Site-directed mutagenesis at aspartate and glutamate residues of xylanase from Bacillus pumilus.
To elucidate the reaction mechanism of xylanase, the identification of amino acids essential for its catalysis is of importance. Studies have indicated the possibility that the reaction mechanism of xylanase is similar to that of hen's egg lysozyme, which involves acidic amino acid residues. On the basis of this assumption, together with the three-dimensional structure of Bacillus pumilus xylanase and its amino acid sequence similarity to other xylanases of different origins, three acidic amino acids, namely Asp-21, Glu-93 and Glu-182, were selected for site-directed mutagenesis. The Asp residue was altered to either Ser or Glu, and the Glu residues to Ser or Asp. The purified mutant xylanases D21E, D21S, E93D, E93S, E182D and E182S showed single protein bands of about 26 kDa on SDS/PAGE. C.d. spectra of these mutant enzymes show no effect on the secondary structure of xylanase, except that of D21E, which shows a little variation. Furthermore, mutations of Glu-93 and Glu-182 resulted in a drastic decrease in the specific activity of xylanase as compared with mutation of Asp-21. On the basis of these results we propose that Glu-93 and Glu-182 are the best candidates for the essential catalytic residues of xylanase.
Ko
E P
EP
Department of Biotechnology, Faculty of Engineering, Osaka University, Japan.
Akatsuka
H
H
Moriyama
H
H
Shinmyo
A
A
Hata
Y
Y
Katsube
Y
Y
Urabe
I
I
Okada
H
H
D001224:Aspartic Acid / Q000235:genetics*; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002942:Circular Dichroism; D003198:Computer Simulation; D005971:Glutamates / Q000235:genetics*; D018698:Glutamic Acid; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism; D005779:Immunodiffusion; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D043325:Xylan Endo-1,3-beta-Xylosidase
D005971:Glutamates; D001224:Aspartic Acid; D018698:Glutamic Acid; D006026:Glycoside Hydrolases; D043325:Xylan Endo-1,3-beta-Xylosidase