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

Research output: Contribution to journalArticle

85 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

Fingerprint

Chitinases
Chitin
Bacilli
Bacillus
Hydrolysis
Crystalline materials
Substrates
Mutation
Acetylglucosamine
Mutagenesis
Site-Directed Mutagenesis
Oligosaccharides
Catalytic Domain

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Watanabe, Takeshi ; Ariga, Yumiko ; Sato, Urara ; Toratani, Tadayuki ; Hashimoto, Masayuki ; Nikaidou, Naoki ; Kezuka, Yuichiro ; Nonaka, Takamasa ; Sugiyama, Junji. / Aromatic residues within the substrate-binding cleft of Bacillus circulans chitinase A1 are essential for hydrolysis of crystalline chitin. In: Biochemical Journal. 2003 ; Vol. 376, No. 1. pp. 237-244.
@article{84407f9a48144b17acc5b8bba33a1366,
title = "Aromatic residues within the substrate-binding cleft of Bacillus circulans chitinase A1 are essential for hydrolysis of crystalline chitin",
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.",
author = "Takeshi Watanabe and Yumiko Ariga and Urara Sato and Tadayuki Toratani and Masayuki Hashimoto and Naoki Nikaidou and Yuichiro Kezuka and Takamasa Nonaka and Junji Sugiyama",
year = "2003",
month = "11",
day = "15",
doi = "10.1042/BJ20030419",
language = "English",
volume = "376",
pages = "237--244",
journal = "Biochemical Journal",
issn = "0264-6021",
publisher = "Portland Press Ltd.",
number = "1",

}

Aromatic residues within the substrate-binding cleft of Bacillus circulans chitinase A1 are essential for hydrolysis of crystalline chitin. / Watanabe, Takeshi; Ariga, Yumiko; Sato, Urara; Toratani, Tadayuki; Hashimoto, Masayuki; Nikaidou, Naoki; Kezuka, Yuichiro; Nonaka, Takamasa; Sugiyama, Junji.

In: Biochemical Journal, Vol. 376, No. 1, 15.11.2003, p. 237-244.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Aromatic residues within the substrate-binding cleft of Bacillus circulans chitinase A1 are essential for hydrolysis of crystalline chitin

AU - Watanabe, Takeshi

AU - Ariga, Yumiko

AU - Sato, Urara

AU - Toratani, Tadayuki

AU - Hashimoto, Masayuki

AU - Nikaidou, Naoki

AU - Kezuka, Yuichiro

AU - Nonaka, Takamasa

AU - Sugiyama, Junji

PY - 2003/11/15

Y1 - 2003/11/15

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0344825227&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0344825227&partnerID=8YFLogxK

U2 - 10.1042/BJ20030419

DO - 10.1042/BJ20030419

M3 - Article

VL - 376

SP - 237

EP - 244

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - 1

ER -