Aromatic residues within the substrate-binding cleft of Bacillus circulans chitinase A1 are essential for hydrolysis of crystalline chitin

Takeshi Watanabe, Yumiko Ariga, Urara Sato, Tadayuki Toratani, Masayuki Hashimoto, Naoki Nikaidou, Yuichiro Kezuka, Takamasa Nonaka, Junji Sugiyama

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95 Citations (Scopus)

Abstract

Bacillus circulans chitinase A1 (ChiA1) has a deep substrate-binding cleft on top of its (β/α)8-barrel catalytic domain and an interaction between the aromatic residues in this cleft and bound oligosaccharide has been suggested. To study the roles of these aromatic residues, especially in crystalline-chitin hydrolysis, site-directed mutagenesis of these residues was carried out. Y56A and W53A mutations at subsites - 5 and - 3, respectively, selectively decreased the hydrolysing activity against highly crystalline β-chitin. W164A and W285A mutations at subsites + 1 and + 2, respectively, decreased the hydrolysing activity against crystalline β-chitin and colloidal chitin, but enhanced the activities against soluble substrates. These mutations increased the Km-value when reduced (GlcNAc)5 (where GlcNAc is N-acetylglucosamine) was used as the substrate, but decreased substrate inhibition observed with wild-type ChiA1 at higher concentrations of this substrate. In contrast with the selective effect of the other mutations, mutations of W433 and Y279 at subsite - 1 decreased the hydrolysing activity drastically against all substrates and reduced the Kcat-value, measured with 4-methylumbelliferyl chitotrioside to 0.022% and 0.59% respectively. From these observations, it was concluded that residues Y56 and W53 are only essential for crystalline-chitin hydrolysis. W164 and W285 are very important for crystalline-chitin hydrolysis and also participate in hydrolysis of other substrates. W433 and Y279 are both essential for catalytic reaction as predicted from the structure.

Original languageEnglish
Pages (from-to)237-244
Number of pages8
JournalBiochemical Journal
Volume376
Issue number1
DOIs
Publication statusPublished - 2003 Nov 15

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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