Probing the catalytically essential residues of the {alpha}-L-arabinofuranosidase from Thermobacillus xylanilyticus

Takoua Debeche1, Christophe Bliard2, Philippe Debeire1 and Michael J. O'Donohue1,3

1 Unité de Fractionnement des Agro-ressources et Emballage, Institut National de la Recherche Agronomique, Centre de Recherche Agronomique, 2, esplanade R. Garros, BP 224, 51686 Reims Cedex 02 and 2 Centre National de la Recherche Scientifique, UMR 6013, Bât. 18, BP 1039, 51687 Reims Cedex 02, France


    Abstract
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 Abstract
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 Materials and methods
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The {alpha}-L-arabinofuranosidase D3 from Thermobacillus xylanilyticus is an arabinoxylan-debranching enzyme which belongs to family 51 of the glycosyl hydrolase classification. Previous studies have indicated that members of this family are retaining enzymes and may form part of the 4/7 superfamily of glycosyl hydrolases. To investigate the active site of {alpha}-L-arabinofuranosidase D3, we have used sequence alignment, site-directed mutagenesis and kinetic analyses. Likewise, we have shown that Glu28, Glu176 and Glu298 are important for catalytic activity. Kinetic data obtained for the mutant Glu176->Gln, combined with the results of chemical rescue using the mutant Glu176->Ala, have shown that Glu176 is the acid-base residue. Moreover, NMR analysis of the arabinosyl-azide adduct, which was produced by chemical rescue of the mutant Glu176->Ala, indicated that {alpha}-L-arabinofuranosidase D3 hydrolyses glycosidic bonds with retention of the anomeric configuration. The results of similar chemical rescue studies using other mutant enzymes suggest that Glu298 might be the catalytic nucleophile and that Glu28 is a third member of a catalytic triad which may be responsible for modulating the ionization state of the acid-base and implicated in substrate fixation. Overall, these findings support the hypothesis that {alpha}-L-arabinofuranosidase D3 belongs to the 4/7 superfamily and provide the first experimental evidence concerning the catalytic apparatus of a family 51 arabinofuranosidase.

Keywords: arabinofuranosidase/catalytic site/chemical rescue/family 51/site-directed mutagenesis


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
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{alpha}-L-Arabinofuranosidases (EC 3.2.1.55) catalyse the hydrolysis of non-reducing {alpha}-L-arabinofuranosidic linkages in various arabinofuranose-containing polysaccharides (Beldman et al., 1997Go). Presently, at least 50 sequences of {alpha}-L-arabinofuranosidases (verified and hypothetical) are present in GenBank. Within the glycosyl hydrolase classification (Henrissat, 1991Go), which groups enzymes into families on the basis of amino acid sequence similarity, the majority of these sequences are grouped within families 43, 51 and 54. Currently, the largest of these is family 51 which contains 18 {alpha}-L-arabinofuranosidase sequences. Although the DNA sequences of several family 51 enzymes have been cloned and expressed (Schwarz et al., 1990Go; Flipphi et al., 1993Go; Debeche et al., 2000Go; Matsuo et al., 2000Go) and several biotechnological applications employ {alpha}-L-arabinofuranosidases (Saha, 2000Go), only a few studies have so far attempted to characterize the catalytic mechanism of these enzymes (Selwood and Sinnott, 1988Go; Sinnott, 1990Go; Pitson et al., 1996Go) and no structural information is available.

A great many studies have led to the observation that all glycosyl hydrolases appear to cleave the glycosidic bond via one of two different mechanisms which result in either inversion or net retention of the anomeric configuration (McCarter and Withers, 1994Go). Both mechanisms involve general acid-base catalysis and require two essential residues, which in most glycosyl hydrolases are aspartate and/or glutamate residues. Inverting glycosidases catalyse hydrolysis of the glycosidic bond via a single displacement reaction. One of the catalytic residues, acting as a general acid, provides protonic assistance to the departing glycosidic oxygen while the second, acting as a general base, activates a water molecule which effects a direct displacement at the anomeric centre. Retaining enzymes, perform catalysis via a double displacement mechanism. In the first step (glycosylation), one catalytic residue (acid-base), acting as a general acid, provides protonic assistance to the departing glycosidic oxygen while a second residue, behaving as a nucleophile, attacks the substrate at its anomeric centre thus generating a covalently linked glycosyl-enzyme transition state. In the second step, the acid-base, now functioning as a general base, activates a water molecule which attacks on the opposite face of the anomeric centre, forming a product with the same anomeric configuration as the substrate. Although these catalytic mechanisms have been elucidated mainly through the study of glycopyranosyl hydrolases, they would also appear to apply to glycofuranosyl hydrolases. Indeed, a study of the stereochemical course of hydrolyses catalysed by several arabinofuranosyl hydrolases has previously provided evidence which suggests that family 51 and family 54 {alpha}-L-arabinofuranosidases are retaining enzymes (Pitson et al., 1996Go). With regard to the catalytic machinery, attempts to identify the catalytically essential residues of {alpha}-L-arabinofuranosidases have thus far been limited to a sequence alignment study performed by Zverlov et al. (Zverlov et al., 1998Go). These authors showed that family 51 enzymes contain two conserved regions which are also present in other retaining enzyme families which belong to the 4/7 superfamily (Jenkins et al., 1995Go). Significantly, all enzymes in this superfamily possess a (ß/{alpha})8-barrel fold in which the two conserved regions mentioned above contain the catalytically essential glutamates and are localized at the C-terminal ends of ß-strands 4 and 7. Therefore, it was concluded that in family 51 arabinofuranosidases Glu176 and Glu298 (AbfD3 numbering) are the acid-base and nucleophile, respectively, and that these enzymes belong to the 4/7 superfamily.

Recently, we cloned and expressed in Escherichia coli an {alpha}-L-arabinofuranosidase (AbfD3) from Thermobacillus xylanilyticus D3 (Debeche et al., 2000Go). As a first step in a structure/function study, we wish to identify the essential catalytic residues of this enzyme. Several strategies for the identification of such residues in other glycosyl hydrolases have been described. For retaining glycosidases, fluorinated glycoside inhibitors coupled with mass spectral analyses have proved to be a powerful strategy (Withers and Aebersold, 1995Go; Williams and Withers, 2000Go). However, when a recombinant enzyme is available, a simpler strategy, based on site-directed mutagenesis and kinetic analysis can also provide an accurate identification of the catalytic residues (MacLeod et al., 1996Go). Therefore, we now describe the use of such an approach for the experimental identification of the essential catalytic residues of AbfD3.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
Substrate and general reagents

The substrate para-nitrophenyl-{alpha}-L-arabinofuranoside (pNP-ara) and all general reagents were obtained from Sigma Chemical Co. (St. Quentin Fallavier, France).

Mutagenesis, protein expression and purification

The plasmid containing the complete coding sequence of AbfD3 (GenBank accession number Y16849), pET24-AbfD3, was used as a template for in vitro mutagenesis using the QuickChangeTM kit (Stratagene, Amsterdam, the Netherlands). The forward mutagenic primers were (bold lettering indicates mismatch positions):

Glu28->Ala,5'-GGCCATTTCTCGGCACATCTCGGGCG-3'

Glu28->Asp,5'-CCATTTCTCGGACCATCTCGGGCGATG-3'

Glu28->Gln,5'-CGGCCATTTCTCGCAACATCTCGGGCG-3'

Asp55->Ala,5'-GGGATCCGAAACGCCGTGCTCGAGGCG-3'

Glu112->Ala,5'-CCATTTCGGCACCCATGCGTTCATGATGCTGTGC-3'

Glu143->Ala,5'-CCGAATGGGTCGCGTACATTACGTTCGACG-3'

Glu176->Ala,5'-GCGTCGGCAACGCGAACTGGGGCTGC-3'

Glu176->Asp,5'-GCGTCGGCAACGACAACTGGGGCTGC-3'

Glu176->Gln,5'-GCGTCGGCAACCAGAACTGGGGCTGC-3'

Glu298->Ala,5'-CTGATCGTGGACGCATGGGGCACGTGG-3'

Glu298->Asp,5'-CTGATCGTGGACGACTGGGGCACGTGG-3'

Glu298->Gln,5'-CTGATCGTGGACCAATGGGGCACGTGG-3'

All mutations were confirmed by DNA sequencing. Expression and subsequent purification of wild-type or mutated AbfD3, produced in E.coli BL21(DE3)pLysS (Stratagene), were performed as previously described (Debeche et al., 2000Go).

Enzyme assays

{alpha}-L-Arabinofuranosidase activity of each mutant was determined by continuous measurement of p-nitrophenol (pNP) release. Normally, an aliquot (900 µl) of 5 mM pNP-ara in 50 mM sodium acetate (pH 5.8) was mixed in a cuvette with 100 µl of enzyme and pre-incubated at 60°C for 5 min. After, hydrolysis of pNP-ara was monitored over a 15 min period at 401 nm (in these conditions {varepsilon}(pNP) = 2126 M-1). To calculate the specific activity of each mutated enzyme, the molecular extinction coefficient was assumed to be the same as that of the wild-type AbfD3 ({varepsilon}280nm = 85 230 M-1). For determination of Km, the standard assay was performed in triplicate using various substrate (pNP-ara) concentrations in the range 0.01–5 mM. Km values were determined by analysing the slopes of Lineweaver–Burk plots.

The thermal stability of the mutant enzymes was determined by incubating the enzymes at defined temperatures in a buffered solution. Aliquots were removed at regular time intervals and were used to perform the assay described above.

To analyse the effect of anions on hydrolysis, the kinetic parameters of reactions catalysed by wild-type or mutant AbfD3 were determined in the presence of various concentrations of sodium azide or sodium formate, using the previously described assay.

The pH dependence of enzyme activity was studied using a discontinuous enzyme assay. For this method, solutions of pNP-ara (5 mM) were prepared by dissolving the substrate in universal buffers (Britton and Robinson type) (McKenzie, 1969Go) having pH values in the range 3.5–10.5. Hydrolysis was initiated by the addition of 100 µl of enzyme to 900 µl of each of the pNP-ara solutions. Reactions were terminated by the addition of one volume of 1 M sodium carbonate. One international unit (IU) of activity was defined as the amount of enzyme releasing 1 µmol of pNP per minute.

Protein analysis

All AbfD3 variants were analysed in order to verify that all mutant enzymes displayed physicochemical characteristics similar to those of wild-type AbfD3. SDS–PAGE (12% w/v) was performed essentially according to the standard method (Laemmli, 1970Go). Molecular mass was determined using a PE Biosystems Voyager DE (Framingham, USA) MALDI-ToF mass spectrometer. Spectra were recorded in positive ion mode using sinapinic acid as the matrix. Protein secondary structure was examined using a JASCO J-810 spectropolarimeter. Spectra were measured at 20°C using 10 µM protein solutions in pure water.

Analysis of the products of azide-reactivated hydrolyses

Initial product analysis was performed using TLC. Samples were applied to 0.2 mm silica gel plates (K60, Merck, Nogent-sur-Marne, France) and migration was performed at room temperature using 9:0.5:0.5 (v/v/v) acetonitrile/triethylamine/water as the mobile phase. After migration, the plates were air-dried and vaporized with 0.2% (v/v) orcinol dissolved in H2SO4 (20% v/v), then heated at 100°C until the reaction products were visible.

Azide-reactivated hydrolyses of pNP-ara were further analysed using 1H- and 13C-NMR. In order to exchange 1H for D, a solution of pNP-ara (18.45 mM) in 50 mM sodium acetate buffer pH 5.8 was lyophilized and then re-dissolved in 5 ml of D2O (99.8 atom %). This was repeated three times. For hydrolysis, 700 µl of D2O-exchanged pNP-ara was mixed with 200 µl of 5 M sodium azide dissolved in D2O, and 100 µl of enzyme (1 mg/ml in 20 mM Tris–HCl, pH 8.0). Reactions were incubated for 16 h and then spectral data were recorded at 293 K using a Brucker DRX spectrometer, at 500.13 MHz for 1H-NMR and 125.75 MHz for 13C-NMR. Spectra were calibrated on the buffer acetate methyl proton at 1.89 p.p.m. The spectra of the pure products (arabinose, pNP and pNP-ara) were recorded using the same conditions (pH, temperature, concentration, solvent) as those for the enzymatic reactions.

The presence of an azide moiety in the product of the azide-reactivated reaction catalysed by Glu176->Ala was indicated using IR spectrometry. For analysis, a solution of the pure product (in acetonitrile) was placed in a demountable cell (pathlength 0.1 mm) equipped with a CaF2 IR window and spectra were collected using a Protégé 460 FT-IR spectrometer (Nicolet, Madison, WI, USA).


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Preliminary identification of the catalytic residues by alanine mutagenesis

Since the alignment reported by Zverlov et al. (Zverlov et al., 1998Go), was composed of only seven family 51 arabinofuranosidase sequences, we generated a new alignment which includes 17 sequences (Figure 1Go). The significant sequence differences between the arabinofuranosidases of eukaryotic origin (two sequences from Arabidopsis thaliana and one from Aspergillus niger) and those of prokaryotic origin, prevented the simple alignment of all of these sequences. However, by separately aligning the two sequence groups it was possible to identify several highly conserved motifs which facilitated the manual merger of the two alignments. Analysis of the final alignment confirmed the conserved nature of Glu176 and Glu298 (Zverlov et al., 1998Go) and, in addition, revealed four other invariant acidic residues (Glu28, Asp55, Glu112 and Glu143). In order to investigate the importance of all six residues, each one was independently substituted by an alanine. Biochemical analyses revealed that all of the mutants migrated on SDS–PAGE as single species and displayed molecular masses which were similar to that of the wild-type AbfD3 (56 102 ± 9.3 Da). In addition, the mutant enzymes appeared to be correctly folded as evidenced by wild-type-like thermal stabilities and circular dichroism spectra. Measurement of the specific activities of each of the mutants towards pNP-ara revealed that Asp55->Ala, Glu112->Ala and Glu143->Ala displayed specific activities (347, 356 and 361 IU/mg, respectively) similar to that of the wild-type AbfD3 (357 IU/mg). In contrast, Glu28->Ala, Glu176->Ala and Glu298->Ala were essentially inactive towards pNP-ara. The specific activities of these mutant enzymes were decreased 5950-, 8925- and 178 500-fold, respectively, compared to AbfD3. In a second mutagenesis experiment, the double mutant Glu28->Ala/Glu176->Ala was created and subsequently expressed and purified. The analysis of the specific activity of this enzyme failed to reveal any measurable activity.



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Fig. 1. Local similarities in the amino acid sequences of family 51 arabinofuranosidase. For clarity only those sequence blocks which contain conserved carboxylate residues are shown. Each sequence is numbered individually and dots indicate gaps which were introduced into the sequence in order to achieve good alignment. The inverted closed triangle symbol indicates the positions of the carboxylate residues which were mutated during the course of this work. Regions where sequence identity is 100% are indicated by white characters in black boxing. Highly conserved regions are indicated by grey shading. The sequences used for the alignment were:TXY-ABFD3, AbfD3 from T.xylanilyticus (O69262); BST_ABF1, AbfA from Bacillus stearothermophilus (AAD45520); BSU_ABFA and BSU_ABFB, Abf1 and Abf2 from B.subtilis (P94531 and P94552, respectively); SLI_ABFA, Abf from Streptomyces lividans (P53627); BOV_ABF1 and BOV_ABF2, Abf1 and Abf2 from Bacteroides ovatus (Q59218 and Q59219, respectively); CST_ABF, Abf from Clostridium stercorarium (O08457); CXY_ABF1 and CXY_ABF2, AbfI and AbfII from Cytophaga xylanolytica (O68278 and O68279, respectively); TM_ABF, Abf from Thermotoga maritima (AAD35366); SCO_ARA and SCO_ABF2, Ara and AbfA from Streptomyces coelicolor A3(2) (O88043 and CAB86096, respectively); SCH_ABFA, AbfI from Streptomyces chartreusis (BAA90771); ATH_ABFA and ATH_ABFB, T1N24.13 protein and Abf from A.thaliana (Q9XH04 and Q9SG80, respectively); ANG_ABF, AbfA from A.niger (P42254).

 
Kinetic analysis of mutant enzymes

To provide further insight into the catalytic roles of Glu28, Glu176 or Glu298, these residues were further substituted either by glutamine or aspartate. As before, biochemical analyses of all the mutant enzymes (Glu28->Ala/Asp/Gln, Glu176->Ala/Asp/Gln and Glu298->Ala/Asp/Gln) indicated that the overall structural integrity of the AbfD3 variants was not perturbed. Michaelis–Menten parameters for the hydrolysis of pNP-ara were determined for reactions catalysed by each mutant (Table IGo), with the exception of Glu29->Ala and the double mutant Glu28->Ala/Glu176->Ala, which had no detectable activity. While the majority of the mutant enzymes displayed Km values very similar to that of AbfD3, the Km value displayed by Glu28->Gln was very high. As a result, the values for specific activity and kcat of this enzyme were underestimated. The Glu176->Gln mutant, which exhibited the highest activity (its specific activity was only 100-fold lower than that of AbfD3), showed only a 66-fold reduction of the kcat/Km value and a 110-fold reduction of the kcat value. Likewise, among the three Glu298 mutants, Glu298->Gln also exhibited the highest residual activity. The kcat values for Glu28->Ala and Glu176->Ala were decreased 6510- and 10 510-fold, respectively, while those of Glu28->Asp, Glu176->Asp and Glu298->Asp were decreased 3800-, 6310- and 7735-fold, respectively.


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Table I. Kinetic parameters for the hydrolysis of pNP-ara by wild-type and AbfD3 mutantsa
 
pH dependence of mutated enzyme activity

To investigate pH dependence of mutated AbfD3, we chose the isosteric mutants Glu28->Gln, Glu176->Gln and Glu298->Gln. For each mutant enzyme, the specific activity at different pH values was determined. Figure 2Go shows the relative activities of the mutants as a function of pH compared to that of AbfD3. Wild-type activity was described by a typical pH dependent curve, with maximum activity being obtained between pH 5.6 and 6.2. However, hydrolysis of pNP-ara by Glu176->Gln was characterized by a marked insensitivity to pH while Glu28->Gln and Glu298->Gln displayed an increased pH sensitivity and lower pH optima.



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Fig. 2. pH dependence of enzymatic activity of wild-type and mutant AbfD3 forms. Activity measurements were performed using a discontinuous assay method in which hydrolysis of pNP-ara was catalysed either by wild-type AbfD3 (closed squares) or one of the mutant AbfD3 forms (Glu28->Gln, closed triangle; Glu176->Gln, closed diamond; Glu298->Gln, closed circle). All measurements were performed in the presence of universal buffer adjusted to different pH values in the range 3.5–10.5. The maximum activity for each enzyme was determined and defined as 100% relative activity.

 
Effect of exogenous nucleophiles on mutant enzyme activity

It has been shown that the addition of exogenous nucleophiles to reactions catalysed by mutant glycosyl hydrolases, where the nucleophile or acid-base have been eliminated, can lead to reactivation (MacLeod et al., 1994Go). Therefore, the sodium salts of azide and formate were employed in separate experiments in order to reactivate the AbfD3 mutants, Glu28->Ala, Glu176->Ala and Glu298->Ala. Likewise, large concentration-dependent increases in activity were observed with both anions, whereas no such reactivation was observed with wild-type AbfD3. For Glu28->Ala, increasing concentrations of sodium formate induced a progressive but very significant rise in activity (60-fold increase with 4 M sodium formate), which did not reach saturation in the concentration range that was tested. Similarly, increasing concentrations of sodium formate enhanced the activity of the mutant Glu176->Ala, although the overall enhancement was much less significant (4-fold increase at 4 M sodium formate). In contrast, the mutant Glu298->Ala, was significantly reactivated (36-fold increase) using much lower concentrations (in the range 0.5–1.0 M) of sodium formate, while higher concentrations led to inhibition. Sodium azide had a similar, significant effect on the three mutants, although much lower concentrations were required to achieve almost maximal reactivation. Indeed, 6- (Glu28->Ala), 7- (Glu176->Ala) and 67-fold (Glu298->Ala) increases in activity were observed up to 0.5 M sodium azide. However, beyond a concentration of 1 M the activity of the mutants Glu176->Ala and Glu298->Ala was inhibited, whereas that of Glu28->Ala was increased further, but in a more progressive manner (9-fold increase at 4 M sodium azide). In order to further investigate the reactivation process, the kinetic parameters for AbfD3 and the mutants Glu28->Ala, Glu176->Ala and Glu298->Ala in the presence of azide and formate salts were determined (Table IIGo). The presence of either anion (2 M) in reactions catalysed by the mutant Glu28->Ala resulted in very significant increases in the kcat value (32- and 430-fold, respectively, compared to control reactions), but also provoked large increases in the Km values. In the case of the mutant Glu176->Ala, the presence of azide or formate (1 M) caused only small increases in the kcat values, while diminishing the Km values. For Glu298->Ala, the absence of reliable kinetic parameters for this mutant in the absence of exogenous nucleophiles precluded any comparison of kcat and Km values. However, the Km values determined in the presence of azide or formate were similar to that of AbfD3 and the kcat values were of the same order of magnitude when compared to that of the relatively active mutant, Glu176->Ala, in the same conditions.


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Table II. Kinetic parameters for the hydrolysis of pNP-ara by wild-type AbfD3 and mutant enzymes in the presence of exogenous nucleophiles
 
Stereochemical course of azide-reactivated hydrolyses

Often reactivation using exogenous nucleophiles can lead to the production of stable glycosyl intermediates which can be identified by NMR spectroscopy (MacLeod et al., 1996Go). In order to detect the formation of arabinosyl-azide in azide-reactivated hydrolyses of pNP-ara the reactions products were analysed using a combination of analytical techniques. For the reaction catalysed by Glu28->Ala, both TLC and NMR analyses revealed that complete hydrolysis had occurred and that two products, pNP and arabinose, had been generated. In contrast, the products of azide-reactivated reactions catalysed by Glu176->Ala or Glu298->Ala contained, in addition to pNP, a new product in place of arabinose. In the case of Glu176->Ala, hydrolysis was rapid and led to the complete hydrolysis of pNP-ara which facilitated analysis of the new unidentified product by NMR and IR spectroscopy. The chemical shifts of the H-1 (5.41 p.p.m.) and C-1 (95.72 p.p.m.) for this product were almost identical to those of 2,3,5-tri-O-benzyl-{alpha}-D-arabinofuranosyl-azide (5.41 and 94.45 p.p.m., respectively) and the coupling constants of both compounds were very similar (Stimac and Kobe, 2000Go) (Table IIIGo). Likewise comparison of the 13C chemical shifts of pNP-ara of this compound revealed that, with the exception of the C-1 signal, these spectra could be superimposed (Figure 3Go). In order to further characterize the new product IR spectroscopy was employed. This method revealed a band at 2111cm-1 characteristic of an azide moiety (Stimac and Kobe, 2000Go). With regard to the azide-reactivated reaction involving Glu298->Ala, this reaction was much slower than that catalysed by Glu176->Ala and was incomplete even after 72 h. During this prolonged incubation at 60°C, hydrolysis of pNP-ara did occur, but similar slow hydrolysis also occurred in the control experiment which was devoid of enzyme. Following this observation, further attempts at analysis were abandoned.


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Table III. Chemical shifts of the unidentified compound generated by the azide-restored reaction catalysed by the mutant Glu176->Ala and the two reference compounds
 


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Fig. 3. (A) Comparison of the 13C-NMR spectrum of the product generated by azide-reactivation of hydrolysis catalysed by the mutant Glu176->Ala with that of pNP-ara. (B) pNP-ara arabinosyl-azide adduct generated by azide-reactivation of the reaction catalysed by the mutant Glu176->Ala.

 

    Discussion
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 Abstract
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 Materials and methods
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 References
 
It is now a widely held concept that glycosyl hydrolases which belong to the same family employ the same catalytic mechanism for glycosidic bond cleavage. Therefore, since a previous study has demonstrated that at least two family 51 arabinofuranosidases (Abf2 from Bacillus subtilis and AbfA from A.niger) are retaining enzymes (Pitson et al., 1996Go), we have sought to identify the acid-base and nucleophile in the family 51 arabinofuranosidase from T.xylanilyticus.

Previously, alignment of selected sequences of several glycosyl hydrolase families (Zverlov et al., 1998Go) has allowed the identification of two potentially essential acidic residues (Glu176 and Glu298 in AbfD3). In this work, we present alignment data that confirms this finding and reveals four other invariant carboxylic acid residues. To probe the functional relevance of these six acidic residues, we have individually replaced them by a neutral alanine. A straightforward activity assay was then sufficient to evaluate the functional consequences of these mutations.

Kinetic analysis of reactions catalysed by Glu28->Ala, Glu176->Ala or Glu298->Ala clearly indicated that all of these residues are important for catalytic activity. Overall, the mutation of either of these residues to alanine, glutamine or aspartate causes activity to decrease by a factor of between 102 and 105. Although these decreases are significant, certain of the residual activities are high when compared to the results of some similar studies (Withers et al., 1992Go; Knetgel et al., 1995Go; Moracci et al., 1998Go). Often, high residual activity has been attributed to small amounts of contaminating wild-type enzyme from undetermined sources (Withers et al., 1992Go; Moracci et al., 1998Go; Wolfgang and Wilson, 1999Go). In the present work, contamination of mutant protein samples is unlikely because the double mutation Glu28->Ala/Glu176->Ala does not exhibit any measurable activity towards pNP-ara and none of the mutants displayed activity towards 1,5-{alpha}-L-arabinobiose, a substrate which possesses a much poorer leaving group than pNP-ara. Instead, for mutants, where glutamate is replaced by glutamine (Glu28->Gln, Glu176->Gln and Glu298->Gln), we believe that the residual activities are due to two factors. In the case of Glu298->Gln, deamidation (Wright, 1991Go; Bischoff and Kolbe, 1994Go) would appear to be the most probable explanation. Indeed, prolonged incubation of this particular mutant at elevated temperatures (from 60°C) leads to a significant restoration of activity (data not shown). Interestingly, Glu176->Gln and Glu28->Gln do not behave in this way, suggesting that the deamidation of Glu298 maybe favoured, or even catalysed, by its neighbouring structural environment. In contrast, the non-negligible residual activities of Glu28->Gln and Glu176->Gln are probably associated with the use of a substrate with an aryl group (pNP) which displays good leaving ability. In this case, the remarkable residual activity of the mutant Glu176->Gln is most consistent with that of a mutant enzyme lacking the catalytic acid-base residue (Zechel and Withers, 2000Go). Indeed, elimination of the acid-base catalyst should not have a profound effect on catalysis since pNP-ara itself should provide the necessary acid catalysis (Wang et al., 1995Go; MacLeod et al., 1996Go).

The unexpected behaviour of the glutamine substitutions precluded a comparison of these mutants with their aspartate homologues (Glu28->Asp, Glu176->Asp and Glu298->Asp). However, in agreement with the results of studies performed on other glycosidases, altering side-chain length while maintaining the ionizable group did lead to severe reductions in catalytic activity (Trimbur et al., 1992Go; Withers et al., 1992Go; Wakarchuk et al., 1994Go; Bolam et al., 1996Go). Therefore, in AbfD3, the correct positioning of the glutamate residues with respect to each other and to the substrate must be a critical factor for efficient catalysis.

The shape of the curve describing the pH dependence of retaining glycosyl hydrolases is mainly determined by the ionization of the acid-base residue (Wang et al., 1995Go; Moracci et al., 1996Go; Zechel and Withers, 2000Go). Therefore, elimination of this residue should lead to a significant alteration of the pH dependence curve. For AbfD3, a major modification of the pH dependence curve was observed in the case of the mutation of Glu176, supporting the view that this residue is the acid-base catalyst. In addition, this loss of pH dependence is further proof that the residual activity is not due to contaminating wild-type activity.

It has been shown that the addition of exogenous nucleophiles to reactions catalysed by mutant glycosyl hydrolases, where the nucleophile or the acid-base have been eliminated, can lead to reactivation (MacLeod et al., 1994Go; Wang et al., 1994Go, 1995Go; Moracci et al., 1998Go; Viladot et al., 1998Go; Islam et al., 1999Go; Zechel and Withers, 2000Go; Bravman et al., 2001Go). Usually, the use of such compounds will preferentially reactivate enzymes which lack the nucleophile, although reactivation of enzymes lacking the acid-base or another conserved active site residue is also observed (MacLeod et al., 1994Go; Wang et al., 1995Go; Cottaz et al., 1996Go; MacLeod et al., 1996Go; Islam et al., 1999Go; Bravman et al., 2001Go). Among the alanine mutants, Glu298->Ala displayed the highest reactivation in the presence of exogenous nucleophiles, indicating that Glu298 is probably the catalytic nucleophile. To a much lesser extent, the mutants Glu176->Ala and Glu28->Ala were also reactivated by exogenous nucleophiles. For Glu176->Ala, the putative acid-base residue, reactivation by formate or azide was accompanied by a decrease in the Km values and a very modest increase in the kcat values. This is surprising since, for a mutant which can not perform base-catalysed deglycosylation, one would expect that the use of exogenous anions should alleviate the accumulation of the glycosyl-enzyme intermediate (MacLeod et al., 1994Go; Zechel and Withers, 2000Go). However, similar results obtained by Viladot et al. led these authors to suggest that azide may also intervene at the glycosylation step (Viladot et al., 1998Go). Inversely, the behaviour of Glu28->Ala in the presence of anions (increased Km and kcat values) indicates that substrate fixation problems, which are not remedied by nucleophiles, are coupled to an increase in deglycosylation rate. Therefore, Glu28 is probably the third member of a catalytic triad and, like equivalent residues in other glycosyl hydrolases, maybe responsible for both the modulation of the ionization state of the acid-base residue and substrate fixation (Damude et al., 1995Go; Knetgel et al., 1995Go; McCarter and Withers, 1996Go; Brzozowski and Davies, 1997Go).

Previously, it has been shown that anion rescue of mutated glycosidases can provide the basis for a simple strategy to identify both the nucleophile and the acid-base residues (MacLeod et al., 1996Go). Indeed, following azide-reactivation of mutated glycosidases, glycosyl-azide adducts are formed exclusively when the nucleophile or acid-base residues have been eliminated. When the nucleophile is mutated, azide should perform direct displacement at the anomeric centre and provoke inversion of the anomeric configuration. In contrast, reactivation of an acid-base mutant should lead to a modified double displacement mechanism, where azide replaces the general base-activated water molecule for the deglycosylation step. In this case, the anomeric configuration of the glycosyl-azide adduct is identical to that of the substrate. So far, this strategy has been mainly employed for the study of glucosyl hydrolases, probably due to the availability of NMR data for the two glucosyl-azide anomers (Withers et al., 1992Go; Wang et al., 1995Go ; MacLeod et al., 1996Go; Moracci et al., 1998Go; Viladot et al., 1998Go). In this work, we have exploited certain elements of this strategy in order to enhance our study of the AbfD3 mutants. TLC and 1H-NMR analyses of the products of azide-rescued reactions catalysed by Glu28->Ala failed to reveal the formation of azide adducts and therefore allowed the elimination of Glu28 as a potential catalytic residue (MacLeod et al., 1996Go). In contrast, in the azide-reactivated reaction catalysed by Glu176->Ala, pNP-ara was hydrolysed to pNP and a second compound which upon IR spectroscopic analysis was shown to contain azide. In order to further analyse this azide adduct, NMR data were recorded and compared to those of reference compounds. Since a thorough data search failed to reveal any data concerning {alpha}- or ß-L-arabinofuranosyl azides, the reference compounds we chose were the substrate, pNP-ara, and {alpha}- and ß-tri-O-benzyl-D-arabinofuranosyl-azide (Stimac and Kobe, 2000Go). In this way, we were able to infer that the azide adduct possesses an {alpha}-L-furanose conformation and, in consequence, that chemical rescue of the mutant Glu176->Ala leads to the formation of {alpha}-L-arabinofuranosyl azide. Therefore, together with the other data presented in this work, we can confirm that Glu176 is the acid-base catalyst.

With regard to the elucidation of the exact role of Glu298, the use of chemical rescue proved to be inappropriate. Indeed, the extremely feeble activity of the mutant Glu298->Ala towards pNP-ara necessitated the use of very long incubation periods at 60°C to achieve hydrolysis. In these conditions, azide reacted directly with pNP-ara leading to the formation of an azide adduct in the absence of enzyme. A similar direct nucleophilic attack of a pNP-glycoside by azide has already been described for the study of the thermostable ß-glycosidase from Sulfolobus solfatricus (Moracci et al., 1998Go).

In conclusion, this work represents the first detailed study of the catalytic machinery of a glycofuranosyl hydrolase by site-directed mutagenesis. Our data provide strong evidence to support the hypothesis that Glu176 is the catalytic acid-base and, in addition, indicate that Glu298 is the nucleophile. A hitherto unidentified residue, Glu28, has also been shown to form part of a catalytic triad. In a previous report, Zverlov et al. (Zverlov et al., 1998Go) proposed that the family 51 arabinofuranosidases all belong to the 4/7 superfamily of glycosyl hydrolases (Jenkins et al., 1995Go). This claim was based on the prediction that the conserved motif G-N-E (residues 174–176 in AbfD3), which should be localized at the end of ß4, contains the acid-base and that a second residue (equivalent to Glu298 in AbfD3), which should be localized at the C-terminal end of ß7, is the catalytic nucleophile. Our data support this hypothesis and thus provide a vital key towards the attribution of a molecular architecture to the family 51 enzymes.


    Notes
 
3 To whom correspondence should be addressed. E-mail: michael.odonohue{at}univ-reims.fr Back


    Acknowledgments
 
The authors would like to thank Béatrice Hermant and Francioise Charton for their skilful technical assistance, Professor A.J.P.Alix for allowing us to perform the circular dichroism measurements, and Drs H.Driguez and S.Cottaz for their useful suggestions. This work was partially funded by a grant from the consortium Europol'Agro.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received April 17, 2001; revised September 6, 2001; accepted September 27, 2001.