[en] Xyl1 from Streptomyces sp. S38 belongs to the low molecular mass family 11 of endo--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.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
De Lemos Esteves, Frédéric ; Université de Liège - ULiège > Département des sciences de la vie > Physiologie végétale
Ruelle, Virginie
Lamotte-Brasseur, Josette ; Université de Liège - ULiège > Unité de cristallographie - Centre d'ingénierie des protéines
Quinting, Birgit
Frère, Jean-Marie ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Language :
English
Title :
Acidophilic adaptation of family 11 endo--1,4-xylanases: Modeling and mutational analysis
Publication date :
01 February 2004
Journal title :
Protein Science: A Publication of the Protein Society
ISSN :
0961-8368
eISSN :
1469-896X
Publisher :
Cold Spring Harbor Laboratory Press, Woodbury, United States - New York
Chen, Y.L., Tang, T.Y., and Cheng, K.J. 2001. Directed evolution to produce an alkalophilic variant from a Neocallimastix patriciarum xylanase. Can. J. Microbiol. 47: 1088-1094.
Collins, T., Meuwis, M.A., Stals, I., Claeyssens, M., Feller, G., and Gerday, C. 2002. A novel family 8 xylanase, functional and physicochemical characterization. J. Biol. Chem. 277: 35133-35139.
Fushinobu, S., Ito, K., Konno, M., Wakagi, T., and Matsuzawa, H. 1998. Crystallographic and mutational analyses of an extremely acidophilic and acid-stable xylanase: Biased distribution of acidic residues and importance of Asp37 for catalysis at low pH. Protein Eng. 11: 1121-1128.
Georis, J., Giannotta, F., Lamotte-Brasseur, J., Devreese, B., Van Beeumen, J., Granier, B., and Frere, J.M. 1999. Sequence, overproduction and purification of the family 11 endo-β-1,4-xylanase encoded by the xyl1 gene of Streptomyces sp. S38. Gene 237: 123-133.
Georis, J., de Lemos Esteves, F., Lamotte-Brasseur, J., Bougnet, V., Devreese, B., Giannotta, F., Granier, B., and Frere, J.M. 2000. An additional aromatic interaction improves the thermostability and thermophilicity of a mesophilic family 11 xylanase: Structural basis and molecular study. Protein Sci. 9: 466-475.
Gilkes, N.R., Henrissat, B., Kilburn, D.G., Miller Jr., R.C., and Warren, R.A. 1991. Domains in microbial β-1, 4-glycanases: Sequence conservation, function, and enzyme families. Microbiol. Rev. 55: 303-315.
Gruber, K., Klintschar, G., Hayn, M., Schlacher, A., Steiner, W., and Kratky, C. 1998. Thermophilic xylanase from Thermomyces lanuginosus: High-resolution X-ray structure and modeling studies. Biochemistry 37: 13475-13485.
Harris, G.W., Pickersgill, R.W., Connerton, I., Debeire, P., Touzel, J.P., Breton, C., and Perez, S. 1997. Structural basis of the properties of an industrially relevant thermophilic xylanase. Proteins 29: 77-86.
Henrissat, B. and Davies, G. 1997. Structural and sequence-based classification of glycoside hydrolases. Curr. Opin. Struct. Biol. 7: 637-644.
Hopwood, D.A., Bidd, M.J., Chater, K.F., Kieser, T., Bruton, C.J., Kieser, H.M., Lydiate, D.J., Smith, C.P., Ward, J.M., and Shrempf, H. 1985. Genetic manipulation of Streptomyces. A laboratory manual. The John Innes Foundation, Norwich, UK.
Ito, K., Iwashita, K., and Iwano, K. 1992. Cloning and sequencing of the xynC gene encoding acid xylanase of Aspergillus kawachii. Biosci. Biotechnol. Biochem. 56: 1338-1340.
Joshi, M.D., Sidhu, G., Pot, I., Brayer, G.D., Withers, S.G., and McIntosh, L.P. 2000. Hydrogen bonding and catalysis: A novel explanation for how a single amino acid substitution can change the pH optimum of a glycosidase. J. Mol. Biol. 299: 255-279.
Joshi, M.D., Sidhu, G., Nielsen, J.E., Brayer, G.D., Withers, S.G., and McIntosh, L.P. 2001. Dissecting the electrostatic interactions and pH-dependent activity of a family 11 glycosidase. Biochemistry 40: 10115-10139.
Kluepfel, D., Vats-Mehta, S., Aumont, F., Shareck, F., and Morosoli, R. 1990. Purification and characterization of a new xylanase (xylanase B) produced by Streptomyces lividans 66. Biochem. J. 267: 45-50.
Krengel, U., and Dijkstra, B.W. 1996. Three-dimensional structure of endo-1,4-β-xylanase I from Aspergillus niger: Molecular basis for its low pH optimum. J. Mol. Biol. 263: 70-78.
Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.
Mazy-Servais, C., Moreau, A., Gerard, C., and Dusart, J. 1996. Cloning and nucleotide sequence of a xylanase-encoding gene from Streptomyces sp. strain EC3. DNA Seq. 6: 147-158.
Mehler, E.L. and Guarnieri, F. 1999. A self-consistent, microenvironment modulated screened coulomb potential approximation to calculate pH-dependent electrostatic effects in proteins. Biophys. J. 77: 3-22.
Miller, G.L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31: 426-428.
Morosoli, R., Bertrand, J.L., Mondou, F., Shareck, F., and Kluepfel, D. 1986. Purification and properties of a xylanase from Streptomyces lividans. Biochem. J. 239: 587-592.
Muilu, J., Torronen, A., Perakyla, M., and Rouvinen, J. 1998. Functional conformational changes of endo-1,4-xylanase II from Trichoderma reesei: A molecular dynamics study. Proteins 31: 434-444.
Pace, C.N. 1986. Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol. 131: 266-280.
Pearlman, D., Case, D.A., Caldwell, J.A., Ross, W.S., Cheathman, T.E., Ferguson, D.M., Seibel, G.L., Singh, U.C., Weiner, P.K., and Kollman, P.A. 1995. AMBER 4.1. University of California, San Francisco, CA.
Rekker, R.F. 1977. The hydrophobic fragmental constant. Elsevier, Amsterdam.
-. 1979. The hydrophobic fragmental constant: An extension to a 1000 data point set. Eur. J. Med. Chem. 14: 479-488.
Sabini, E., Sulzenbacher, G., Dauter, M., Dauter, Z., Jorgensen, P.L., Schulein, M., Dupont, C., Davies, G.J., and Wilson, K.S. 1999. Catalysis and specificity in enzymatic glycoside hydrolysis: A 2,5B conformation for the glycosyl-enzyme intermediate revealed by the structure of the Bacillus agaradhaerens family 11 xylanase. Chem. Biol. 6: 483-492.
Sabini, E., Wilson, K.S., Danielsen, S., Schulein, M., and Davies, G.J. 2001. Oligosaccharide binding to family 11 xylanases: Both covalent intermediate and mutant product complexes display (2,5)B conformations at the active centre. Acta Crystallogr. D Biol. Crystallogr. 57: 1344-1347.
Sapag, A., Wouters, J., Lambert, C., de Ioannes, P., Eyzaguirre, J., and Depiereux, E. 2002. The endoxylanases from family 11: Computer analysis of protein sequences reveals important structural and phylogenetic relationships. J. Biotechnol. 95: 109-131.
Shih, H.H., Brady, J., and Karplus, M. 1985. Structure of proteins with single-site mutations: A minimum perturbation approach. Proc. Natl. Acad. Sci. 82: 1697-1700.
Sidhu, G., Withers, S.G., Nguyen, N.T., McIntosh, L.P., Ziser, L., and Brayer, G.D. 1999. Sugar ring distortion in the glycosyl-enzyme intermediate of a family G/11 xylanase. Biochemistry 38: 5346-5354.
Singh, S., Madlala, A.M., and Prior, B.A. 2003. Thermomyces lanuginosus: Properties of strains and their hemicellulases. FEMS Microbiol. Rev. 27: 3-16.
Sunna, A. and Antranikian, G. 1997. Xylanolytic enzymes from fungi and bacteria. Crit. Rev. Biotechnol. 17: 39-67.
Torronen, A. and Rouvinen, J. 1995. Structural comparison of two major endo-1,4-xylanases from Trichoderma reesei. Biochemistry 34: 847-856.
-. 1997. Structural and functional properties of low molecular weight endo-1,4-β-xylanases. J. Biotechnol. 57: 137-149.
Torronen, A., Mach, R.L., Messner, R., Gonzalez, R., Kalkkinen, N., Harkki, A., and Kubicek, C.P. 1992. The two major xylanases from Trichoderma reesei: Characterization of both enzymes and genes. Biotechnology 10: 1461-1465.
Torronen, A., Kubicek, C.P., and Henrissat, B. 1993. Amino acid sequence similarities between low molecular weight endo-1,4-β-xylanases and family H cellulases revealed by clustering analysis. FEBS Lett. 321: 135-139.
Torronen, A., Harkki, A., and Rouvinen, J. 1994. Three-dimensional structure of endo-1,4-β-xylanase II from Trichoderma reesei: Two conformational states in the active site. EMBO J. 13: 2493-2501.
Wakarchuk, W.W., Campbell, R.L., Sung, W.L., Davoodi, J., and Yaguchi, M. 1994. Mutational and crystallographic analyses of the active site residues of the Bacillus circulans xylanase. Protein Sci. 3: 467-475.
Wong, K.K.Y. and Saddler, J.N. 1992. Trichoderma xylanases, their properties and application. Crit. Rev. Biotechnol. 12: 413-435.
Wong, K.K., Tan, L.U., and Saddler, J.N. 1988. Multiplicity of β-1,4-xylanase in microorganisms: Functions and applications. Microbiol. Rev. 52: 305-317.
Wouters, J., Georis, J., Engher, D., Vandenhaute, J., Dusart, J., Frere, J.M., Depiereux, E., and Charlier, P. 2001. Crystallographic analysis of family 11 endo-β-1,4-xylanase Xyl1 from Streptomyces sp. S38. Acta Crystallogr. D Biol. Crystallogr. 57: 1813-1819.
Yang, R.C., MacKenzie, C.R., Bilous, D., and Narang, S.A. 1989. Hyperexpression of a Bacillus circulans xylanase gene in Escherichia coli and characterization of the gene product. Appl. Environ. Microbiol. 55: 1192-1195.
