Protein Engineering, Vol. 12, No. 3, 251-257,
March 1999
© 1999 Oxford University Press
Characterization of active-site aromatic residues in xylanase A from Streptomyces lividans
Centre de Microbiologie et Biotechnologie, INRS–Institut Armand-Frappier, Laval, Québec, H7N 4Z3, Canada
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Abstract |
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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 KM 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.
Keywords: family 10 hydrolase/glycosyl hydrolase/site-directed mutagenesis/structure–function/xylanase
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Introduction |
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Xylanases (EC 3.2.1.8) catalyze the hydrolysis of xylan, a complex heteropolymer found in plant cell walls. On the basis of sequence homology of their catalytic domains, xylanases have been classified into families 10 and 11 of the 66 families of glycosyl hydrolases (Henrissat and Bairoch, 1998
). Xylanase A from Streptomyces lividans (XlnA) is a 47 kDa secreted protein composed of two domains: a 33 kDa catalytic domain responsible for the xylanolytic activity and a 14 kDa xylan-binding domain involved in the specific binding to the insoluble substrate (Dupont et al., 1998). The catalytic domain of XlnA belongs to family 10 of glycosyl hydrolases (Shareck et al., 1991) and folds into an (
/ß)8 motif (Derewenda et al., 1994). The catalytic domain of XlnA, along with members of 11 other different families of glycosyl hydrolases, is also classified into clan GH-A on the basis of structural and mechanistic information (Henrissat and Bairoch, 1998). Xylanases hydrolyze ß-1,4-glycosidic bonds with net retention of the configuration of the anomeric carbon via a double displacement mechanism (Sinnott, 1990). The side chains of two glutamic acids are involved, one acting as a nucleophile and the other as an acid–base catalyst. These two amino acids were identified in XlnA by site-directed mutagenesis (Moreau et al., 1994) and confirmed with the structure of the catalytic domain (Derewenda et al., 1994).
Available structural information on family 10 xylanases has allowed the identification of the residues present in the active site along with their possible involvement in the catalytic function of this type of protein. For example, substrate binding in the active site of family 10 xylanases has been investigated with the use of co-crystal structures. In one of these studies, the structure of XynA from Pseudomonas fluorescens subsp. cellulosa has been obtained in the presence of xylopentaose (Harris et al., 1994). This permitted the identification of the residues forming six substrate-binding subsites (–2 to +4; Davies, et al. 1997) of this enzyme. In another study, White et al. (1996) obtained the crystal structure of Cex from Cellulomonas fimi covalently complexed with a 2-fluorocellobioside, an analogue of the catalytic intermediate in the double displacement mechanism. They identified many interactions which could be of crucial importance in the binding and stabilization of the catalytic intermediate in the active site of family 10 xylanases. All these structural data show the implication of different aromatic residues in the binding of the substrate and/or the catalytic intermediate. The aromatic amino acids would allow specific and stable binding of the carbohydrate moieties by partial stacking of the sugar ring (Quiocho, 1986) and/or by hydrogen bonding to hydroxyl groups of the pyranose ring.
The role of aromatic residues in the function of xylanases has been studied through chemical modification. For example, the inactivation of a family 11 xylanase from Streptomyces T7 with N-bromosuccinimide suggested the importance of a tryptophan side chain in the active site of the enzyme (Keskar et al., 1989). Moreover, for family 11 xylanase XynA from Schizophilum commune, chemical modification of tyrosyl side chains by tetranitromethane suggested that Y97, a conserved residue in this family of enzyme, plays an essential role in substrate binding (Bray and Clarke, 1995).
The active site of XlnA contains many aromatic residues, four of which are exposed to solvent and could be involved in substrate binding (W85, Y172, W266 and W274). These residues are highly conserved in family 10 glycosyl hydrolases, suggesting their importance in the function of this type of enzyme (Figure 1). Moreover, the equivalents of these amino acids were shown to be part of the substrate-binding subsites surrounding the cleavage site that contain the two catalytic residues, –1 and +1 (Harris et al., 1994). Site-directed mutagenesis was used to replace these aromatic residues in XlnA and the purified mutant proteins were characterized.
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Materials and methods |
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Site-directed mutagenesis
Site-directed mutagenesis of xlnA was performed according to the method of Kunkel (1985) on phagemid pIAF217 or pAM19.1 (Moreau et al., 1994). The following oligonucleotides were used for mutagenesis: W85A, 5'-CTG GGA GTG GGCGGC CAG GGT-3'; W85F, 5'-CTG GGA GTG GAA GGC CAG GGT-3'; W85H, 5'-CTG CTG GGA GTG GTG GGC CAG GGT GTG-3'; Y172A, 5'-CTC GAC GTT GGC GTC GTT GTA-3'; Y172F, 5'-CTC GAC GTT GAA GTC GTT GTA-3'; Y172S, 5'-CTC GAC GTT GGA GTC GTT GTA-3'; W266A, 5'-GCG CAC ACC GGC GAC GGT GAT-3'; W266F, 5'-GCG CAC ACC GAA GAC GGT GAT-3'; W266H, 5'-GCG CAC ACC GTG GAC GGT GAT-3'; W274A, 5'-CTC CGA CCG GGC GGA GTC GCT-3'; W274F, 5'-CTC CGA CCG GAA GGA GTC GCT-3'; W274H 5'-CTC CGA CCG GTG GGA GTC GCT-3' [underlining indicates the substituted nucleotide(s)]. Screening and subcloning of the mutated genes from pIAF217 or pAM19.1 into plasmids pIAF18 and pIAF18.1, respectively, were done as described previously (Moreau et al., 1994; Roberge et al., 1997a).
Enzyme production and purification
XlnA was obtained as described previously (Bertrand et al., 1989). Proteins in the supernatant of S.lividans culture were first concentrated by ultrafiltration with a 3 kDa cut-off membrane (Omega). The concentrated proteins were then precipitated with ammonium sulfate at 65% saturation and, after centrifugation, the precipitate was dissolved in 50 mM sodium citrate buffer, pH 6.0. Samples of 100 mg of protein were loaded on a Phenyl-Sepharose column (Pharmacia) in 50 mM sodium citrate buffer, pH 5.6, containing 1 M ammonium sulfate. Proteins were eluted with a decreasing linear gradient to 0 M ammonium sulfate, followed by an increasing linear gradient to 50% ethylene glycol. Protein concentration was measured by UV absorption at 280 nm. The fractions containing XlnA were pooled, dialysed against Milli-Q water and freeze-dried. Further purification to apparent homogeneity as indicated by SDS–PAGE analysis (>95% by Coomassie Brilliant Blue staining), was achieved by separation on a Superdex HR75 beaded column (3x60 cm) (Pharmacia) with 100 mM sodium citrate, pH 6.0, as the eluent. The purified XlnA-containing fractions were pooled, dialyzed and freeze-dried.
Enzymatic activity determinations
The specific activity was determined by incubating the enzymes with 4.5 mg/ml birchwood xylan (Sigma) in 50 mM sodium citrate buffer, pH 6.0, at 60°C for 10 min. The released reducing sugars were determined by the p-hydroxybenzoic acid hydrazide method (Lever et al., 1984) adapted for microtiter plates. For the determination of Michaelis–Menten constants, the initial velocities of the enzymes were measured at 60°C in 50 mM sodium citrate buffer, pH 6.0, with birchwood xylan concentrations increasing from 0.045 to 4.5 mg/ml. The kinetic parameters were calculated with GraFit software version 3.09b. All enzymatic activities were expressed in international units (IU), where 1 IU represents the amount of enzyme releasing 1 µmol/min of reducing sugars using xylose as standard.
Enzyme stability
For the determination of the stability, solutions of 500 µg/ml of enzyme were incubated at 60°C. Samples were taken at regular intervals and kept on ice for 30 min prior to determination of residual activity. The half-life was obtained by plotting the natural logarithm of the residual activity as a function of incubation time. Stability was also determined in the presence of substrate by incubating the enzyme with 7.2 mg/ml birchwood xylan in 50 mM sodium citrate buffer, pH 6.0. At regular intervals, aliquots were withdrawn and analyzed for their reducing sugar contents. The XlnA half-lives were determined by non-linear analysis of the released reducing sugars versus time plots.
Circular dichroism (CD)
Solutions of 500 µg/ml xylanase in 10 mM sodium phosphate buffer, pH 6.0, were analyzed, at room temperature, using a 0.05 cm jacketed cell on a Jasco J-710 spectropolarimeter interfaced with an IBM computer. Data were averaged from 10 acquisitions between 250 and 190 nm at a scan rate of 100 nm/min. Thermal denaturation of XlnA was done by heating 500 µg/ml samples in a 0.05 cm jacketed cell at a rate of 0.5°C/min from 50 to 85°C using a Neslab 110 water-bath. Changes in XlnA structure due to unfolding were followed at 210 nm at every 0.2°C. The values were then transformed into fraction of unfolded protein according to the following equation, assuming a two-state unfolding mechanism:
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where Xn is the value for folded protein, Xd the value for unfolded protein and X the value observed (Pace et al., 1989).
HPLC analysis of hydrolysis products
Wild-type XlnA (14.8 nM) or the mutant proteins (equivalent amount of enzymatic units) were incubated with birchwood xylan (4.5 mg/ml) or xylopentaose (4.5 mM) in 50 mM sodium citrate buffer, pH 6.0, at 60 or 45°C, respectively. Because of their low thermal stability, the hydrolysis of xylan by mutants W266A and W266F was performed at 45°C along with the wild-type enzyme for comparison purposes. At time intervals, aliquots were withdrawn, boiled for 5 min and diluted 5- or 41-fold in water for xylan or xylopentaose hydrolysis, respectively, prior to HPLC analysis. Samples of 100 µl were injected on to a Dionex CarboPac PA1 (4x250 mm) anion-exchange column installed on a Dionex DX-500 HPLC system equipped with a pulsed-electrochemical detector interfaced to an IBM computer and a Thermo Separation Products AS3500 autosampler with 150 mM NaOH (1 ml/min) as the eluent. The oligosaccharides were separated by applying a linear gradient to 500 mM sodium acetate in 150 mM NaOH. The identification and concentration determination of the oligoxylosides produced were achieved by comparison with different concentrations of purified standards (X1 to X9) analyzed under the same conditions.
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Results |
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To study the role of W85, Y172, W266 and W274 in XlnA, three mutant proteins were generated at each position: W85A/F/H, Y172A/F/S, W266A/F/H and W274A/F/H. Far-UV CD was first used to determine whether any of these mutations would affect the structure of the enzyme. None of the mutations significantly modified the CD spectrum of the enzyme, indicating that the secondary structure content remained the same after mutation (data not shown). Therefore, loss of function and differences with the wild-type protein characteristics described thereafter may only be due to minor local structural changes undetectable by far-UV CD.
To evaluate the consequences of the mutations on the enzyme, the specific activity against xylan was measured. Only mutation Y172F did not alter the activity and all the others decreased it by more than 74% (Table I). Particularly, the three mutations at position W266 (W266A/F/H) dramatically reduced the specific activity by 99.7, 85.7 and 97.5%, respectively, suggesting that this residue is important for the catalytic properties of the protein (Table I). Most of these mutant proteins retained enough activity for accurate evaluation of their kinetic parameters. The catalytic constant (kcat) of the enzyme was reduced by values similar to the specific activity (Table I). Under the conditions used, the specific activity is a measure of the apparent kcat since a saturating concentration of substrate was used. However, the kcat value provides a more accurate measure of the catalytic efficiency of XlnA, because enzyme stability cannot influence this parameter. As expected, the binding affinity of XlnA for xylan was reduced by most of the mutations, confirming that these residues play a role in substrate binding. Only mutation W85F did not alter this parameter. Interestingly, replacement of the three tryptophan and the tyrosine residues by a phenylalanine is less detrimental to the affinity of XlnA for xylan (lower KM values) than other mutations (Table I), suggesting that the aromatic nature of these residues is important. The results show that mutation of these aromatic residues caused a decrease of 61.0–99.3% in the specificity of XlnA for xylan, which is reflected by losses in binding energy [
(G)] (Wilkinson et al., 1983) ranging from 2.61 to 13.8 kJ/mol (Table I).
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The role of these aromatic residues for the thermal stability of XlnA was investigated by activity measurements and CD. The transition temperatures (Tm) of wild-type and mutant XlnA were determined from the thermal unfolding curves obtained from CD measurements at 210 nm, the wavelength where the difference in ellipticity between the native and unfolded states of XlnA is maximal. Under these conditions, this experiment provides a way to quantify the effects of the point mutations on the stability of the secondary structures present in XlnA. The three mutations introduced at each position have different effects on the thermal stability of XlnA. Mutations W85H, Y172F and W274F/H did not significantly modify the Tm of XlnA, while mutations W85A, Y172S and W274A increased it by 2.0, 1.6 and 4.1°C, respectively. All the other mutations decreased the Tm by values ranging from 3.3 to 7.3°C (Table II
). These variations in Tm translate into differences in free energy of unfolding ranging from a stabilization of 9.5 kJ/mol to a destabilization of 16.7 kJ/mol. However, if one generalizes these results, mutations at positions W85, Y172 and particularly W266 tend to decrease the thermal stability of XlnA, whereas mutations at position W274 have no effect or slightly increase the Tm of the protein (Table II).
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The thermal stability of mutated proteins was also evaluated by measuring the half-life of the xylanolytic activity at 60°C in absence or in presence of xylan. Since the activity of the enzyme is measured, these assays provide a way to evaluate the effect of the mutation on the thermal stability of the active site loops of XlnA. Therefore, these results yield additional information on the role of these amino acids located in the active site of XlnA. In absence of substrate, the half-life of XlnA at 60°C was decreased by all the mutations. Replacement of W266 had the worst effect, reducing the half-life by 91–98.5%, whereas mutations at position W274 had a less pronounced effect than the others decreasing the half-life of the enzyme by 56–79% (Table II
). In presence of xylan, no protection against thermal inactivation of XlnA was observed for mutations Y172A and W266F, while the other enzymes were protected to levels ranging from 13 to 112% of the wild-type value (Table II). The degree of protection against thermal inactivation provided by the presence of xylan, in comparison with the wild-type protein, is dependent on the enzyme affinity for xylan, i.e. the half-life in the presence of substrate is proportional to the affinity for xylan, as suggested previously (Roberge et al., 1998). This, however, cannot explain the subtle variations observed for some mutations such as Y172A. In that particular case, one would expect Y172A to be more stable than Y172S since its KM is lower, but this is not the case (Table II). This deviation from the general rule could be explained by the important decrease in stability of the structure of that mutant as measured by CD, probably caused by local structural perturbations (Table II). Nevertheless, these and previous results (Roberge et al., 1998) support the fact that stabilization provided by the presence of xylan is dependent on the affinity of the enzyme for its substrate.
In order to verify whether the mode of action of XlnA was affected by any of the mutations studied, the hydrolysis products of birchwood xylan and X5 were analyzed by HPLC. Figure 2 shows the results obtained with wild-type XlnA. In our HPLC analysis, oligoxylosides with a degree of polymerization <10 were well resolved and linearly produced throughout the experiment. The major oligoxylosides produced in the hydrolysis of birchwood xylan by XlnA are X3 and X4 (Figure 2A). No xylose is seen after 30 min of hydrolysis, while XlnA also produced many other oligoxylosides such as X2, X5–X8 and other oligoxylosides with degrees of polymerization up to 20 (Figure 2A). From the HPLC analysis at different intervals of incubation, a time course of oligoxylosides production was constructed and the results for wild-type XlnA are shown in Figure 2C. Oligoxylosides with a degree of polymerization <9 were analyzed and their rates of production decreased in the order X3 > X4 > X5 > X2 = X6 > X7 > X8. Using the same method, hydrolysis of xylan by the different mutant proteins was also analyzed. No major differences from the wild-type profile were observed (data not shown). However, X2 was produced by all the mutant proteins at a rate 20–60% slower than that of the wild-type XlnA.
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(4.2 min); X3, 
(6.0 min); X4, 
(9.3 min); X5, 
(14.2 min); X6, 
(18.9 min); X7, 
(21.1 min); X8, 
(22.2 min); X9, 
(22.9 min).Hydrolysis of X5 mainly produces X3 but also significant amounts of X2 and X4 and only very little X1 is observed after 30 min of hydrolysis (Figure 2B
). Incubation of XlnA with a saturating amount of X5 also gives rise to transglycosylation products with degrees of polymerization up to 9 (Figure 2D, inset). In the hydrolysis of X5, the increase in the concentrations of released oligoxylosides was not linear over time during the 30 min period used in our experiment (Figure 2D) and it was impossible to calculate the rate of production for each oligoxyloside as in the case with the hydrolysis of xylan. The amount of the transglycosylation products X7–X9 increased in the first 5 min and was stable for the remainder of the 30 min period, while the concentration of X6 increased with time during the whole period (Figure 2D). Since X1 is not readily produced from the hydrolysis of X5, the transglycosylation product X6 was not observed at first, but was probably generated by the assembly of two X3 or one X2 and one X4 in the course of the reaction. Neither the hydrolysis nor the transglycosylation products of the reaction with X5 were significantly altered by any of the mutations studied, indicating that the mode of action of XlnA was not affected (data not shown). However, when X5 was hydrolyzed with the same quantity of enzymatic units, mutations Y172A, Y172S, W266F W274A and W274H caused a reduction in hydrolysis efficiency by 88, 72, 91, 73 and 51%, respectively (data not shown). |
Discussion |
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S.lividans XlnA is a well characterized member of family 10 glycosyl hydrolases for which the three-dimensional structure of the catalytic domain is known (Derewenda et al., 1994
). There are over 30 enzymes in this family, four of which have known three-dimensional structures (Henrissat and Bairoch, 1998). Thus, primary and tertiary structure comparisons are very useful tools in the identification of potentially important residues. These comparisons have shown the presence of conserved aromatic residues in the active site of this family that could be important in substrate binding. Site-directed mutagenesis was used to replace specifically four of these aromatic amino acids shown to be located in subsites –1 and +1 of the active site of family 10 xylanases (Harris et al., 1994). While mutation of either of these four active residues did not alter the hydrolysis pattern of the enzyme with either xylan or xylopentaose as substrates, significant variations in kinetic and stability parameters from the wild-type were observed, allowing a better understanding of the roles of these four aromatic residues in the structure–function relationships of family 10 xylanases. Role of tryptophan 85
In the structure of XlnA, W85 (N
1) is hydrogen-bonded to E128 (O1), the acid–base catalyst and to N127 (O
1) (Figure 3). This pattern was also observed in the structures of other family 10 xylanases, which suggested its importance for catalysis by the enzyme (Dominguez et al., 1995; Harris et al., 1996; White et al., 1996). From the kinetic results, the 70% average loss in activity due to the three mutations tested (Table I) could be attributed to secondary effects involving one or both of these residues, which have been shown to be very important in the catalytic efficiency of XlnA (Moreau et al., 1994; Roberge et al., 1997b). Moreover, the aromatic nature of W85 appears to play a role in substrate binding, as expected. Increases in KM were obtained by replacing W85 by non-aromatic residues (A and H), while the mutant enzyme W85F maintained its affinity for xylan (Table I). This suggests that W85 is involved in stacking interactions with the substrate. Furthermore, this residue is important for the thermal stability of the protein, as shown by the decrease in mutant enzyme half-lives at 60°C in the absence of substrate (Table II). In that case, the aromatic nature of W85 is probably not the only factor involved since the phenylalanine substitution seems to have the worst effect on that parameter. These results suggest that hydrogen bonding involving W85 is important for the stability of the protein.
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Role of tyrosine 172
The hydroxyl group of Y172 is involved in hydrogen-bonding interactions with D132 (O1), R139 (N
1) and an active-site water molecule (Figure 3). However, these interactions do not seem to be important for enzyme activity since Y172F mutant protein, without hydrogen-bonding possibility, is as active as the wild-type protein (Table I). The aromatic nature of this residue is important in substrate binding, since its replacement by non-aromatic residues decreases the affinity for xylan (Table II) and the apparent rate of hydrolysis of xylopentaose, while the Y172F mutation behaves similarly to the wild-type. Our results also show that Y172 is important for enzyme stability, particularly in the absence of substrate at 60°C (Table II). Again, the Y172F replacement is less detrimental to enzyme stability in the presence of substrate, strengthening the hypothesis that the protection gained against thermal inactivation is highly dependent on substrate affinity (Roberge et al., 1998). However, results for the Tm of the proteins suggest that hydrogen-bonding interactions are not important for the structural stability of XlnA, since similar Tm values were obtained for both the Y172F mutant and wild-type proteins (Table II). In this case, it is difficult to draw conclusions on the role of the aromatic nature of Y172 without knowing the structure of the mutant protein, since the Y172S mutation did not decrease the Tm of XlnA while significantly reducing the half-lives with and without substrate (Table II).
Role of tryptophan 266
Amino acid W266 is also highly conserved in family 10 xylanases. Its N1 atom is hydrogen-bonded to two active-site water molecules. However, W266 is also stacked to E236, the residue acting as a nucleophile in the hydrolysis of substrate in XlnA (Figure 3). This interaction was proposed to be of importance in other families of glycosyl hydrolases using the same mechanism of action (Dominguez et al., 1995). Our results confirm that these structural features are indeed important for the function of this type of enzyme and comparison of the specific activity of the different mutant proteins at this position suggests that the aromatic nature of W266 is involved in the activity of the protein.
The equivalents of residue W266 were shown to be involved in substrate binding in two other family 10 xylanase structures when complexed with substrates (Harris et al., 1996; White et al., 1996). Our kinetic analysis of W266F mutant protein clearly shows that this amino acid is involved in substrate binding and supports the previously published structural analysis. While mutant W266F retains a certain activity level, it is still 66% less active than the wild-type protein (Table I), indicating that W266 probably serves to position the nucleophile in the active site for efficient binding and catalysis. The large variation in affinity for xylan and the important reduction in activity towards xylopentaose exhibited by mutant W266F probably reflect a bad positioning of E236. This would weaken one of the major stabilizing interactions between O2 of E236 and OH-2 of the substrate at the transition state (White et al., 1996). Moreover, the modest effect on the kcat of the enzyme caused by the W266F mutation suggests that the hydrogen bond observed between N1 of the W266 equivalent and the catalytic intermediate analog in C.fimi Cex (White et al., 1996; Notenboom et al., 1998), which is missing in W266F, plays a minor role in the stabilization of the intermediate. Finally, W266 is very important in the thermal stability of XlnA, as shown by the large decreases in half-lives at 60°C and in Tm exhibited by all the mutant proteins studied (Table II). Again this supports the concept that the residues forming the complex hydrogen bonding network in the active site of the enzyme are involved in interactions crucial for the integrity of the structure of family 10 xylanases.
Role of tryptophan 274
In contrast to the three other amino acids studied here, W274 is not involved in hydrogen bonding interactions with other residues (Figure 3). In fact, W274 (N) is only hydrogen bonded to two active-site water molecules. Our kinetic analysis of the three mutant proteins at position W274 showed that this residue is involved in substrate binding (Table I). This supports the observations that the equivalent of W274 in the binding of xylopentaose at subsite –1 in XynA from P. fluorescens subsp. cellulosa has a high B value in the refined structure of this enzyme (Harris et al., 1994). The authors suggested that this residue could be important for the binding of large xylan substrates (Harris et al., 1994, 1996). Our results support this hypothesis, although the significant decrease in activity towards xylopentaose caused by mutations W274A and W274H, but not by W274F, indicates that the aromatic nature of this residue is also important for efficient catalysis of smaller oligoxylosides. Comparing the hydrolysis patterns of both xylan and xylopentaose by the three mutants showed that the aromatic nature of this residue plays a large role in substrate binding, suggesting that stacking interactions between W274 and the substrate are involved. From the decreases in kcat obtained (Table I), W274 also seems to play a role in the stabilization of the catalytic intermediate. This result was expected after analysis of the complexed structures of other family 10 xylanases that suggested W274 to be important for the specificity between glucosyl and xylosyl substrates (White et al., 1996; Notenboom et al., 1998). Finally, our results on the Tm values have shown that W274 is not crucial to the stability of the XlnA structure. Still, replacement of this active site residue reduced the half-life in absence of substrate by up to 79% (Table II), suggesting that the active site environment is affected by these mutations.
In conclusion, this study on four conserved aromatic residues in family 10 xylanases using site-directed mutagenesis has demonstrated their role in substrate binding and catalysis by this type of enzyme. As expected for sugar-binding proteins, the aromatic nature of W85, Y172 and W274 is particularly important in substrate binding. W266 is also involved in the catalytic efficiency of the protein and the four residues were shown to be important for the thermal stability of XlnA. This kinetic evidence supports the structural observations that were made on other family 10 xylanases and thus improves our knowledge of the structure–function relationship of this type of protein.
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Acknowledgments |
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This work was supported by grants from the Natural Sciences and Engineering Council of Canada (NSERC) and by the Fonds pour la Formation de Chercheurs et l'Aide à la Recherche du Québec (FCAR) and by a scholarship to M.R. from the Fondation Armand-Frappier. We thank Liette Biron, Lisette Duval and Christine Jacques for their assistance in the production and purification of some of the mutant proteins.
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Notes |
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1 Present address: Protein Engineering Department, Genentech Inc.,South San Francisco, CA 94080, USA
2 To whom correspondence should be addressed. E-mail: claude_dupont{at}iaf.uquebec.ca
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Received September 21, 1998; revised December 2, 1998; accepted December 7, 1998.
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