Identification of amino acid residues essential for the substrate specificity of Flavobacterium sp. Endo-β-N-acetylglucosaminidase

Kiyotaka Fujita, Ryo Ich Nakatake, Kayo Yamabe, Akira Watanabe, Yasuhiko Asada, Kaoru Takegawa

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

The gene encoding the endo-β-N-acetylglucosaminidase from Flavobacterium sp. (Endo-Fsp) was sequenced. The Endo-Fsp gene was overexpressed in Escherichia coli cells, and was purified from inclusion bodies after denaturation by 8 M urea. The renatured Endo-Fsp had the same optimum pH and substrate specificity as the native enzyme. Endo-Fsp had 60% sequence identity with the endo-β-N-acetylglucosaminidase from Streptomyces plicatus (Endo-H), and the putative catalytic residues were conserved. Site-directed mutagenesis was done at conserved residues based on the three-dimensional structure and mutagenesis of Endo-H. The mutant of Glu-128, corresponding to Glu-132 in Endo-H and identified as an active site residue, was inactivated. Mutagenesis around the predicted active site of Endo-Fsp reduced the enzymatic activity. Moreover, the hydrolytic activity toward hybrid-type oligosaccharides was decreased compared to that toward high-mannose type oligosaccharides by mutagenesis of Asp-126 and Asp-127. Therefore, site-directed mutagenesis of some of these conserved residues indicates that the predicted active sites are essential to the enzymatic activity of Endo-Fsp, and may have similar roles in catalysis as their counterparts in Endo-H.

Original languageEnglish
Pages (from-to)1542-1548
Number of pages7
JournalBioscience, Biotechnology and Biochemistry
Volume65
Issue number7
DOIs
Publication statusPublished - Jul 1 2001
Externally publishedYes

Fingerprint

Flavobacterium
Acetylglucosaminidase
Mutagenesis
Essential Amino Acids
Substrate Specificity
Catalytic Domain
Site-Directed Mutagenesis
Oligosaccharides
Amino Acids
Substrates
Inclusion Bodies
Streptomyces
Mannose
Catalysis
Genes
Urea
Denaturation
Gene encoding
Escherichia coli
Enzymes

All Science Journal Classification (ASJC) codes

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

Cite this

Identification of amino acid residues essential for the substrate specificity of Flavobacterium sp. Endo-β-N-acetylglucosaminidase. / Fujita, Kiyotaka; Nakatake, Ryo Ich; Yamabe, Kayo; Watanabe, Akira; Asada, Yasuhiko; Takegawa, Kaoru.

In: Bioscience, Biotechnology and Biochemistry, Vol. 65, No. 7, 01.07.2001, p. 1542-1548.

Research output: Contribution to journalArticle

Fujita, Kiyotaka ; Nakatake, Ryo Ich ; Yamabe, Kayo ; Watanabe, Akira ; Asada, Yasuhiko ; Takegawa, Kaoru. / Identification of amino acid residues essential for the substrate specificity of Flavobacterium sp. Endo-β-N-acetylglucosaminidase. In: Bioscience, Biotechnology and Biochemistry. 2001 ; Vol. 65, No. 7. pp. 1542-1548.
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