Extremophiles

, Volume 12, Issue 1, pp 61–68

The α-l-fucosidase from Sulfolobus solfataricus

Authors

    • Institute of Protein BiochemistryConsiglio Nazionale delle Ricerche
  • Fiorella Conte
    • Institute of Protein BiochemistryConsiglio Nazionale delle Ricerche
  • Mosè Rossi
    • Institute of Protein BiochemistryConsiglio Nazionale delle Ricerche
    • Dipartimento di Biologia Strutturale e FunzionaleUniversità di Napoli “Federico II”
  • Marco Moracci
    • Institute of Protein BiochemistryConsiglio Nazionale delle Ricerche
Review

DOI: 10.1007/s00792-007-0105-y

Cite this article as:
Cobucci-Ponzano, B., Conte, F., Rossi, M. et al. Extremophiles (2008) 12: 61. doi:10.1007/s00792-007-0105-y

Abstract

Glycoside hydrolases form hyperthermophilic archaea are interesting model systems for the study of catalysis at high temperatures and, at the moment, their detailed enzymological characterization is the only approach to define their role in vivo. Family 29 of glycoside hydrolases classification groups α-l-fucosidases involved in a variety of biological events in Bacteria and Eukarya. In Archaea the first α-l-fucosidase was identified in Sulfolobus solfataricus as interrupted gene expressed by programmed −1 frameshifting. In this review, we describe the identification of the catalytic residues of the archaeal enzyme, by means of the chemical rescue strategy. The intrinsic stability of the hyperthermophilic enzyme allowed the use of this method, which resulted of general applicability for β and α glycoside hydrolases. In addition, the presence in the active site of the archaeal enzyme of a triad of catalytic residues is a rather uncommon feature among the glycoside hydrolases and suggested that in family 29 slightly different catalytic machineries coexist.

Keywords

Glycoside hydrolaseChemical rescueNucleophileAcid/baseCatalytic triad

Introduction

Carbohydrates serve as structural components and energy source of the cell and are involved in a variety of molecular recognition processes in intercellular communication (Varki 1993; Sears and Wong 1996). Consequently, glycoside hydrolases play important roles in biological systems ranging from the degradation of polysaccharides as food source through to the modification of glycoconjugates on the surfaces of proteins and cells. Among the available glycoside hydrolases, the enzymes from hyperthermophiles are of particular interest for both basic and applied research. In fact, the function of the glycoconjugates identified in hyperthermophiles and of the enzymes involved in their synthesis and degradation is still largely unknown (Lower and Kennelly 2002). In addition, hyperthermophilic glycosidases are interesting model systems in basic research for the study of protein adaptation to heat and, since they catalyze single substrate reactions by following well-known mechanisms, they are the ideal candidates for the study of catalysis at high temperatures. Furthermore, they are particularly appealing for industrial applications as they show peculiar enzymological properties and can withstand the harsh operational conditions adopted in industrial applications. Beside this, the unique substrate specificities or reduced substrate/product inhibition allow the synthesis of new products that are not produced by their mesophilic counterparts (Fischer et al. 1996). On the other hand, the harsh conditions of growing of these organisms have hindered microbiological and genetic studies in vivo; therefore, the isolation of the genes encoding for hyperthermophilic glycosidases and the detailed enzymological characterization of these enzymes is the only approach to define their role in vivo.

Glycoside hydrolases are classified on the basis of their amino acid sequence similarity in about 100 families (http://www.cazy.org) and 14 clans showing conserved three-dimensional (3D) structures. These enzymes follow two distinct mechanisms which are termed retaining or inverting if the enzymatic cleavage of the glycosidic bond liberates a sugar hemiacetal with the same or the opposite anomeric configuration of the glycosidic substrate, respectively. Inverting glycoside hydrolases operate with a one step, single-displacement mechanism with the assistance of a general acid and a general base group in the active site (McCarter and Withers 1994). Instead, retaining enzymes follow a two-step mechanism with formation of a covalent glycosyl-enzyme intermediate (Fig. 1). The carboxyl group in the active centre functions as a general acid/base catalyst, and the carboxylate functions as the nucleophile of the reaction (Koshland 1953). In the first step (glycosylation step), the nucleophile attacks the anomeric group of the substrate, while the acid/base catalyst, acting in this step as a general acid, protonates the glycosidic oxygen, thereby assisting the leaving of the aglycon moiety. The concerted action of the two amino acids leads to the formation of a covalent glycosyl-enzyme intermediate (Sinnot 1990; McCarter and Withers 1994). In the second step (deglycosylation step), the glycosyl-enzyme intermediate is cleaved by a water molecule that acts as nucleophile being polarized by the general base catalyst. The product of the reaction retained the anomeric configuration of the substrate. When an acceptor different from water, such as an alcohol or a sugar, intercepts the reactive glycosyl-enzyme intermediate, retaining enzymes work in transglycosylation mode; the interglycosidic linkage of the product maintains the same anomeric configuration of the substrate. This property makes the retaining glycosyl hydrolases interesting tools for the synthesis of carbohydrates. Despite the differences, the two mechanisms show significant similarities: they both operate via transition states with substantial oxocarbenium ion character and have a catalytic dyad with a pair of carboxylic acids directly involved in catalysis.
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Fig. 1

Retaining reaction mechanism of α-l-fucosidases

The determination of the reaction mechanism and the identification of key active-site residues in glycoside hydrolases are crucial issues to allow the classification of these enzymes (Henrissat and Bairoch 1993; Henrissat and Davies 1997), to unravel the catalytic machinery (McCarter and Withers 1994; Zechel and Withers 2000), and to produce enzymes with novel characteristics (Perugino et al. 2004).

Family 29 of glycoside hydrolases (GH29) groups α-l-fucosidases (EC 3.2.1.51) from plants, vertebrates, and pathogenic microbes of plants and humans (Henrissat 1991). α-l-fucosidases are exo-glycosidases capable of cleaving α-linked l-fucose residues from glycoconjugates, in which the most common linkages are α-(1-2) to galactose and α-(1-3), α-(1-4), and α-(1-6) to N-acetylglucosamine residues. These compounds are involved in a variety of biological events as growth regulators and as the glucidic part of receptors in signal transduction, cell–cell interactions, and antigenic response (Vanhooren and Vandamme 1999). The central role of fucose derivatives in biological processes explains the interest in α-l-fucosidase and fucosyl-transferase activities. α-l-fucosidases in higher plants and in mammals are associated with different mechanisms of cell growth and regulation, since they are involved in the modification of fucosylated glucans (Staudacher et al. 1999). In plants, α-l-fucosylated oligosaccharides derived from xyloglucan have been shown to regulate auxin- and acid pH-induced growth (de La Torre et al. 2002). In mammals, oligosaccharides containing fucose are reported to play important roles in a variety of physiological and pathological events (Xiang and Bernstein 1992; Wiese et al. 1997; Listinsky et al. 1998; Mori et al. 1998; Noda et al. 1998; Russell et al. 1998; Michalski and Klein 1999; Rapoport and Pendu 1999).

Here, the characterization of the reaction mechanism and the identification of the catalytic residues of the first archaeal α-l-fucosidase identified in the hyperthermophile Sulfolobus solfataricus are briefly reviewed.

General features of the α-l-fucosidase

The first archaeal α-l-fucosidase has been identified and characterized recently (Cobucci-Ponzano et al. 2003a). The analysis of the genome of the hyperthermophilic archaeon S. solfataricus (She et al. 2001) revealed the presence of two open reading frames (ORFs), SSO11867 and SSO3060, encoding for 81 and 426 amino acid polypeptides that are homologous to the N- and the C-terminal parts, respectively, of full-length bacterial and eukaryal GH29 fucosidases (Henrissat 1991). The two ORFs are separated by a −1 frameshift and, to produce a single polypeptide, a single base was inserted by site-directed mutagenesis in the region of overlap between SSO11867 and SSO3060, restoring a single reading frame between the ORFs. The single ORF obtained was used to express the enzyme in Escherichia coli (Cobucci-Ponzano et al. 2003a). The recombinant enzyme, Ssα-fuc, is a nonamer of 57 kDa molecular mass subunits in solution and is highly active and specific for 4NP-α-l-fucoside (4-NP-α-l-Fuc) at 65°C (Cobucci-Ponzano et al. 2003a; Rosano et al. 2004). Moreover, Ssα-fuc is thermoactive and thermostable, as expected for an enzyme from a hyperthermophilic microorganism. The optimal temperature of Ssα-fuc is 95°C and the enzyme displayed high stability maintaining 60% of the residual activity after 2 h at 80°C (Cobucci-Ponzano et al. 2003a). It is worth noting that the mutation inserted to obtain the recombinant Ssα-fuc was designed on the basis of a mechanism of regulation of gene expression known as programmed −1 frameshifting (Farabaugh 1996). Very recently it was found that the two ORFs express in vivo a full length protein by programmed −1 frameshifting, demonstrating, for the first time, that this mechanism of gene expression, known so far only in Eukarya and Bacteria (Baranov et al. 2001) is used to regulate the expression of this gene in S. solfataricus (Cobucci-Ponzano et al. 2006).

In the framework of our mechanistic studies on glycoside hydrolases, the reaction mechanism of Ssα-Fuc was studied in detail and the residues directly involved in catalysis were identified. The retaining reaction mechanism was demonstrated, for the first time in GH29, by using Ssα-fuc. In fact, the enzyme is able to function in transfucosylation mode as reported for several mesophilic α-fucosidases (Murata et al. 1999; Farkas et al. 2000); its synthetic ability was demonstrated by using 4-NP-α-l-Fuc and 4-NP-α-d-glucoside (4-NP-α-d-Glc) as donor and acceptor, respectively. The fucosylated products were disaccharides of the acceptor in which the α-l-fucose moiety of the donor is attached at positions 2 and 3 of Glc (α-l-Fucp-(1-2)-α-d-Glc-O-4-NP and α-l-Fucp-(1-3)-α-d-Glc-O-4-NP) (Cobucci-Ponzano et al. 2003a). The α-anomeric configuration of the interglycosidic linkages in the products demonstrated that GH29 α-fucosidases follow a retaining reaction mechanism (Cobucci-Ponzano et al. 2003a). The hydrolytic activity of Ssα-fuc on the disaccharide α-l-Fuc-(1-3)-α-l-Fuc-O-4-NP revealed that the enzyme is an exo-glycosyl hydrolase that attacks the substrates from their non-reducing end (Cobucci-Ponzano et al. 2003a).

Identification of the nucleophile of the reaction

The active-site residues of retaining α- and β-glycosidases have been identified with a variety of methods, including mechanism-based inhibitors labelling the catalytic nucleophile and inspection of X-ray structures (McCarter and Withers 1996; Vocadlo et al. 2000, 2001; Tarling et al. 2003). An alternative approach often exploited for retaining glycoside hydrolases consists in the mutation of aspartic/glutamic acid residues identified by sequence analysis and conserved in the family of interest. Mutations of the catalytic residues with non-nucleophilic amino acids lead to the strong reduction or even abolition of the enzymatic activity (Ly and Withers 1999). However, these mutants can be reactivated in the presence of external nucleophiles such as sodium azide. The isolation of glycosyl-azide products with an anomeric configuration opposite to that of the substrate allows the identification of the catalytic nucleophile of the reaction (Fig. 2) (Ly and Withers 1999). This methodology is termed chemical rescue. Once the reaction mechanism and the active-site residues of a particular enzyme have been experimentally determined, they can be easily extended to all the homologous enzymes by following the classification in families.
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Fig. 2

Identification, by chemical rescue, of the nucleophile residue in retaining α-fucosidases. The α-l-fucoside substrate is converted in β-l-fucosyl-azide

The nucleophile of GH29 α-l-fucosidases was identified, for the first time, by reactivating the Ssα-fuc D242G inactive mutant in the presence of sodium azide and by analyzing the anomeric configuration of the fucosyl-azide product (Cobucci-Ponzano et al. 2003b). The D242G mutant showed a turnover number (kcat) of 0.24 s−1 on 4-NP-α-l-Fuc, which is 1.2 × 10−3 times that of the wild type activity (287 s−1), indicating that the D242G mutation affected a residue involved in catalysis in Ssα-fuc. In the presence of 2 M sodium azide the mutant revealed a kcat value of 9.66 s−1, indicating a 40-fold re-activation by azide (Table 1). The fucosyl-azide product obtained by the D242G mutant was found in the inverted (β-l) configuration when compared with the substrate (Fig. 2). This finding allowed, for the first time, the unambiguous assignment of Asp242 and its homologous residues as the nucleophilic catalytic residues of GH29 α-l-fucosidases (Cobucci-Ponzano et al. 2003b). The activity of the mutant D242G was also rescued on 4-NP-α-l-Fuc in the presence of sodium formate. The steady-state kinetic constants of D242G, determined in the presence of external nucleophiles, revealed that sodium azide and sodium formate produced about 0.5 and 0.056% of reactivation of the mutant, respectively, which was calculated by taking as 100% the kcat/KM of the wild type (Table 1) (Cobucci-Ponzano et al. 2003b). The higher nucleophilicity of sodium azide, when compared with formate, explains the higher reactivation produced by the former. The specific activity of the mutant increases with temperature up to 80°C, indicating that, despite the mutation, the reactivated enzyme maintains its thermophilicity.
Table 1

Steady-state kinetic constants of wild type and D242G mutant at 65°C

 

kcat (s−1)

KM (mM)

kcat/KM (s−1 mM−1)

Wild type

287 ± 11

0.028 ± 0.004

10,250

D242G

NAa

NAa

+NaN3 2M

9.7 ± 0.3

0.19 ± 0.02

51.5

+NaCOOH 0.1 M

5.9 ± 0.2

1.0 ± 0.1

5.8

aNA (not measurable activity) means that, using 20 μg of enzyme in the assay, the rates of change in absorbance at substrate concentration below 0.1 mM did not vary in the experimental conditions

This was the first example of the application of the chemical rescue method to α-(d/l)-glycosidases as it has been used so far only for β-d-glycosidases. Later, by following a similar approach, the corresponding residue was also identified in the α-fucosidase from Thermotoga maritima (Tmα-fuc) (Tarling et al. 2003). These results on GH29 enzymes demonstrated that chemical rescue could be of general applicability for retaining enzymes.

Identification of the acid/base catalyst of the reaction

The approach utilized for the identification of the acid/base catalyst of retaining glycosyl hydrolases is less straightforward if compared to the nucleophile. In fact, the use of specific inhibitors for the acid/base catalyst is still elusive and successful results are less common (Tull et al. 1996; Vocadlo et al. 2002). For these reasons, the acid/base catalyst of several retaining glycosidases was identified through 3D structure inspection and detailed characterization of mutants in which conserved aspartic and glutamic acid residues have been replaced by isosteric and non-ionizable amino acids as asparagine, glutamine, alanine, or glycine (Ly and Withers 1999). Replacing the acid/base catalyst with the small non-ionizable glycine residue generally reduces dramatically the activity of the mutant and modifies its pH profile. In fact, when the acid/base is removed, the basic limb of the typical bell-shaped pH dependence curve is severely affected (Ly and Withers 1999). The chemical rescue of the activity of the inactive mutant is also a useful tool. In fact, as described above for the mutant in the residue acting as the nucleophile of the reaction, the presence of the glycine generates a room in the active site allowing the access of a small nucleophilic ion. However, this time, the external nucleophile (i.e. azide) occupies the cavity formed by mutation after the formation of the glycosyl-enzyme intermediate. In these cases, the rate enhancement and the isolation of a glycosyl-azide product with the same anomeric configuration of the substrate resulted in the most effective method to unequivocally identify the acid/base catalyst (MacLeod et al. 1996; Viladot et al. 1998; Ly and Withers 1999; Vallmitjana et al. 2001; Debeche et al. 2002; Li et al. 2002; Rydberg et al. 2002; Shallom et al. 2002; Vocadlo et al. 2002; Bravman et al. 2003; Zechel et al. 2003; Paal et al. 2004; Sulzenbacher et al. 2004).

By following this line of approach, in Ssα-fuc several aminoacids among highly conserved histidine, aspartic, and glutamic acid residues were picked and mutated into a glycine; the mutants H46G, E58G H123G, D124G, D146G were characterized in detail. Furthermore, the mutant E292G was added to this collection since the corresponding residue was identified as the acid/base catalyst in Tmα-fuc. Surprisingly, this residue falls in a region of alignment scarcely conserved in GH29 (Sulzenbacher et al. 2004). The preliminary kinetic characterization of the Ssα-fuc mutants on 4-NP-α-l-Fuc, reported in Table 2, revealed that D124 and D146 were not directly involved in catalysis since the activity was not significantly affected by the mutations. Instead, the affinity for the substrate of H46G and H123G was remarkably different from that of the wild type: the mutation of His46 produced a 607-fold increase in the KM, while no saturation was observed with the mutant H123G. Also the mutation of the residues Glu58 and Glu292 affected catalysis severely (Table 2); again, no saturation could be observed with the former residue, while E292G showed unchanged affinity for 4-NP-α-l-Fuc but a 154-fold reduction in the turnover number (Cobucci-Ponzano et al. 2005).
Table 2

Steady-state kinetic constants of wild type and mutants at 65°C

 

kcat (s−1)

KM (mM)

kcat/KM (s−1 mM−1)

Wild type

287 ± 11

0.028 ± 0.004

10,250

D124G

240 ± 7

0.09 ± 0.01

2,740

D146G

224 ± 5

0.033 ± 0.003

6,791

H46G

419 ± 99

17.0 ± 5.7

25

H123G

NDa

NDa

3.5

E58G

NDa

NDa

1.9

E292G

1.86 ± 0.09

0.06 ± 0.01

33

The specificity constants were calculated by using 4-NP-α-l-Fuc in the range 1–7 mM and by plotting (velocity) vs. (S)

aND not determined. No saturation was observed on up to 25 mM 4-NP-α-l-Fuc

This preliminary characterization indicated that the residues His46, His123, Glu58, and Glu292 are involved in substrate binding or in catalysis; however, experiments of chemical rescue of the enzymatic activity on the mutants H46G and H123G allowed us to exclude their involvement in catalysis (Cobucci-Ponzano et al. 2005). Furthermore, the inspection of the crystal structure of Tmα-fuc suggested that His46 and His122, which correspond to His34 and His128 in Tmα-fuc, respectively, stabilize the 4-hydroxyl group of fucose.

The characterization of the mutants E58G and E292G, compared to the data collected on the corresponding residues in Tmα-fuc (Glu66 and Glu262, respectively), gave unexpected results, suggesting that in GH29 two catalytic machineries coexist. The analysis of the 3D structure of the Tmα-fuc and the kinetic characterization of the mutants clearly indicated that, in this enzyme, Glu66 and Glu266 were involved in substrate binding and in the acid/base catalysis, respectively (Sulzenbacher et al. 2004). In particular, the mutation in the residue Glu66 produced a tenfold drop of activity while the mutation in Glu266 determined the absence of saturation by the substrate. Intriguingly, in Ssα-fuc the mutant E58G mirrored the behaviour of the Tmα-fuc mutant in the Glu266 residue (lack of saturation) while the Ssα-fuc mutant E292G showed a marked inactivation as observed for the Tmα-fuc mutant in Glu66 (Table 2). These results suggested that in Ssα-fuc Glu58 is the acid/base catalyst. This conclusion was further supported by the analysis of the pH dependence of wild type and mutants Ssα-fuc. Wild type enzyme has a peculiar pH dependence, which is not bell-shaped, but shows a reproducible increase of activity at pH 8.6 (Fig. 3), suggesting that more than two ionizable groups are involved in catalysis (Debeche et al. 2002). The pH dependence of the mutant E292G is similar to that of the wild type enzyme; instead, this pH profile was dramatically changed in the E58G mutant, producing a typical bell-shaped curve with a pH optimum at 4.6 sharper than that of the wild type (3.0–5.0) (Fig. 3). These data suggested that the removal of Glu58 unmasked the ionization of a group responsible for the basic limb (pKa 5.3) and possibly increased the pKa of the nucleophile of the reaction mainly determining the acidic limb. Unfortunately, the pH dependence of the wild type and mutant Tmα-fuc was not reported, precluding a detailed comparison.
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Fig. 3

pH Dependence of wild type, E58G and E292G. Data are reported as percent of maximal activity on 4-NP-α-l-Fuc at 65°C

To try to better define the nature of the acid/base catalyst in Ssα-fuc the chemical rescue, which, as described above, is one of the most definite tools to assign this role in glycosidases, was exploited. Remarkably, E58G was activated by more than 70-fold in the presence of sodium azide, formate and acetate (Table 3). In addition, it was observed that the sodium azide anion activated E58G only in the presence of larger ions (phosphate and citrate) while this effect was much reduced in acetate and formate, which already activate the mutant. Presumably, in phosphate and citrate buffers, azide has full access to the small cavity created by the mutation in the active site. Instead, acetate and formate, occupying this space, could activate the reaction hampering the access of azide. In striking contrast, the activity of E292G could not be rescued by any of the nucleophiles used. These results made it very unlikely that Glu292 is the acid/base catalyst of Ssα-fuc, and allowed to assign this role to Glu58.
Table 3

Steady-state kinetic constants of wild type, E58G and E292G mutants in different reaction conditions

 

kcat (s−1)

KM (mM)

kcat/KM (s−1 mM−1)

pH

Reaction conditiona

Wild type

287 ± 11

0.028 ± 0.004

10,250

6.3

Sodium phosphate

430 ± 49

0.26 ± 0.09

1,624

5.0

Sodium citrate

E58G

NDb

NDb

1.9

6.3

Sodium phosphate

143 ± 8

1.6 ± 0.3

89

4.6

Sodium citrate

586 ± 43

0.6 ± 0.2

950

4.6

Sodium acetate

846 ± 46

1.1 ± 0.2

790

4.6

Sodium formate

679 ± 82

2.9 ± 0.4

229

6.3

Sodium phosphate + NaN3c

E292G

1.86 ± 0.09

0.06 ± 0.01

33

6.3

Sodium phosphate

1.86 ± 0.17

0.09 ± 0.02

21

4.6

Sodium acetate

The specificity constants were calculated by using 4-NP-α-l-Fuc in the range 1–7 mM and by plotting (velocity) vs. (S)

aAssays were performed at 65°C in the reported buffers in 0.05 M concentration on 4-NP-α-l-Fuc

bNot determined; no saturation was observed with up to 25 mM 4-NP-α-l-Fuc

cSodium azide was used at the concentration of 0.15 M

These data demonstrated that the Glu58 is the acid-base catalyst and suggested that the Glu292 has a relevant role in catalysis presumably modulating the pKa of the latter, thereby affecting the pH optimum of the enzyme. Intriguingly, the behaviour of the catalytic residues of Ssα-fuc is different from that of Tmα-fuc (Sulzenbacher et al. 2004). Nevertheless, considering that among the amino acid sequences of GH29 the predicted acid/base residues are not invariant, it would not be surprising that the enzymes show structural differences in the active site explaining the different catalytic machineries.

Conclusions

The first archaeal α-l-fucosidase was identified in S. solfataricus and is encoded by an interrupted gene. The recombinant enzyme Ssα-fuc, obtained by site directed mutagenesis, is fully active and thermostable and allowed the first detailed study on the retaining reaction mechanism of GH29 glycoside hydrolases. Interestingly, the inspection of the catalytic machinery of Ssα-fuc revealed the presence in the active site of a triad of catalytic residues, namely Asp242, Glu58, and Glu292. This is a rather uncommon feature among the glycoside hydrolases and the comparison with the Tmα-fuc suggested that in GH29 slightly different catalytic machineries coexist. It is worth noting that the use of the chemical rescue method at harsh pHs and ionic strengths was possible because of the intrinsic stability of Ssα-fuc and resulted of general applicability for β and α glycoside hydrolases.

The body of this work demonstrates that the α-l-fucosidase from S. solfataricus is an interesting model system to uncover new mechanisms of gene expression in Archaea and to study the reaction mechanisms of glycoside hydrolases. In addition, the transfucosylating activity of Ssα-fuc and the availability of several mutants in the active site could be the starting points for the biotechnological exploitation of this enzyme in the synthesis of fucosylated oligosaccharides.

Acknowledgments

This work was supported by MIUR project “Folding di proteine: l’altra metà del codice genetico” RBAU015B47_006. The IBP-CNR belongs to the Centro Regionale di Competenza in Applicazioni Tecnologico-Industriali di Biomolecole e Biosistemi.

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© Springer 2007