Site-directed mutagenesis at aspartate and glutamate residues of xylanase from Bacillus pumilus

E. P. Ko, H. Akatsuka, Hideaki Moriyama, A. Shinmyo, Y. Hata, Y. Katsube, I. Urabe, H. Okada

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)117-121
Number of pages5
JournalBiochemical Journal
Volume288
Issue number1
DOIs
StatePublished - Jan 1 1992

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Acidic Amino Acids
Mutagenesis
Bacilli
Viperidae
Site-Directed Mutagenesis
Aspartic Acid
Glutamic Acid
Mutation
Essential Amino Acids
Catalysis
Polyacrylamide Gel Electrophoresis
Amino Acid Sequence
Amino Acids
Enzymes
Proteins
Bacillus pumilus
hen egg lysozyme

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Site-directed mutagenesis at aspartate and glutamate residues of xylanase from Bacillus pumilus. / Ko, E. P.; Akatsuka, H.; Moriyama, Hideaki; Shinmyo, A.; Hata, Y.; Katsube, Y.; Urabe, I.; Okada, H.

In: Biochemical Journal, Vol. 288, No. 1, 01.01.1992, p. 117-121.

Research output: Contribution to journalArticle

Ko, EP, Akatsuka, H, Moriyama, H, Shinmyo, A, Hata, Y, Katsube, Y, Urabe, I & Okada, H 1992, 'Site-directed mutagenesis at aspartate and glutamate residues of xylanase from Bacillus pumilus', Biochemical Journal, vol. 288, no. 1, pp. 117-121. https://doi.org/10.1042/bj2880117
Ko, E. P. ; Akatsuka, H. ; Moriyama, Hideaki ; Shinmyo, A. ; Hata, Y. ; Katsube, Y. ; Urabe, I. ; Okada, H. / Site-directed mutagenesis at aspartate and glutamate residues of xylanase from Bacillus pumilus. In: Biochemical Journal. 1992 ; Vol. 288, No. 1. pp. 117-121.
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