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

被引:4
作者
Fujita, K [1 ]
Nakatake, R [1 ]
Yamabe, K [1 ]
Watanabe, A [1 ]
Asada, Y [1 ]
Takegawa, K [1 ]
机构
[1] Kagawa Univ, Fac Agr, Dept Life Sci, Miki, Kagawa 7610795, Japan
关键词
endo-beta-N-acetylglucosaminidase; Flavobacterium sp; site-directed mutagenesis; substrate specificity;
D O I
10.1271/bbb.65.1542
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The gene encoding the endo-beta -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-beta -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.
引用
收藏
页码:1542 / 1548
页数:7
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