The α-l-fucosidase from Sulfolobus solfataricus
- First Online:
- Cite this article as:
- Cobucci-Ponzano, B., Conte, F., Rossi, M. et al. Extremophiles (2008) 12: 61. doi:10.1007/s00792-007-0105-y
- 154 Views
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.
KeywordsGlycoside hydrolaseChemical rescueNucleophileAcid/baseCatalytic triad
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.
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 188.8.131.52) 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
Steady-state kinetic constants of wild type and D242G mutant at 65°C
kcat/KM (s−1 mM−1)
287 ± 11
0.028 ± 0.004
9.7 ± 0.3
0.19 ± 0.02
+NaCOOH 0.1 M
5.9 ± 0.2
1.0 ± 0.1
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).
Steady-state kinetic constants of wild type and mutants at 65°C
kcat/KM (s−1 mM−1)
287 ± 11
0.028 ± 0.004
240 ± 7
0.09 ± 0.01
224 ± 5
0.033 ± 0.003
419 ± 99
17.0 ± 5.7
1.86 ± 0.09
0.06 ± 0.01
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.
Steady-state kinetic constants of wild type, E58G and E292G mutants in different reaction conditions
kcat/KM (s−1 mM−1)
287 ± 11
0.028 ± 0.004
430 ± 49
0.26 ± 0.09
143 ± 8
1.6 ± 0.3
586 ± 43
0.6 ± 0.2
846 ± 46
1.1 ± 0.2
679 ± 82
2.9 ± 0.4
Sodium phosphate + NaN3c
1.86 ± 0.09
0.06 ± 0.01
1.86 ± 0.17
0.09 ± 0.02
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.
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.
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.