Role of tryptophan residues in a class V Chitinase from Nicotiana tabacum

Naoyuki Umemoto, Takayuki Ohnuma, Henri Urpilainen, Takanori Yamamoto, Tomoyuki Numata, Tamo Fukamizo

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Tryptophan residues located in the substrate-binding cleft of a class V chitinase from Nicotiana tabacum (NtChiV) were mutated to alanine and phenylalanine (W190F, W326F, W190F/W326F, W190A, W326A, and W190A/W326A), and the mutant enzymes were characterized to define the role of the tryptophans. The mutations of Trp326 lowered thermal stability by 5- 7 °C, while the mutations of Trp190 lowered stability only by 2-4 °C. The Trp326 mutations strongly impaired enzymatic activity, while the effects of the Trp190 mutations were moderate. The experimental data were rationalized based on the crystal structure of NtChiV in a complex with (GlcNAc)4, in which Trp190 is exposed to the solvent and involved in face-to-face stacking interaction with the +2 sugar, while Trp326 is buried inside but interacts with the -2 sugar through hydrophobicity. HPLC analysis of anomers of the enzymatic products suggested that Trp190 specifically recognizes the β-anomer of the +2 sugar. The strong effects of the Trp326 mutations on activity and stability suggest multiple roles of the residue in stabilizing the protein structure, in sugar residue binding at subsite -2, and probably in maintaining catalytic efficiency by providing a hydrophobic environment for proton donor Glu115.

Original languageEnglish
Pages (from-to)778-784
Number of pages7
JournalBioscience, Biotechnology and Biochemistry
Volume76
Issue number4
DOIs
Publication statusPublished - Apr 30 2012
Externally publishedYes

Fingerprint

Chitinases
Tryptophan
Sugars
Tobacco
Mutation
Hydrophobicity
Phenylalanine
Alanine
Protons
Thermodynamic stability
Hydrophobic and Hydrophilic Interactions
Crystal structure
Hot Temperature
High Pressure Liquid Chromatography
Substrates
Enzymes
Proteins

All Science Journal Classification (ASJC) codes

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

Cite this

Role of tryptophan residues in a class V Chitinase from Nicotiana tabacum. / Umemoto, Naoyuki; Ohnuma, Takayuki; Urpilainen, Henri; Yamamoto, Takanori; Numata, Tomoyuki; Fukamizo, Tamo.

In: Bioscience, Biotechnology and Biochemistry, Vol. 76, No. 4, 30.04.2012, p. 778-784.

Research output: Contribution to journalArticle

Umemoto, Naoyuki ; Ohnuma, Takayuki ; Urpilainen, Henri ; Yamamoto, Takanori ; Numata, Tomoyuki ; Fukamizo, Tamo. / Role of tryptophan residues in a class V Chitinase from Nicotiana tabacum. In: Bioscience, Biotechnology and Biochemistry. 2012 ; Vol. 76, No. 4. pp. 778-784.
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