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 journalArticlepeer-review

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 - 2012
Externally publishedYes

All Science Journal Classification (ASJC) codes

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

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