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Protein Science (2004), 13:1209-1218. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Acidophilic adaptation of family 11 endo-{beta}-1,4-xylanases: Modeling and mutational analysis

Frédéric de Lemos Esteves1, Virginie Ruelle2, Josette Lamotte-Brasseur1, Birgit Quinting3 and Jean-Marie Frère1

1 Centre d’Ingénierie des Protéines, Institut de Chimie, B6a, and
2 Laboratoire de Spectrométrie de Masse, Université de Liège, Sart Tilman, B-4000 Liège, Belgium
3 Centre d’Economie Rurale, Division Immunologie Animale, Marloie, B-6900, Belgium

(RECEIVED December 10, 2003; FINAL REVISION January 22, 2004; ACCEPTED February 1, 2004)



Abstract

Xyl1 from Streptomyces sp. S38 belongs to the low molecular mass family 11 of endo-{beta}-1,4-xylanases. Its three-dimensional structure has been solved at 2.0 Å and its optimum temperature and pH for enzymatic activity are 60°C and 6.0, respectively. Aspergillus kawachii xylanase XynC belongs to the same family but is an acidophilic enzyme with an optimum pH of 2.0. Structural comparison of Xyl1 and XynC showed differences in residues surrounding the two glutamic acid side chains involved in the catalysis that could be responsible for the acidophilic adaptation of XynC. Mutations W20Y, N48D, A134E, and Y193W were introduced by site-directed mutagenesis and combined in multiple mutants. Trp 20 and Tyr 193 are involved in substrate binding. The Y193W mutation inactivated Xyl1 whereas W20Y decreased the optimum pH of Xyl1 to 5.0 and slightly increased its specific activity. The N48D mutation also decreased the optimum pH of Xyl1 by one unit. The A134E substitution did not induce any change, but when combined with N48D, a synergistic effect was observed with a 1.4 unit decrease in the optimum pH. Modeling showed that the orientations of residue 193 and of the fully conserved Arg 131 are different in acidophilic and "alkaline" xylanases whereas the introduced Tyr 20 probably modifies the pKa of the acid–base catalyst via residue Asn 48. Docking of a substrate analog in the catalytic site highlighted striking differences between Xyl1 and XynC in substrate binding. Hydrophobicity calculations showed a correlation between acidophilic adaptation and a decreased hydrophobicity around the two glutamic acid side chains involved in catalysis.

Keywords: endo-{beta}-1,4-xylanase; acidophilicity; site-directed mutagenesis; structural analysis; hydrophobicity; docking

Reprint requests to: Jean-Marie Frère, Centre d’Ingénierie des Protéines, Institut de Chimie, B6a, Université de Liège, Sart Tilman, B-4000 Liège, Belgium: e-mail: jmfrere{at}ulg.ac.be; fax: 00-32-4-366-33-64.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03556104.


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