular Dynamics Simulation
Site-Directed Mutagenesis

All Science Journal Classification (ASJC) codes

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

Cite this

Mutational analysis of amino acid residues involved in catalytic activity of a family 18 chitinase from tulip bulbs. / Suzukawa, Keisuke; Yamagami, Takeshi; Ohnuma, Takayuki; Hirakawa, Hideki; Kuhara, Satoru; Aso, Yoichi; Ishiguor, Masatsune.

In: Bioscience, Biotechnology and Biochemistry, Vol. 67, No. 2, 01.01.2003, p. 341-346.

Research output: Contribution to journalArticle

Suzukawa, Keisuke ; Yamagami, Takeshi ; Ohnuma, Takayuki ; Hirakawa, Hideki ; Kuhara, Satoru ; Aso, Yoichi ; Ishiguor, Masatsune. / Mutational analysis of amino acid residues involved in catalytic activity of a family 18 chitinase from tulip bulbs. In: Bioscience, Biotechnology and Biochemistry. 2003 ; Vol. 67, No. 2. pp. 341-346.
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