Characterization of active-site aromatic residues in xylanase A from Streptomyces lividans.
The role of four aromatic residues (W85, Y172, W266 and W274) in the structure-function relationship in xylanase A from Streptomyces lividans (XlnA) was investigated by site-directed mutagenesis where each residue was subjected to three substitutions (W85A/H/F; W266A/H/F; W274A/H/F and Y172A/F/S). These four amino acids are highly conserved among family 10 xylanases and structural data have implicated them in substrate binding at the active site. Far-UV circular dichroism spectroscopy was used to show that the overall structure of XlnA was not affected by any of these mutations. High-performance liquid chromatographic analysis of the hydrolysis products of birchwood xylan and xylopentaose showed that mutation of these aromatic residues did not alter the enzyme's mode of action. As expected, though, it did reduce the affinity of XlnA for birchwood xylan. A comparison of the kinetic parameters of different mutants at the same position demonstrated the importance of the aromatic nature of W85, Y172 and W274 in substrate binding. Replacement of these residues by a phenylalanine resulted in mutant proteins with a K(M) closer to that of the wild-type protein in comparison with the other mutations analyzed. The kinetic analysis of the mutant proteins at position W266 indicated that this amino acid is important for both substrate binding and efficient catalysis by XlnA. These studies also demonstrated the crucial role of these active site aromatic residues for the thermal stability of XlnA.
Roberge
M
M
Centre de Microbiologie et Biotechnologie, INRS-Institut Armand-Frappier, Laval, Québec, Canada.
Shareck
F
F
Morosoli
R
R
Kluepfel
D
D
Dupont
C
C
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D002942:Circular Dichroism; D017931:DNA Primers; D043364:Endo-1,4-beta Xylanases; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet; D013302:Streptomyces / Q000201:enzymology*; D014995:Xylosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D014995:Xylosidases; D043364:Endo-1,4-beta Xylanases