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

研究成果: ジャーナルへの寄稿学術誌査読

4 被引用数 (Scopus)

抄録

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.

本文言語英語
ページ(範囲)1542-1548
ページ数7
ジャーナルBioscience, Biotechnology and Biochemistry
65
7
DOI
出版ステータス出版済み - 7月 2001
外部発表はい

!!!All Science Journal Classification (ASJC) codes

  • バイオテクノロジー
  • 分析化学
  • 生化学
  • 応用微生物学とバイオテクノロジー
  • 分子生物学
  • 有機化学

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