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 Flavobacteriu m sp. (Endo-Fsp) was sequenced. The Endo-Fsp gene was overexpressed in Esch erichia coli cells, and was purified from inclusion bodies after denaturati on by 8 m urea. The renatured Endo-Fsp had the same optimum pH and substrat e 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 mutagen esis was done at conserved residues based on the three-dimensional structur e and mutagenesis of Endo-H. The mutant of Glu-128, corresponding to Glu-13 2 in Endo-H and identified as an active site residue, was inactivated. Muta genesis around the predicted active site of Endo-Fsp reduced the enzymatic activity. Moreover, the hydrolytic activity toward hybrid-type oligosacchar ides was decreased compared to that toward high-mannose type oligosaccharid es by mutagenesis of Asp-126 and Asp-127. Therefore, site-directed mutagene sis of some of these conserved residues indicates that the predicted active sites are essential to the enzymatic activity of Endo-Fsp, and may have si milar roles in catalysis as their counterparts in Endo-H.