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Difference between revisions of "Glycoside Hydrolase Family 29"
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== Three-dimensional structures == | == Three-dimensional structures == | ||
− | Very few structures are available for GH29 enzyme. The first crystal structure being solved is the one for the α-L-fucosidase from ''T. maritima'', Tmα-fuc. The simultaneous solution of the structures of an enzyme-product complex and of a glycosyl-enzyme intermediate allowed the unambiguous identification of the [[general acid/base]] <cite> | + | Very few structures are available for GH29 enzyme. The first crystal structure being solved is the one for the α-L-fucosidase from ''T. maritima'', Tmα-fuc. The simultaneous solution of the structures of an enzyme-product complex and of a glycosyl-enzyme intermediate allowed the unambiguous identification of the [[general acid/base]] <cite>8</cite>, as described above. Tmα-fuc assembles as a hexamer and displays a two-domain fold, composed of a catalytic (β/α)<sub>8</sub>-like domain and a C-terminal β-sandwich domain. The two key active site residues are located at the C-terminal ends of strands of β-strands 4 (nucleophile) and 6 (acid/base). |
Revision as of 09:12, 12 January 2010
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- Author: ^^^Gerlind Suzlenbacher^^^
- Responsible Curator: ^^^Steve Withers^^^
Glycoside Hydrolase Family GH 29 | |
Clan | none |
Mechanism | retaining |
Active site residues | known |
CAZy DB link | |
http://www.cazy.org/fam/GH29.html |
Substrate specificities
The glycoside hydrolases of this family are exo-acting α-fucosidases from archaeal, bacterial and eukaryotic origin. No other activities have been observed for GH29 family members. So fare the only other CAZY family containing α-fucosidases is family GH95. The human enzyme FucA1 is of medical interest because its deficiency leads to fucosidosis, an autosomal recessive lysosomal storage disease [1].
Kinetics and Mechanism
GH29 α-fucosidases are retaining enzymes following a classical Koshland double-displacement mechanism, as first proposed in 1987 for human liver α-fucosidase via burst kinetics experiments and using methanol as an alternative glycone acceptor to produce methyl-α-L-fucoside [2]. This has been further confirmed by 1H NMR monitoring of the reaction catalyzed by a α-L-fucosidase from Thermus sp. [3], and a α-L-fucosidase from the marine mollusc Pecten maximus[4], as well as by COSY and 1H-13C NMR spectroscopy analysis of the interglycosidic linkage of disaccharides formed by the transglycosylation action of Sulfolobus solfataricus α-L-fucosidase, Ssα-fuc [5]. GH95 α-fucosidases, in contrast, operate with inversion of the anomeric configuration.
Catalytic Residues
The catalytic nucleophile in GH29 was first identified in the Sulfolobus solfataricus α-L-fucosidase, Ssα-fuc, as Asp242 in the sequence VYFDWWI via chemical rescue of an inactive mutant with sodium azide [6]. Concomitantly the catalytic nucleophile of Thermotoga maritima α-L-fucosidase, Tmα-fuc, was confirmed to be Asp224 in the sequence LWNDMGW through trapping of the 2-deoxy-2-fluorofucosyl-enzyme intermediate and subsequent peptide mapping via LC-MS/MS technologies, as well as by chemical rescue of an inactive mutant [7]. The trapping of the 2-deoxy-2-fluorofucosyl-enzyme intermediate in Tmα-fuc was corroborated by crystallographic studies [8]. The catalytic nucleophile of the human enzyme FucA1 has recently been identified as being Asp225 [9].
Whereas the catalytic nucleophile in GH29 has been shown to be a conserved aspartate residue, the identity of the general acid/base is still controversial. Structural and mutagenesis studies of Tmα-fuc provided strong evidence for the variant Glu266 being the general acid/base [8]. In the crystal structure the carboxyl function of this residue is 5.5 Å apart from that of the catalytic nucleophile Asp224, a distance commonly observed in retaining glycosidases proceeding via a classical Koshland double-displacement mechanism. Although multiple sequence alignments show that Glu266 is not conserved within GH29, the residue is structurally conserved in two 3-D structures of α-L-fucosidases from Bacteroides thetaiotaomicron sp., recently deposited in the Protein Data Bank (accession numbers 3eyp and 3gza). Studies of Ssα-fuc demonstrated that mutation of the Glu residue corresponding in sequence to Tmα-fuc Glu266 scarcely impaired the catalytic activity of the enzyme, whereas the E58G mutant yielded a 4000-fold reduction of kcat/KM and could be chemically rescued [10]. In the crystal structure of Tmα-fuc in complex with fucose [8], the residue corresponding to Ssα-fuc Glu58, Glu66, is found 7.5 Å distant form the catalytic nucleophile Asp224 and hydrogen bond to the C-3 hydroxyl group of fucose, which altogether makes this residue an unlikely candidate for the function of the general acid/base. A recent study of the human α-L-fucosidase FucA1, carefully done combining bioinformatics, structural modelling, mutagenesis, chemical rescue with azide and 1H NMR spectral analysis, identified Glu289 as the general acid/base [9]. The equivalent residue in Tmα-fuc, Glu281, as inferred from sequence alignment of FucA1 and Tmα-fuc, points to the interior of the (β/α)8 barrel and lies about 15 Å apart form the catalytic centre.
Altogether it appears that family GH29 is a quite exceptional CAZy family in that multiple sequence alignments do not allow an unambiguous assignment of the general acid/base.
Three-dimensional structures
Very few structures are available for GH29 enzyme. The first crystal structure being solved is the one for the α-L-fucosidase from T. maritima, Tmα-fuc. The simultaneous solution of the structures of an enzyme-product complex and of a glycosyl-enzyme intermediate allowed the unambiguous identification of the general acid/base [8], as described above. Tmα-fuc assembles as a hexamer and displays a two-domain fold, composed of a catalytic (β/α)8-like domain and a C-terminal β-sandwich domain. The two key active site residues are located at the C-terminal ends of strands of β-strands 4 (nucleophile) and 6 (acid/base).
Family Firsts
- First stereochemistry determination
- Cite some reference here, with a short (1-2 sentence) explanation [11].
- First catalytic nucleophile identification
- Cite some reference here, with a short (1-2 sentence) explanation [12].
- First general acid/base residue identification
- Cite some reference here, with a short (1-2 sentence) explanation [13].
- First 3-D structure
- Cite some reference here, with a short (1-2 sentence) explanation [3].
References
- O'Brien JS, Willems PJ, Fukushima H, de Wet JR, Darby JK, Di Cioccio R, Fowler ML, and Shows TB. (1987). Molecular biology of the alpha-L-fucosidase gene and fucosidosis. Enzyme. 1987;38(1-4):45-53. DOI:10.1159/000469189 |
- White WJ Jr, Schray KJ, Legler G, and Alhadeff JA. (1987). Further studies on the catalytic mechanism of human liver alpha-L-fucosidase. Biochim Biophys Acta. 1987;912(1):132-8. DOI:10.1016/0167-4838(87)90256-1 |
- Eneyskaya EV, Kulminskaya AA, Kalkkinen N, Nifantiev NE, Arbatskii NP, Saenko AI, Chepurnaya OV, Arutyunyan AV, Shabalin KA, and Neustroev KN. (2001). An alpha-L-fucosidase from Thermus sp. with unusually broad specificity. Glycoconj J. 2001;18(10):827-34. DOI:10.1023/a:1021163720282 |
- Berteau O, McCort I, Goasdoué N, Tissot B, and Daniel R. (2002). Characterization of a new alpha-L-fucosidase isolated from the marine mollusk Pecten maximus that catalyzes the hydrolysis of alpha-L-fucose from algal fucoidan (Ascophyllum nodosum). Glycobiology. 2002;12(4):273-82. DOI:10.1093/glycob/12.4.273 |
- Cobucci-Ponzano B, Trincone A, Giordano A, Rossi M, and Moracci M. (2003). Identification of an archaeal alpha-L-fucosidase encoded by an interrupted gene. Production of a functional enzyme by mutations mimicking programmed -1 frameshifting. J Biol Chem. 2003;278(17):14622-31. DOI:10.1074/jbc.M211834200 |
- Cobucci-Ponzano B, Trincone A, Giordano A, Rossi M, and Moracci M. (2003). Identification of the catalytic nucleophile of the family 29 alpha-L-fucosidase from Sulfolobus solfataricus via chemical rescue of an inactive mutant. Biochemistry. 2003;42(32):9525-31. DOI:10.1021/bi035036t |
- Tarling CA, He S, Sulzenbacher G, Bignon C, Bourne Y, Henrissat B, and Withers SG. (2003). Identification of the catalytic nucleophile of the family 29 alpha-L-fucosidase from Thermotoga maritima through trapping of a covalent glycosyl-enzyme intermediate and mutagenesis. J Biol Chem. 2003;278(48):47394-9. DOI:10.1074/jbc.M306610200 |
- Sulzenbacher G, Bignon C, Nishimura T, Tarling CA, Withers SG, Henrissat B, and Bourne Y. (2004). Crystal structure of Thermotoga maritima alpha-L-fucosidase. Insights into the catalytic mechanism and the molecular basis for fucosidosis. J Biol Chem. 2004;279(13):13119-28. DOI:10.1074/jbc.M313783200 |
- Liu SW, Chen CS, Chang SS, Mong KK, Lin CH, Chang CW, Tang CY, and Li YK. (2009). Identification of essential residues of human alpha-L-fucosidase and tests of its mechanism. Biochemistry. 2009;48(1):110-20. DOI:10.1021/bi801529t |
- Cobucci-Ponzano B, Mazzone M, Rossi M, and Moracci M. (2005). Probing the catalytically essential residues of the alpha-L-fucosidase from the hyperthermophilic archaeon Sulfolobus solfataricus. Biochemistry. 2005;44(16):6331-42. DOI:10.1021/bi047495f |