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 journalArticlepeer-review

4 Citations (Scopus)


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
Issue number7
Publication statusPublished - Jul 2001
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Medicine(all)


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