Specific Characterization of Substrate and Inhibitor Binding Sites of a Glycosyl Hydrolase Family 11 Xylanase fromAspergillus niger
2002; Elsevier BV; Volume: 277; Issue: 46 Linguagem: Inglês
10.1074/jbc.m205657200
ISSN1083-351X
AutoresTariq A. Tahir, Jean‐Guy Berrin, Ruth H. Flatman, Alain Roussel, Peter Roepstorff, Gary Williamson, Nathalie Juge,
Tópico(s)Studies on Chitinases and Chitosanases
ResumoThe importance of aromatic and charged residues at the surface of the active site of a family 11 xylanase fromAspergillus niger was evaluated using site-directed mutagenesis. Ten mutant proteins were heterologously produced inPichia pastoris, and their biochemical properties and kinetic parameters were determined. The specific activity of the Y6A, Y10A, Y89A, Y164A, and W172A mutant enzymes was drastically reduced. The low specific activities of Y6A and Y89A were entirely accounted for by a change in k cat and K m, respectively, whereas the lower values of Y10A, Y164A, and W172A were due to a combination of increased K m and decreasedk cat. Tyr6, Tyr10, Tyr89, Tyr164, and Trp172 are proposed as substrate-binding residues, a finding consistent with structural sequence alignments of family 11 xylanases and with the three-dimensional structure of the A. niger xylanase in complex with the modeled xylobiose. All other variants, D113A, D113N, N117A, E118A, and E118Q, retained full wild-type activity. Only N117A lost its sensitivity toxylanase inhibitor protein I (XIP-I), a protein inhibitor isolated from wheat, and this mutation did not affect the fold of the xylanase as revealed by circular dichroism. The N117A variant showed kinetics, pH stability, hydrolysis products pattern, substrate specificity, and structural properties identical to that of the wild-type xylanase. The loss of inhibition, as measured in activity assays, was due to abolition of the interaction between XIP-I and the mutant enzyme, as demonstrated by surface plasmon resonance and electrophoretic titration. A close inspection of the three-dimensional structure of A. niger xylanase suggests that the binding site of XIP-I is located at the conserved "thumb" hairpin loop of family 11 xylanases. The importance of aromatic and charged residues at the surface of the active site of a family 11 xylanase fromAspergillus niger was evaluated using site-directed mutagenesis. Ten mutant proteins were heterologously produced inPichia pastoris, and their biochemical properties and kinetic parameters were determined. The specific activity of the Y6A, Y10A, Y89A, Y164A, and W172A mutant enzymes was drastically reduced. The low specific activities of Y6A and Y89A were entirely accounted for by a change in k cat and K m, respectively, whereas the lower values of Y10A, Y164A, and W172A were due to a combination of increased K m and decreasedk cat. Tyr6, Tyr10, Tyr89, Tyr164, and Trp172 are proposed as substrate-binding residues, a finding consistent with structural sequence alignments of family 11 xylanases and with the three-dimensional structure of the A. niger xylanase in complex with the modeled xylobiose. All other variants, D113A, D113N, N117A, E118A, and E118Q, retained full wild-type activity. Only N117A lost its sensitivity toxylanase inhibitor protein I (XIP-I), a protein inhibitor isolated from wheat, and this mutation did not affect the fold of the xylanase as revealed by circular dichroism. The N117A variant showed kinetics, pH stability, hydrolysis products pattern, substrate specificity, and structural properties identical to that of the wild-type xylanase. The loss of inhibition, as measured in activity assays, was due to abolition of the interaction between XIP-I and the mutant enzyme, as demonstrated by surface plasmon resonance and electrophoretic titration. A close inspection of the three-dimensional structure of A. niger xylanase suggests that the binding site of XIP-I is located at the conserved "thumb" hairpin loop of family 11 xylanases. Endo-(1,4)-β-xylanases (EC 3.2.1.8) depolymerize the xylan backbone by cleaving the β-(1,4) glycosidic bonds betweend-xylose residues in the main chain to produce short xylooligosaccharides (1Biely P. Trends Biotechnol. 1985; 3: 286-290Abstract Full Text PDF Scopus (799) Google Scholar, 2Biely P. Coughlan M.P. Hazlewood G.P. Hemicellulose and hemicellulases. Portland Press, London1993: 29-51Google Scholar). Based on amino acid sequence similarities, the endoxylanases have been grouped into two classes: family 10 and family 11 (3–5). The two families have different molecular structures, molecular weights, and catalytic properties (6Jeffries T.W. Curr. Opin. Biotechnol. 1996; 7: 337-342Crossref PubMed Scopus (107) Google Scholar, 7Biely P. Vrsansaka M. Tenkanen M. Kluepfel D. J. Biotechnol. 1997; 57: 151-166Crossref PubMed Scopus (491) Google Scholar, 8Sapag A. Wouters J. Lambert C. de Ioannes P. Eyzaguirre J. Depiereux E. J. 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The active site contains two conserved glutamate residues located on either side of the extended open cleft, which have been identified as the nucleophilic and acid/base catalysts (28Törrönen A. Rouvinen J. J. Biotechnol. 1997; 57: 137-149Crossref PubMed Scopus (151) Google Scholar). Only a few ligand-enzyme complexes (13Wakarchuk W.W. Campbell R.L. Sung W.L. Davoodi J. Yaguchi M. Protein Sci. 1994; 3: 467-475Crossref PubMed Scopus (283) Google Scholar, 16Sabini E. Sulzenbacher G. Dauter M. Dauter Z. Jorgensen P.L. Schulein M. Dupont C. Davies G.J. Wilson K.S. Chem. Biol. 1999; 6: 483-492Abstract Full Text PDF PubMed Scopus (127) Google Scholar,17Sabini E. Wilson K.S. Danielsen S. Schulein M. Davies G.J. Acta Crystallogr. Sect. D. 2001; 57: 1344-1347Crossref PubMed Scopus (44) Google Scholar, 29Havukainen R. Törrönen A. Laitinen T. Rouvinen J. Biochemistry. 1996; 35: 9617-9624Crossref PubMed Scopus (72) Google Scholar, 30Sidhu G. Withers S.G. Nguyen N.T. McIntosh L.P. Ziser L. Brayer G.D. Biochemistry. 1999; 38: 5346-5354Crossref PubMed Scopus (171) Google Scholar) have been crystallized. The subsites that bind the glycone or aglycone regions of the substrate are prefixed by − and +, respectively, and the number that identifies each subsite is related to the proximity to the site of bond cleavage (31Davies G.J. Wilson K. Henrissat B. Biochem. J. 1997; 321: 557-559Crossref PubMed Scopus (839) Google Scholar). The cleavage by definition takes place between subsites −1 and +1. Subsites −3, −2, and −1 are well characterized by the inspection of the complex structures (13Wakarchuk W.W. Campbell R.L. Sung W.L. Davoodi J. Yaguchi M. Protein Sci. 1994; 3: 467-475Crossref PubMed Scopus (283) Google Scholar, 16Sabini E. Sulzenbacher G. Dauter M. Dauter Z. Jorgensen P.L. Schulein M. Dupont C. Davies G.J. Wilson K.S. Chem. Biol. 1999; 6: 483-492Abstract Full Text PDF PubMed Scopus (127) Google Scholar, 17Sabini E. Wilson K.S. Danielsen S. Schulein M. Davies G.J. Acta Crystallogr. Sect. D. 2001; 57: 1344-1347Crossref PubMed Scopus (44) Google Scholar), but the characterization of the aglycone subsites is based only on modeling (22Törrönen A. Rouvinen J. Biochemistry. 1995; 34: 847-856Crossref PubMed Scopus (216) Google Scholar, 26Gruber K. Klintschar G. Hayn M. Schlacher A. Steiner W. Kratky C. Biochemistry. 1998; 37: 13475-13485Crossref PubMed Scopus (130) Google Scholar). Aspergillus niger xylanase is a 20-kDa family 11 xylanase with a pI and a pH optimum of 3.5 (32Hessing J.G.M. van Rotterdam C. Verbakel J.M.A. Roza M. Maat J. van Gorcom R.F.M. van den Hondel C.A.M.J.J. Curr. Genet. 1994; 26: 228-232Crossref PubMed Scopus (52) Google Scholar, 33Berrin J.G. Williamson G. Puigserver A. Chaix J.C. McLauchlan W.R. Juge N. Protein Expression Purif. 2000; 19: 179-187Crossref PubMed Scopus (71) Google Scholar) for which an x-ray crystal structure is available (24Krengle U. Dijkstra B.W. J. Mol. Biol. 1996; 263: 70-78Crossref PubMed Scopus (158) Google Scholar). The active site is located within a deep and long cleft, which is lined with many aromatic amino acid residues and is large enough to accommodate at least four xylose residues (24Krengle U. Dijkstra B.W. J. Mol. Biol. 1996; 263: 70-78Crossref PubMed Scopus (158) Google Scholar). The two conserved catalytic residues, Glu79 and Glu170, face each other on opposite sides (24Krengle U. Dijkstra B.W. J. Mol. Biol. 1996; 263: 70-78Crossref PubMed Scopus (158) Google Scholar). This fungal enzyme has been studied for its role as a bread improver (32Hessing J.G.M. van Rotterdam C. Verbakel J.M.A. Roza M. Maat J. van Gorcom R.F.M. van den Hondel C.A.M.J.J. Curr. Genet. 1994; 26: 228-232Crossref PubMed Scopus (52) Google Scholar, 34Hessing, J. G. M., van Gorcom, R. F. M., Verbakel, J. M. A., Roza, M., and Maat, J. (December 26, 1991) PCT International Patent Application WO/91/19782.Google Scholar, 35Maat J. Roza M. Verbakel J. Stam H. Santos da Silva M.J. Bosse M. Egmond M.R. Hagemans M.L.D. Gorcom R.F.M.V. Hessing J.G.M. van der Hondel C.A.M.J.J. Rotterdam C.V. Visser J. Beldman G. Kusters-van Somerson M.A. Voragen A.G.J. Xylans and Xylanases. Elsevier Science Publishers B.V., Amsterdam1992: 349-360Google Scholar, 36Debyser W. Peumans W.J. Van Damme E.J.M. Delcour J.A. J. Cereal Sci. 1999; 30: 39-43Crossref Scopus (123) Google Scholar), in wheat processing (37Christophersen C. Andersen E. Jakobsen T.S. Wagner P. Starch/Staerke. 1997; 49: 5-12Crossref Scopus (65) Google Scholar), and as a supplement in animal feed (38Bedford M.R. Classen H.L. Visser J. Beldman G. Kusters-van Somerson M.A. Voragen A.G.J. Xylan and Xylanases. Elsevier Science Publishers B. V., Amsterdam1992: 361-370Google Scholar, 39Graham H. Inborr J. Visser J. Beldman G. Kusters-van Someren M.A. Voragen A.G.J. Xylans and Xylanases. Elsevier Science Publishers B. V., Amsterdam1992: 535-538Google Scholar).The presence of protein inhibitors of endo-(1,4)-β-d-xylanase in cereals was first reported in wheat flour protein extracts (40Debyser W. Derdelinckx G. Delcour J.A. J. Am. Soc. Brew. Chem. 1997; 55: 153-156Google Scholar, 41Debyser, W., and Delcour, J. A. (November 5, 1998) WO9849278.Google Scholar, 42Rouau X. Surget A. J. Cereal Sci. 1998; 28: 63-70Crossref Scopus (39) Google Scholar). To date, two types of endoxylanase inhibitors with different structures and specificities have been described. The first type are xylanaseinhibitor protein (XIP) 1The abbreviations used are: XIP-I, xylanase inhibitor protein I; BCX, Bacillus circulans xylanase; HPAEC, high pressure anion exchange chromatography; IC50, inhibitor concentration for 50% inhibition; IEF, isoelectrofocusing; MALDI-MS, matrix-assisted laser desorption ionization/mass spectrometry; RU, resonance units; TAXI, Triticum aestivumL. xylanase inhibitor; xylanase, endo-1,4-β-d-xylanase; XylA, A. niger xylanase; XynA, Thermomyces lanuginosus xylanase; XYNI, Trichoderma reesei xylanase I, XYNII, Trichoderma reesei xylanase II; Xyn11, Bacillus agaradhaerens xylanase 1The abbreviations used are: XIP-I, xylanase inhibitor protein I; BCX, Bacillus circulans xylanase; HPAEC, high pressure anion exchange chromatography; IC50, inhibitor concentration for 50% inhibition; IEF, isoelectrofocusing; MALDI-MS, matrix-assisted laser desorption ionization/mass spectrometry; RU, resonance units; TAXI, Triticum aestivumL. xylanase inhibitor; xylanase, endo-1,4-β-d-xylanase; XylA, A. niger xylanase; XynA, Thermomyces lanuginosus xylanase; XYNI, Trichoderma reesei xylanase I, XYNII, Trichoderma reesei xylanase II; Xyn11, Bacillus agaradhaerens xylanase-like inhibitors and have been isolated from wheat (43McLauchlan W.R. Garcia-Conesa M.T. Williamson G. Roza M. Ravenstein P. Maat J. Biochem. J. 1999; 338: 441-446Crossref PubMed Scopus (169) Google Scholar, 44Hessing, M., and Happe, R. P. (February 16, 2000) EP 0979830 A1.Google Scholar) and rye (45McLauchlan, W. R., Flatman, R. H., Sancho, A. I., Kakuta, J., Faulds, C. B., Elliot, G. O., Kroon, P. A., Furniss, C. S. M., Juge, N., Ravestein, P., Williamson, G., Proceedings of the 2nd European Symposium on Enzymes in Grain Processing, Simoinen, T., Tenkanen, M., 2000, 55, 61, Technical Research Center of Finland (VTT), Helsinki, Finland.Google Scholar). They are monomeric glycosylated proteins with molecular masses of ∼29 kDa and pI values of 8.7–8.9. The second type are the T riticumaestivum L. xylanase inhibitor (TAXI)-like inhibitors (36Debyser W. Peumans W.J. Van Damme E.J.M. Delcour J.A. J. Cereal Sci. 1999; 30: 39-43Crossref Scopus (123) Google Scholar). They are high pI, nonglycosylated proteins with molecular masses of ∼40 kDa. At least two inhibitors of this type (TAXI I and TAXI II) with different pI values (8.8 and ∼9.3, respectively) and varying specificities toward different endoxylanases have been identified in wheat (46Gebruers K. Debyser W. Goesaert H. Proost P. Van Damme J. Delcour J.A. Biochem. J. 2001; 353: 239-244Crossref PubMed Scopus (122) Google Scholar). The N-terminal amino acid sequences of TAXI-I and TAXI-II showed a high degree of identity, but there was no similarity to XIP-I. The TAXI-like inhibitors are believed to be active against bacterial and fungal family 11 endoxylanases but not against family 10 endoxylanases (46Gebruers K. Debyser W. Goesaert H. Proost P. Van Damme J. Delcour J.A. Biochem. J. 2001; 353: 239-244Crossref PubMed Scopus (122) Google Scholar, 47Goesaert H. Debyser W. Gebruers K. Proost P. Van Damme J. Delcour J.A. Cereal Chem. 2001; 78: 453-457Crossref Scopus (48) Google Scholar). XIP-I inhibited both family-10 and 11 fungal xylanases apart from the family 10Aspergillus aculeatus xylanase with K ivalues ranging from 3.4 to 610 nm, but bacterial family 10 and 11 xylanases were not inhibited (48Flatman R. McLauchlan W.R. Juge N. Furniss C.S. Berrin J.G. Hughes R.K. Manzanares P. Ladbury J.E. O'Brien R. Williamson G. Biochem. J. 2002; 365: 773-781Crossref PubMed Google Scholar).We have previously reported the production and characterization of theA. niger xylanase in Pichia pastoris and shown that the recombinant enzyme was similar to the native enzyme and was competitively inhibited by XIP-I with a K i = 350 nm (33Berrin J.G. Williamson G. Puigserver A. Chaix J.C. McLauchlan W.R. Juge N. Protein Expression Purif. 2000; 19: 179-187Crossref PubMed Scopus (71) Google Scholar, 48Flatman R. McLauchlan W.R. Juge N. Furniss C.S. Berrin J.G. Hughes R.K. Manzanares P. Ladbury J.E. O'Brien R. Williamson G. Biochem. J. 2002; 365: 773-781Crossref PubMed Google Scholar). The chosen strategy for mutational analysis ofA. niger xylanase took advantage of data available on theA. niger xylanase-XIP-I complex such as (a) kinetics of inhibition, (b) titration curves, (c) isothermal calorimetry data, and also the availability of (d) the three-dimensional structure of A. nigerxylanase (Protein Data Bank code 1UKR) and (e) an efficient heterologous system for expression of A. niger xylanase. The inhibition mechanism of XIP-I against family 11 fungal A. niger xylanase has been studied in detail (48Flatman R. McLauchlan W.R. Juge N. Furniss C.S. Berrin J.G. Hughes R.K. Manzanares P. Ladbury J.E. O'Brien R. Williamson G. Biochem. J. 2002; 365: 773-781Crossref PubMed Google Scholar). The inhibition is pH-dependent in the range of 4 to 7 as determined by activity assays and titration curves, illustrating the importance of electrostatic interactions in the strength of the interaction. Moreover, isothermal titration calorimetry of the XIP-I:A. niger xylanase complex showed the formation of a complex with a stoichiometry of (1:1) and a heat capacity change of −1.38 kJ/mol, suggesting that the interaction was enthalpy-driven (48Flatman R. McLauchlan W.R. Juge N. Furniss C.S. Berrin J.G. Hughes R.K. Manzanares P. Ladbury J.E. O'Brien R. Williamson G. Biochem. J. 2002; 365: 773-781Crossref PubMed Google Scholar). Aromatic and charged amino acids are believed to play a pivotal role in protein-protein interactions by forming hydrophobic stacking and electrostatic interactions, respectively, with the target ligand. Because the inhibition is competitive, residues were selected near the active site. The three-dimensional structure of the A. nigerxylanase revealed several aromatic and charged residues (Tyr6, Tyr10, Tyr89, Asp113, Asn117, Asp118, Tyr164, and Trp172) on the surface around the binding cleft. The structural analysis of the enzyme showed that these target residues are not part of the hydrogen bond network in the vicinity of the two catalytic residues (Glu79 and Glu170) (24Krengle U. Dijkstra B.W. J. Mol. Biol. 1996; 263: 70-78Crossref PubMed Scopus (158) Google Scholar). To investigate the importance of these residues in binding to XIP-I, appropriate mutations in the xylanase were constructed, and the biochemical properties of the mutated enzymes were evaluated in terms of kinetic properties and ability to interact with XIP-I.Our results indicate that Asn117 is critical for the XIP-I binding capacity of the enzyme. The mutational analysis also allowed a better understanding of the role of individual residues involved in a number of subsites in the active-site cleft of a family 11 glycosyl hydrolase.DISCUSSIONThe active site of glycosyl hydrolases often contain aromatic residues, such as tyrosine and tryptophan, which hydrophobically stack against sugar rings, as well as side chains, which hydrogen bind to hydroxyl groups of the substrate. The replacement of the aromatic amino acid residues, Tyr6, Tyr10, Tyr89, Tyr164, and Trp172 with Ala significantly reduced the enzyme activity of A. niger glycosyl hydrolase family 11 xylanase. These mutations did not affect the fold of the xylanase as revealed by circular dichroism spectra of these variants. Although these residues are not essential for the hydrolytic reactionper se (24Krengle U. Dijkstra B.W. J. Mol. Biol. 1996; 263: 70-78Crossref PubMed Scopus (158) Google Scholar), the present findings indicate that they play an important role in ligand binding and catalysis.In family 11 xylanases, the resolution of a limited number of structures of enzyme-ligand complexes has revealed several active site residues that have the potential to play key roles in the interaction of subsites with xylose moieties. The structure of a catalytically inactive Bacillus circulans xylanase (BCX) mutant incubated with xylotetraose has been characterized, but only a portion of the ligand (a xylobiose moiety) could be observed at the active site (subsites −1 and −2) (13Wakarchuk W.W. Campbell R.L. Sung W.L. Davoodi J. Yaguchi M. Protein Sci. 1994; 3: 467-475Crossref PubMed Scopus (283) Google Scholar). Recently the structure of the glycosyl-enzyme intermediate has been obtained providing more insight into the −1 subsite (30Sidhu G. Withers S.G. Nguyen N.T. McIntosh L.P. Ziser L. Brayer G.D. Biochemistry. 1999; 38: 5346-5354Crossref PubMed Scopus (171) Google Scholar). To evaluate the number of subsites in the active site of Trichoderma reesei xylanases, different xylo-oligomer models were docked to the active site of both XYNI and XYNII (22Törrönen A. Rouvinen J. Biochemistry. 1995; 34: 847-856Crossref PubMed Scopus (216) Google Scholar). XYNI probably has space for three subsites (−2, −1, +1), whereas the active site of XYNII is longer and may have five subsites (−2, −1, +1, +2, +3). Structures of covalently attached epoxyalkyl glycosides to the active site residues of XYNII are also available, but these include only one xylose residue (29Havukainen R. Törrönen A. Laitinen T. Rouvinen J. Biochemistry. 1996; 35: 9617-9624Crossref PubMed Scopus (72) Google Scholar). Modeling studies with a xyloheptaose in the active site of Thermomyces lanuginosusxylanase (XynA) identify residues involved in subsite −2 (26Gruber K. Klintschar G. Hayn M. Schlacher A. Steiner W. Kratky C. Biochemistry. 1998; 37: 13475-13485Crossref PubMed Scopus (130) Google Scholar). Recently the structure of an inactive mutant of the Xyn11 fromBacillus agaradhaerens in complex with xylotriose has been obtained (17Sabini E. Wilson K.S. Danielsen S. Schulein M. Davies G.J. Acta Crystallogr. Sect. D. 2001; 57: 1344-1347Crossref PubMed Scopus (44) Google Scholar). The interactions are very similar to that described at subsites −2 and −1 for the covalent intermediate complex (16Sabini E. Sulzenbacher G. Dauter M. Dauter Z. Jorgensen P.L. Schulein M. Dupont C. Davies G.J. Wilson K.S. Chem. Biol. 1999; 6: 483-492Abstract Full Text PDF PubMed Scopus (127) Google Scholar), with the addition of the interactions for the −3 subsite.The active site residues in the A. niger xylanase correspond remarkably closely in both position and orientation with the residues that contact the sugar rings in these complexes. In Fig.5, structural alignment of the above xylanases with that of A. niger enzyme showed that four of the A. niger xylanase variants (Y10A, Y89A, Y164A, and W172A) have been mutated at positions corresponding to residues that have been involved in discrete subsites of other family 11 xylanases. Tyr10 corresponds to Trp9BCX, Tyr9XYNI, Trp18XYNII, Trp18XynA, and Trp19Xyn11, all involved in stacking interaction with the xylose ring in substite −2 of these xylanases (13Wakarchuk W.W. Campbell R.L. Sung W.L. Davoodi J. Yaguchi M. Protein Sci. 1994; 3: 467-475Crossref PubMed Scopus (283) Google Scholar, 16Sabini E. Sulzenbacher G. Dauter M. Dauter Z. Jorgensen P.L. Schulein M. Dupont C. Davies G.J. Wilson K.S. Chem. Biol. 1999; 6: 483-492Abstract Full Text PDF PubMed Scopus (127) Google Scholar, 17Sabini E. Wilson K.S. Danielsen S. Schulein M. Davies G.J. Acta Crystallogr. Sect. D. 2001; 57: 1344-1347Crossref PubMed Scopus (44) Google Scholar, 22Törrönen A. Rouvinen J. Biochemistry. 1995; 34: 847-856Crossref PubMed Scopus (216) Google Scholar, 26Gruber K. Klintschar G. Hayn M. Schlacher A. Steiner W. Kratky C. Biochemistry. 1998; 37: 13475-13485Crossref PubMed Scopus (130) Google Scholar). Tyr89 corresponds to Tyr96XYNII thought to determine subsite +3 of the Trichoderma reseei enzyme (22Törrönen A. Rouvinen J. Biochemistry. 1995; 34: 847-856Crossref PubMed Scopus (216) Google Scholar). Tyr164 is equivalent to Tyr166BCXand Tyr172XynA, both shown to form hydrogen bonds with the substrate in subsite −2 of the xylanases (13Wakarchuk W.W. Campbell R.L. Sung W.L. Davoodi J. Yaguchi M. Protein Sci. 1994; 3: 467-475Crossref PubMed Scopus (283) Google Scholar, 26Gruber K. Klintschar G. Hayn M. Schlacher A. Steiner W. Kratky C. Biochemistry. 1998; 37: 13475-13485Crossref PubMed Scopus (130) Google Scholar). Trp172 corresponds to Trp166XYNIand Tyr179XYNII, which are thought to determine subsites +1 and +2 of XYNI and XYNII enzyme, respectively (22Törrönen A. Rouvinen J. Biochemistry. 1995; 34: 847-856Crossref PubMed Scopus (216) Google Scholar). Moreover, the oxygen atom OH of residue Tyr6 is in the same position as atom Glu17Xyn11 OE1 (not shown), which, in B. agaradhaerens, is involved in one of the solvent-mediated hydrogen bonds associating the −3 substite sugar (17Sabini E. Wilson K.S. Danielsen S. Schulein M. Davies G.J. Acta Crystallogr. Sect. D. 2001; 57: 1344-1347Crossref PubMed Scopus (44) Google Scholar). These findings are consistent with the substantial decrease in the activity of the Y6A, Y10A, Y89A, Y164A, and W172A mutants against xylan, although these residues do not necessarily play equivalent roles in different family 11 enzymes. In Fig.6, the two xylose rings found at substites −1 and −2 of the BCX mutant complexed with a substrate (13Wakarchuk W.W. Campbell R.L. Sung W.L. Davoodi J. Yaguchi M. Protein Sci. 1994; 3: 467-475Crossref PubMed Scopus (283) Google Scholar) are superimposed to the A. niger xylanase three-dimensional structure. In this view Tyr6, Tyr10, and Tyr164 are in close contact with the ligand, also suggesting their involvement in substites of the A. nigerenzyme. Mutational analyses of active site residues have been carried on other family 11 xylanases, but, to our knowledge, none of the present mutations were reported apart from Tyr166BCX (corresponding to Tyr164in A. niger xylanase), which, replaced by Phe in B. circulans xylanase, led to a small decrease of enzyme activity (13Wakarchuk W.W. Campbell R.L. Sung W.L. Davoodi J. Yaguchi M. Protein Sci. 1994; 3: 467-475Crossref PubMed Scopus (283) Google Scholar). The substrate binding cleft of A. niger xylanase contains at least four xylose-binding subsites (24Krengle U. Dijkstra B.W. J. Mol. Biol. 1996; 263: 70-78Crossref PubMed Scopus (158) Google Scholar). Our data clearly show that Tyr10 and Tyr164 are likely to play an important role in ligand binding in the glycone region of the substrate binding cleft (probably at subsite −2), whereas Trp172 and Tyr89 are involved in the aglycone subsites (probably at subsites +1 and +2, respectively) of A. niger xylanase. Interestingly, the finding that Tyr6played a role in enzyme activity together with its position in the three-dimensional structure might indicate the presence of another subsite (−3) in the substrate binding cleft of A. nigerxylanase.Figure 6Molecular surface representation of theA. niger xylanase active cleft.Two sugar residues have been modeled into the active site by superimposing the structure of B. circulans xylanase complexed with xylobiose (PDB code 1BCX) on top of the A. niger xylanase. The molecular surface of the protein is shown ingray, and the oligosaccharide is represented as a wire model in light green. The target residues for site-directed mutagenesis are highlighted (the aromatic residuesY6, Y10, Y89, Y164, andW172 are shown in yellow and N117, D113, and E118 in turquoise). The nucleophile (E79) and acid/base catalyst (E170, in magenta) are located on opposite sides of the active site cleft. The thumb region (indark green) points back toward the bottom of the cleft. The drawing was generated with SPOCK (62Christopher J.A. SPOCK Manual. Center for Macromolecular Design, Texas A&M University, College Station, TX1998Google Scholar), and single-letter amino acid codes are used.View Large Image Figure ViewerDownload (PPT)Interestingly, all of the fungal xylanases tested so far are inhibited by XIP-I apart from the family 10 A. aculeatus xylanase, whereas none of the bacterial enzymes are (48Flatman R. McLauchlan W.R. Juge N. Furniss C.S. Berrin J.G. Hughes R.K. Manzanares P. Ladbury J.E. O'Brien R. Williamson G. Bioche
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