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Identification of the Catalytic Residues in Family 52 Glycoside Hydrolase, a β-Xylosidase from Geobacillus stearothermophilus T-6

2003; Elsevier BV; Volume: 278; Issue: 29 Linguagem: Inglês

10.1074/jbc.m304144200

1083-351X

Tsafrir Bravman, Valery Belakhov, Dmitry Solomon, G. Shoham, Bernard Henrissat, Timor Baasov, Yuval Shoham,

Enzyme Catalysis and Immobilization

β-d-Xylosidases (EC 3.2.1.37) are exo-type glycoside hydrolases that hydrolyze short xylooligosaccharides to xylose units. The enzymatic hydrolysis of the glycosidic bond involves two carboxylic acid residues, and their identification, together with the stereochemistry of the reaction, provides crucial information on the catalytic mechanism. Two catalytic mutants of a β-xylosidase from Geobacillus stearothermophilus T-6 were subjected to detailed kinetic analysis to verify their role in catalysis. The activity of the E335G mutant decreased ∼106-fold, and this activity was enhanced 103-fold in the presence of external nucleophiles such as formate and azide, resulting in a xylosyl-azide product with an opposite anomeric configuration. These results are consistent with Glu335 as the nucleophile in this retaining enzyme. The D495G mutant was subjected to detailed kinetic analysis using substrates bearing different leaving groups (pK a). The mutant exhibited 103-fold reduction in activity, and the Brønsted plot of log(k cat) versus pK a revealed that deglycosylation is the rate-limiting step, indicating that this step was reduced by 103-fold. The rates of the glycosylation step, as reflected by the specificity constant (k cat/K m), were similar to those of the wild type enzyme for hydrolysis of substrates requiring little protonic assistance (low pK a) but decreased 102-fold for those that require strong acid catalysis (high pK a). Furthermore, the pH dependence profile of the mutant enzyme revealed that acid catalysis is absent. Finally, the presence of azide significantly enhanced the mutant activity accompanied with the generation of a xylosyl-azide product with retained anomeric configuration. These results are consistent with Asp495 acting as the acid-base in XynB2. β-d-Xylosidases (EC 3.2.1.37) are exo-type glycoside hydrolases that hydrolyze short xylooligosaccharides to xylose units. The enzymatic hydrolysis of the glycosidic bond involves two carboxylic acid residues, and their identification, together with the stereochemistry of the reaction, provides crucial information on the catalytic mechanism. Two catalytic mutants of a β-xylosidase from Geobacillus stearothermophilus T-6 were subjected to detailed kinetic analysis to verify their role in catalysis. The activity of the E335G mutant decreased ∼106-fold, and this activity was enhanced 103-fold in the presence of external nucleophiles such as formate and azide, resulting in a xylosyl-azide product with an opposite anomeric configuration. These results are consistent with Glu335 as the nucleophile in this retaining enzyme. The D495G mutant was subjected to detailed kinetic analysis using substrates bearing different leaving groups (pK a). The mutant exhibited 103-fold reduction in activity, and the Brønsted plot of log(k cat) versus pK a revealed that deglycosylation is the rate-limiting step, indicating that this step was reduced by 103-fold. The rates of the glycosylation step, as reflected by the specificity constant (k cat/K m), were similar to those of the wild type enzyme for hydrolysis of substrates requiring little protonic assistance (low pK a) but decreased 102-fold for those that require strong acid catalysis (high pK a). Furthermore, the pH dependence profile of the mutant enzyme revealed that acid catalysis is absent. Finally, the presence of azide significantly enhanced the mutant activity accompanied with the generation of a xylosyl-azide product with retained anomeric configuration. These results are consistent with Asp495 acting as the acid-base in XynB2. β-d-Xylosidases (EC 3.2.1.37) are hemicellulases that hydrolyze short xylooligosaccharides into single xylose units. These enzymes are part of an array of glycoside hydrolases responsible for the complete degradation of xylan (1Shallom D. Shoham Y. Curr. Opin. Microbial. 2003; 6: 219-228Crossref PubMed Scopus (500) Google Scholar). This polymer is the major hemicellulosic polysaccharide in the plant cell wall representing up to 30–35% of the total dry mass (2Beg Q.K. Kapoor M. Mahajan L. Hoondal G.S. Appl. Microbiol. Biotechnol. 2001; 56: 326-338Crossref PubMed Scopus (1054) Google Scholar). Hemicellulases, together with cellulases, have a key role in the carbon cycle in nature, because they are responsible for the complete degradation of the plant biomass to soluble saccharides. These in turn can be used as carbon or energy sources for microorganisms and higher animals. Hemicellulases have attracted much attention in recent years because of their potential industrial uses in biobleaching of paper pulp (2Beg Q.K. Kapoor M. Mahajan L. Hoondal G.S. Appl. Microbiol. Biotechnol. 2001; 56: 326-338Crossref PubMed Scopus (1054) Google Scholar, 3Suurnakki A. Tenkanen M. Buchert J. Viikari L. Adv. Biochem. Eng. Biotechnol. 1997; 57: 261-287Crossref PubMed Google Scholar), bioconversion of lignocellulose material to fermentative products (4Galbe M. Zacchi G. Appl. Microbiol. Biotechnol. 2002; 59: 618-628Crossref PubMed Scopus (866) Google Scholar), improvement of animal feedstock digestibility, and recently in the field of oligosaccharide and thioglycoside synthesis (5Mackenzie L.F. Wang Q. Warren R.A. Withers S.G. J. Am. Chem. Soc. 1998; 120: 5583-5584Crossref Scopus (466) Google Scholar, 6Jahn M. Marles J. Warren R.A. Withers S.G. Angew. Chem. Int. Ed. Engl. 2003; 42: 352-354Crossref PubMed Scopus (130) Google Scholar). The glycosidic bond is one of the most stable bonds in nature, with a half-life of over 5 million years (7Wolfenden R. Lu X. Young G. J. Am. Chem. Soc. 1998; 120: 6814-6815Crossref Scopus (201) Google Scholar). Glycoside hydrolases can accelerate the hydrolysis of the glycosidic bond by more than 1017-fold, making them the most efficient catalysts known. The enzymatic hydrolysis of the glycosidic bonds occurs via two major mechanisms, giving rise to either an overall retention or an inversion of the anomeric configuration. In both mechanisms, the hydrolysis usually requires two carboxylic acids, which are conserved within each glycoside hydrolase family, and proceeds through oxocarbenium ion-like transition states. Inverting glycosidases use a single displacement mechanism with the assistance of general acid and general base residues. Retaining glycosidases follow a two-step double displacement mechanism as shown in Fig. 1, involving two catalytic residues, one functioning as a nucleophile and the other functioning as an acid-base (8Davies G. Sinnott M.L. Withers S.G. Sinnott M.L. Comprehensive Biological Catalysis. Academic Press Ltd., London1998: 119-209Google Scholar). Identification of the key active site residues is of great importance because it provides crucial information regarding the enzymatic catalytic mechanism and allows rational protein design for novel applications, such as for enzymatic synthesis (5Mackenzie L.F. Wang Q. Warren R.A. Withers S.G. J. Am. Chem. Soc. 1998; 120: 5583-5584Crossref Scopus (466) Google Scholar). Candidates for the catalytic residues are primarily identified by searching for conserved carboxylates (Glu or Asp) throughout multiple amino acid sequence alignment. These conserved residues are replaced to a noncarboxylic residue, and the generated mutants are subjected to detailed kinetic analysis using substrates bearing different leaving groups, azide rescue analysis, and pH dependence activity profiles of the mutants and the wild type. In some cases, identification can be accomplished by labeling the catalytic residues using mechanism-based inactivators and affinity labels (9Zechel D.L. Withers S.G. Poulter C.D. Comprehensive Natural Products Chemistry. Vol. 5. Elsevier, New York1999: 279-314Crossref Google Scholar). Based on amino acid sequence similarities, β-d-xylosidases are currently divided into families 3, 39, 43, 52, and 54 of glycoside hydrolases (10Henrissat B. Davies G. Curr. Opin. Struct. Biol. 1997; 7: 637-644Crossref PubMed Scopus (1379) Google Scholar, 11Henrissat B. Bairoch A. Biochem. J. 1996; 316: 695-696Crossref PubMed Scopus (1173) Google Scholar). These families together with all other glycoside hydrolase families can be readily accessed at the constantly updated web site afmb.cnrs-mrs.fr/CAZY. Although the identities of the catalytic residues for most of these β-d-xylosidase families are already known (12Bravman T. Mechaly A. Shulami S. Belakhov V. Baasov T. Shoham G. Shoham Y. FEBS Lett. 2001; 495: 115-119Crossref PubMed Scopus (43) Google Scholar, 13Vocadlo D.J. MacKenzie L.F. He S. Zeikus G.J. Withers S.G. Biochem. J. 1998; 335: 449-455Crossref PubMed Scopus (36) Google Scholar, 14Vocadlo D.J. Wicki J. Rupitz K. Withers S.G. Biochemistry. 2002; 41: 9736-9746Crossref PubMed Scopus (46) Google Scholar, 15Nurizzo D. Turkenburg J.P. Charnock S.J. Roberts S.M. Dodson E.J. McKie V.A. Taylor E.J. Gilbert H.J. Davies G.J. Nat. Struct. Biol. 2002; 9: 665-668Crossref PubMed Scopus (151) Google Scholar, 16Li Y.K. Chir J. Tanaka S. Chen F.Y. Biochemistry. 2002; 41: 2751-2759Crossref PubMed Scopus (35) Google Scholar), no such information is available for family 52. Previously, we reported the cloning and purification of a β-xylosidase from Geobacillus stearothermophilus T-6 (XynB2) showing homology to family 52 glycoside hydrolases. Its stereochemical course of hydrolysis showed that the configuration of the anomeric carbon was retained, indicating that a retaining mechanism prevails in family 52 glycoside hydrolases (17Bravman T. Zolotnitsky G. Shulami S. Belakhov V. Solomon D. Baasov T. Shoham G. Shoham Y. FEBS Lett. 2001; 495: 39-43Crossref PubMed Scopus (37) Google Scholar). Because the β-xylosidase from G. stearothermophilus T-6 can be readily overexpressed and purified, it can serve as an excellent representative of family 52 glycoside hydrolases for the identification of the two key active site residues. This paper describes a detailed kinetic analysis of the putative acid-base and nucleophile mutants of XynB2, using substrates bearing different leaving groups, chemical rescue, and pH dependence profiles. The study provides for the first time unequivocal identification of the two catalytic residues of family 52 glycoside hydrolases. Substrates—The substrates 2,5-dinitrophenyl β-d-xylopyranoside, 3,4-dinitrophenyl β-d-xylopyranoside, 2,4,6-trichlorophenyl β-d-xylopyranoside, and m-nitrophenyl β-d-xylopyranoside were synthesized as described by Ziser et al. (18Ziser L. Setyawati I. Withers S.G. Carbohydr. Res. 1995; 274: 137-153Crossref PubMed Scopus (47) Google Scholar). p-Nitrophenyl β-d-xylopyranoside, o-nitrophenyl β-d-xylopyranoside, and 4-methylumbelliferyl β-d-xylopyranoside were from Sigma. Mutagenesis, Protein Expression, and Purification—The xynB2 gene (GenBank™ accession number AJ305327) from G. stearothermophilus T-6 was cloned in the pET9d vector, overexpressed in Escherichia coli BL21(DE3), and purified as previously reported (17Bravman T. Zolotnitsky G. Shulami S. Belakhov V. Solomon D. Baasov T. Shoham G. Shoham Y. FEBS Lett. 2001; 495: 39-43Crossref PubMed Scopus (37) Google Scholar). Site-directed mutagenesis was performed using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The mutagenic primers for the mutations were as follows (the mutated nucleotides are in bold type): D495G, 5′-GGAAATCACAACGTACGGGAGTTTGGATGTTTCTCTTGG-3′ and 5′-CCAAGAGAAACATCCAAACTCCCGTACGTTGTGATTTCC-3′; E335G, 5′-GCCGATTTGGGTCGTTAACGGCGGCGAGTACCGGATGATG-3′ and 5′-CATCATCCGGTACTCGCCGCCGTTAACGACCCAAATCGGC-3′; E335A, 5′-GCCGATTTGGGTCGTTAACGCCGGCGAGTACCGGATGATG-3′ and 5′-CATCATCCGGTACTCGCCGGCGTTAACGACCCAAATCGGC-3′; and E335Q, 5′-GCCGATTTGGGTCGTTAACCAGGGCGAGTACCGGATGATG-3′ and 5′-CATCATCCGGTACTCGCCCTGGTTAACGACCCAAATCGGC-3′. The mutagenic primers were designed to include the mutation and when possible a restriction site to allow easy identification of the mutation. All of the mutations were created by a double base pair substitution to avoid translational misincorporation during protein synthesis by the host cell. The mutated genes were sequenced to confirm that only the desired mutations were inserted, and the proteins were overexpressed and purified as described for the wild type. Kinetic Studies—Steady state kinetic studies were performed by following the absorbance changes in the UV-visible range, using an Ultrospec 2100 pro spectrophotometer (Amersham Biosciences) equipped with a temperature-stabilized water circulating bath. Initial hydrolysis rates were determined by incubating 500 μl of different substrate concentrations (ranging from 0.1 to 7 K m where applicable) in 100 mm phosphate buffer (pH 7.0) containing 1 mg/ml bovine serum albumin at 40 °C within the spectrophotometer until thermal equilibration was achieved. The exact temperature inside the cuvette was verified using a thermocouple. The reactions were initiated by the addition of 100 μl of appropriately diluted enzyme, and the release of the phenol-derived product was monitored at the appropriate wavelength. For very low K m values, the initial rates were measured with special care. For highly reactive substrates, blank mixtures containing all of the reactants except the enzyme were used to correct for spontaneous hydrolysis of the substrates. Sodium azide and formate were added to the reaction mixtures where mentioned. The extinction coefficients used at pH 7.0 and 40 °C and the wavelength monitored for each substrate were as follows: 2,5-dinitrophenyl, 420 nm, Δ∈ = 3.68 mm–1 cm–1; 3,4-dinitrophenyl, 400 nm, Δ∈ = 11.15 mm–1 cm–1; 2,4,6-trichlorophenyl, 312 nm, Δ∈ = 3.97 mm–1 cm–1; 4-nitrophenyl, 420 nm, Δ∈ = 7.61 mm–1 cm–1; 2-nitrophenyl, 420 nm, Δ∈ = 1.91 mm–1 cm–1; 4-methylumbelliferyl, 355 nm, Δ∈ = 2.87 mm–1 cm–1; 3-nitrophenyl, 380 nm, Δ∈ = 0.455 mm–1 cm–1. The values of K m and k cat were determined by nonlinear regression analysis using the program GraFit 5.0 (19Leatherbarrow R.J. GraFit. 5 Ed. Erithacus Software Ltd., Horley, UK2001Google Scholar). pH dependence studies were carried at 40 °C with pNPX 1The abbreviations used are: pNPX, p-nitrophenyl β-d-xylopyranoside; 2,5-DNPX, 2,5-dinitrophenyl β-d-xylopyranoside; FTIR, Fourier transform infrared spectroscopy. as a substrate. Mixtures containing 600 μl of 1 mg/ml bovine serum albumin and different concentrations of substrate solutions in the appropriate buffer were prewarmed until the reaction was initiated by the addition of 200 μl of appropriately diluted enzyme. The buffers used were at a final concentration of 100 mm and were: citric acid-Na2HPO4 (pH 4.5–6.5), phosphate buffer (pH 6.0–8.0), and Tris-HCl buffer (pH 7.5–8.5). The pH range employed in this study included only pH values for which the enzyme was stable for at least 5 min. The reactions were monitored continuously at 40 °C, and upon completion the actual pH was measured to verify that the pH had not changed. The release of p-nitrophenol was monitored at 400 nm, and the mm extinction coefficients for p-nitrophenolate were determined at pH 4.63, 5.35, 6.02, 6.53, 6.93, 7.61, 8.0, 8.3, and 8.56 as follows: 1.43, 1.89, 4.45, 7.46, 11.2, 16.2, 17.0, 17.3, and 17.6 mm–1 cm–1, respectively. The pK a values assigned to the ionizable groups were determined using the program GraFit 5.0. Isolation and Analysis of Reaction Products in the Presence of Sodium Azide—The enzymatic reactions included 0.4 mg/ml of either XynB2-E335G or XynB2-D495G, 10 mm of 2,5-DNPX, and 1 m sodium azide in a final volume of 10 ml of 100 mm phosphate buffer, pH 7.0. The mixtures were incubated at 40 °C, and the reaction was monitored by TLC. TLC analysis was performed using precoated plates (Silica Gel 60 F254, 0.25 mm; Merck), and MeOH/CHCl3 1:4 as the running solvents. The spots were visualized by charring with a yellow solution containing 120 g of (NH4)Mo7O24·4H2Oand5gof(NH4)2Ce(NO3)6 in 800 ml of 10% H2SO4. After complete hydrolysis of the substrate (∼5 h), the mixtures were lyophilized, and the resulting solid was extracted with methanol (4 × 5 ml). The extracts were combined and evaporated to dryness. The crude material was purified by flash chromatography (MeOH:CHCl3, 1:9) on a silica gel (Merck; 63–200 mesh) column to yield the pure product as a white solid (40 mg). 1H NMR and 13C NMR spectra were recorded at an ambient temperature on a Bruker Avance 500 MHz spectrometer. The mass spectrum was obtained on a TSQ-70B mass spectrometer (Finnigan Mat) by negative chemical ionization in isobutane or on a Bruker Daltomics Apex-III (ICR-MS) by the method of electrospray ionization. Fourier transform infrared spectroscopy (FTIR) was recorded on a Bruker vector 22 spectrometer. Site-directed Mutagenesis—We have recently described the purification and stereochemical course of hydrolysis of a β-xylosidase (XynB2) from family 52 glycoside hydrolases (17Bravman T. Zolotnitsky G. Shulami S. Belakhov V. Solomon D. Baasov T. Shoham G. Shoham Y. FEBS Lett. 2001; 495: 39-43Crossref PubMed Scopus (37) Google Scholar). In that report we suggested that Glu337 and Glu413 are involved in catalysis. However, further examination of these mutants revealed that they are not catalytic residues. To identify the catalytic residues of XynB2, the amino acid sequences of family 52 glycosidases were aligned, and many conserved carboxylic residues were revealed. These were replaced via site-directed mutagenesis to glycine, and the generated mutants were screened for reduction in activity. During the course of the work, it was apparent that part of the mutants exhibited inconsistent behavior. As it turned out, inconsistency arose from two main reasons: (a) contaminations from translational misincorporation as was observed previously (20Shallom D. Belakhov V. Solomon D. Shoham G. Baasov T. Shoham Y. J. Biol. Chem. 2002; 277: 43667-43673Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) and (b) contaminations from the purification procedure. To avoid these potential problems, all of the mutations were created by a double base pair substitution, and special care was taken during all of the purification procedures. Following systematic replacement of the conserved carboxylic residues in XynB2, two mutants, E335G and D495G, that reside in highly conserved regions were promising candidates for the catalytic pair. Therefore, these were overexpressed, purified, and subjected to extensive kinetic analysis as described below. Catalytic Properties of the Glu335 and Asp495 Mutants— Glu335 was replaced with Gly, Ala, or Gln, and the catalytic properties of the E335G, E335A, and E335Q mutants using pNPX and 2,5-DNPX as substrates were determined and summarized in Table I. The k cat values measured for the E335G, E335A, and E335Q mutants were all significantly reduced and are about 10–6-fold of wild type activity with both pNPX and 2,5-DNPX as substrates. The very low activity precluded reliable K m determination.Table Ikcat values for hydrolysis of aryl-β-d-xylosides by XynB2 and its Glu335 mutantsEnzymePhenol substituentkcats-1Wild type4-nitro18Wild type2,5-dinitro64E335G4-nitro2.0 × 10-5E335G2,5-dinitro7.5 × 10-4E335G2,5-dinitro + 1.4 M azide3.3 × 10-1E335G2,5-dinitro + 2.3 M formate6.7 × 10-1E335A4-nitro3.4 × 10-6E335A2,5-dinitro1.7 × 10-4E335A2,5-dinitro + 1.4 M azide5.6 × 10-3E335Q4-nitro8.1 × 10-5E335Q2,5-dinitro4.2 × 10-4E335Q2,5-dinitro + 1.4 M azide1.9 × 10-3 Open table in a new tab Similarly, Asp495 was replaced with Gly, and the kinetic constants of the D495G mutant were determined using different aryl β-d-xylopyranosides with different leaving groups (Table II and Fig. 2). The k cat values of the D495G mutant were 103-fold lower than for the wild type. For both the wild type enzyme and the D495G mutant, k cat values were invariant for hydrolysis of all of the substrates. Although the K m values of the wild type enzyme were roughly similar with all substrates, with the D495G mutant these values increased as the substrate reactivity decreased (increasing pK a). Consequently, the decrease in the specificity constant (k cat/K m) values for the D495G mutant was more pronounced, because the substrates are less reactive.Table IIKinetic parameters for hydrolysis of aryl-β-d-xylosides by XynB2 and by the D495G mutant in the absence and presence of 0.63 m azideAglyconepKaEnzymekcatKmk cat/Kms-1mMs-1 nM-12,5-Dinitrophenyl5.15wtawt, wild type.640.6795D495G0.0570.001734D495G + 0.63 M azide520.0539743,4-Dinitrophenyl5.36wt500.15330D495G0.0880.0003295D495G + 0.63 M azide270.0455942,4,6-Trichlorophenyl6.39wt610.8175D495G0.0630.00163D495G + 0.63 M azide210.181174-Nitrophenyl7.18wt180.13140D495G0.0390.00429.2D495G + 0.63 M azide0.420.019222-Nitrophenyl7.22wt730.4660D495G0.0670.003420D495G + 0.63 M azide2.20.03564-Methylumbelliferyl7.53wt800.35230D495G0.0550.0321.7D495G + 0.63 M azide0.20.121.63-Nitrophenyl8.39wt410.8747D495G0.0210.0610.34D495G + 0.63 M azide0.0260.0990.27a wt, wild type. Open table in a new tab Chemical Rescue of the Catalytic Mutants—Rate acceleration by exogenous nucleophilic anions is the most definitive tool for the identification of the catalytic residues (21Ly H.D. Withers S.G. Annu. Rev. Biochem. 1999; 68: 487-522Crossref PubMed Scopus (266) Google Scholar). In the presence of 1.4 m azide, k cat values increased by ∼4, 33, and 400 times for E335Q, E335A, and E335G mutants, respectively (Table I), indicating that the effect of azide decreases as the side chain is longer. The addition of 2.3 m formate resulted in a 103-fold increase of k cat for the E335G mutants (Table I). Both azide and formate accelerated the reaction in a concentration-dependent manner. The kinetic constants of hydrolysis of 2,5-DNPX by the D495G mutant in the presence of different concentrations of azide were measured and are plotted in Fig. 3. Both k cat and K m increased with increasing azide concentrations until leveling off at about 0.5 m azide. Consequently, the k cat/K m values remained unchanged. The effect of azide was also tested for the hydrolysis of substrates with different leaving groups at substrate saturating conditions (Fig. 4). All of the k cat values increased with increasing concentrations of azide until reaching a plateau. Finally, the kinetic constants for hydrolysis of various aryl β-d-xylopyranosides were determined in the presence of 0.63 m azide (Table II and Fig. 5).Fig. 4k cat values for hydrolysis of aryl β-d-xylopyranosides by XynB2-D495G in the presence of various concentration of sodium azide. ○, 2,5-DNPX; •, 3,4-dinitrophenyl β-d-xylopyranoside; □, 2,4,6-trichlorophenyl β-d-xylopyranoside; ▪, o-nitrophenyl β-d-xylopyranoside; ▵, pNPX. The initial rates were determined at 40 °C in 100 mm phosphate buffer, pH 7.0, using 0.42 mm of each substrate. The pK a values of the leaving group phenolate are indicated in the plot.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5Brønsted plots of the hydrolysis of aryl β-d-xylopyranosides catalyzed by XynB2-D495G in the absence (•) and presence of 0.63 m sodium azide (○). a, plot of log(k cat) versus pK a of the aglycone. b, plot of log(k cat/K m) versus pK a of the aglycone. The initial rates were determined at 40 °C in 100 mm phosphate buffer, pH 7.0.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Characterization of Reaction Products in the Presence of Sodium Azide—The formation of a glycosyl-azide product is a powerful diagnostic tool for identifying the catalytic residues, and determining its anomeric configuration is useful for distinguishing between the acid-base and the nucleophile. TLC analysis of the reaction mixture containing E335G, 2,5-DNPX, and azide revealed the formation of a new product (R f = 0.5) distinct from xylopyranoside (R f = 0.18) and 2,5-DNPX (R f = 0.51). This new product was isolated and identified as α-d-xylopyranosyl azide, as determined by 1H NMR (Fig. 6a), 13C NMR, mass spectrometry, and FTIR: 1H NMR (500 MHz, CD3OD) δ 3.48 (m, 3H, H-2, H-3, H-5), 3.57 (t, 1H, J = 11.0 Hz, H-4), 3.70 (dd, 1H, J = 5.0, 12.0 Hz, H-5′), 5.24 (d, 1H, J = 2.5 Hz, H-1); 13C NMR (125.8 MHz, CD3OD) δ 65.4, 71.0, 73.3, 74.6, 91.6 (C-1); negative CIMS m/z 173.9 (M-H–, C5H9O4N3 requires 175.1); FTIR (mineral oil) ν 2116 cm–1 (N3). Likewise, TLC analysis of the reaction mixture containing D495G, 2,5-DNPX, and azide revealed the formation of a new product (R f = 0.42) distinct from xylopyranoside (R f = 0.18) and 2,5-DNPX (R f = 0.51). This new product was isolated and identified as β-d-xylopyranosyl azide, as determined by 1H NMR (Fig. 6b), 13C NMR, mass spectrometry, and FTIR: 1H NMR (500.1 MHz, CD3OD) δ 3.03 (t, 1H, J 2,3 = 9.0 Hz, H-2), 3.21 (t, 1H, J 5a,5b = 11.5 Hz, H-5a), 3.23 (t, 1H, J 3,4 = 9.0 Hz, H-3), 3.39 (ddd, 1H, J 4,5a = 9.5 Hz, J 4,5b = 5.5 Hz, H-4), 3.84 (dd, 1H, H-5b), 4.33 (d, 1H, J 1,2 = 8.1 Hz, H-1). 13C NMR (125.8 MHz, CD3OD) δ 69.0 (C-5), 70.8 (C-4), 74.7 (C-2), 78.1 (C-3), 92.7 (C-1); electrospray ionization m/z: 198.1 (M+ + Na, C5H9O4N3, requires 175.1); FTIR (mineral oil) ν 2116 cm–1 (N3). pH Dependence—The k cat values of the wild type enzyme and the D495G mutant for hydrolysis of pNPX were determined at different pH values in the range of 4.5–8.5 (Fig. 7). The pH activity profile of the wild type enzyme showed strong dependence upon pH changes with pK a values of <4 and 7.3. Conversely, no such dependence is observed for the mutant enzyme within these pH values. The activity of the mutant at pH levels lower than 4 could not be determined because the enzyme was insoluble. Glu335 Is the Catalytic Nucleophile—In retaining glycoside hydrolases, the nucleophilic residue is extremely important for carrying out efficient catalysis. The nucleophile attacks the anomeric carbon to form a covalent enzyme intermediate, which is sufficiently stable to permit the diffusion of the leaving group from the active site and subsequently the entrance of a water molecule. Furthermore, the nucleophile is important for maintaining the correct ionization state of the acid-base catalyst, and in some cases, for assistance in stabilizing the oxocarbenium ion-like transition state by creating strong hydrogen bond with the sugar 2-hydroxyl (22Zechel D.L. Withers S.G. Acc. Chem. Res. 2000; 33: 11-18Crossref PubMed Google Scholar). Thus, replacing the nucleophile with a noncarboxylic residue usually severely affects the enzymatic catalysis and in some cases leads to nondetectable activity (21Ly H.D. Withers S.G. Annu. Rev. Biochem. 1999; 68: 487-522Crossref PubMed Scopus (266) Google Scholar). The k cat values measured for the E335G, E335A, and E335Q mutants were all drastically reduced with activities of about 106-fold lower than the wild type for hydrolysis of both pNPX and 2,5-DNPX. This magnitude of decrease in activity is typical for nucleophile mutants and was also observed in other retaining glycoside hydrolases (21Ly H.D. Withers S.G. Annu. Rev. Biochem. 1999; 68: 487-522Crossref PubMed Scopus (266) Google Scholar). However, the substantial decrease in the catalytic activity is insufficient for the unequivocal assignment of Glu335 as the catalytic nucleophile. There are several examples where single mutations in proposed catalytic residues of glycoside hydrolases resulted in reduced or undetectable activity (23Sierks M.R. Ford C. Reilly P.J. Svensson B. Protein Eng. 1990; 3: 193-198Crossref PubMed Scopus (119) Google Scholar, 24Totsuka A. Nong V.H. Kadokawa H. Kim C.S. Itoh Y. Fukazawa C. Eur. J. Biochem. 1994; 221: 649-654Crossref PubMed Scopus (38) Google Scholar), whereas crystallographic and biochemical analyses showed that these residues are not involved directly in catalysis (25Harris E.M. Aleshin A.E. Firsov L.M. Honzatko R.B. Biochemistry. 1993; 32: 1618-1626Crossref PubMed Scopus (149) Google Scholar, 26Mikami B. Degano M. Hehre E.J. Sacchettini J.C. Biochemistry. 1994; 33: 7779-7787Crossref PubMed Scopus (121) Google Scholar). To unambiguously identify Glu335 as the catalytic nucleophilic residue of XynB2, the rescue methodology was applied. In this procedure (Fig. 8a), the catalytic activities of the putative catalytic mutants are monitored in the presence of small nucleophilic anions, such as azide or formate. The small anion can enter the vacant place created by the elimination of the nucleophilic residue and attack the anomeric carbon of the sugar substrate to form a glycosyl-azide product (when azide is added) with inverted anomeric configuration. Rate acceleration of the mutant in the presence of external anions is a strong indication that the mutation is indeed in the catalytic residue. Although no rate enhancement was observed for the wild type enzyme, the hydrolysis rates of 2,5-DNPX by the E335G mutant significantly accelerated with increasing concentrations of azide and formate. The presence of 2.3 m formate increased k cat up to 103-fold, only 100 times lower from the wild type activity. Interestingly, in the presence of 1.4 m azide, the rate of hydrolysis by the E335G mutant was accelerated by about 4 × 102-fold, whereas hydrolysis by the E335A and E335Q mutants with the same azide concentration resulted in only 32- and 4-fold rate enhancement, respectively. Hence, as the length of the side chain increases, the vacant place is smaller, preventing azide facile penetration. Interestingly, formate had a greater effect on rate acceleration, although it exhibits lower nucleophilicity. This was also observed with the nucleophile-less mutants of the β-glucosidase from Agrobacterium faecalis (27Wang Q. Graham