Identification of two acidic residues involved in the catalysis of xylanase A from Streptomyces lividans.
On the basis of similarities between known xylanase sequences of the F family, three invariant acidic residues of xylanase A from Streptomyces lividans were investigated. Site-directed-mutagenesis experiments were carried out in Escherichia coli after engineering the xylanase A gene to allow its expression. Replacement of Glu-128 or Glu-236 by their isosteric form (Gln) completely abolished enzyme activity with xylan and p-nitrophenyl beta-D-cellobioside, indicating that the two substrates are hydrolysed at the same site. These two amino acids probably represent the catalytic residues. Immunological studies, which showed that the two mutants retained the same epitopes, indicate that the lack of activity is the result of the mutation rather than misfolding of the protein. Mutation D124E did not affect the kinetic parameters with xylan as substrate, but D124N reduced the Km 16-fold and the Vmax. 14-fold when compared with the wild-type enzyme. The mutations had a more pronounced effect with p-nitrophenyl beta-D-cellobioside as the substrate. Mutation D124E increased the Km and decreased the Vmax. 5-fold each, while D124N reduced the Km 4.5-fold and the Vmax. 75-fold. The mutations had no effect on the cleavage mode of xylopentaose.
Moreau
A
A
Centre de Recherche en Microbiologie Appliquée, Institut Armand-Frappier, Laval, Québec, Canada.
Roberge
M
M
Manin
C
C
Shareck
F
F
Kluepfel
D
D
Morosoli
R
R
D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D043364:Endo-1,4-beta Xylanases; D004926:Escherichia coli; D005971:Glutamates / Q000235:genetics; D018698:Glutamic Acid; D005998:Glycine / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins; D013302:Streptomyces / Q000201:enzymology*
D017931:DNA Primers; D005971:Glutamates; D011994:Recombinant Proteins; D018698:Glutamic Acid; D006026:Glycoside Hydrolases; D043364:Endo-1,4-beta Xylanases; D005998:Glycine