Identification of amino acid residues essential for the substrate specificity of Flavobacterium sp. endo-beta-N-acetylglucosaminidase.
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.
Fujita
K
K
Department of Life Sciences, Faculty of Agriculture, Kagawa University, Japan.
Nakatake
R
R
Yamabe
K
K
Watanabe
A
A
Asada
Y
Y
Takegawa
K
K
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005417:Flavobacterium / Q000201:enzymology* / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D007700:Kinetics; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D004269:DNA, Bacterial; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase