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Difference between revisions of "Glycoside Hydrolase Family 29"
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The [[catalytic nucleophile]] in GH29 was first identified in the ''Sulfolobus solfataricus'' α-L-fucosidase, Ssα-fuc, as Asp242 in the sequence VYF<u>'''D'''</u>WWI via chemical rescue of an inactive mutant with sodium azide <cite>6</cite>. Concomitantly the [[catalytic nucleophile]] of ''Thermotoga maritima'' α-L-fucosidase, Tmα-fuc, was confirmed to be Asp224 in the sequence LWN<u>'''D'''</u>MGW 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 <cite>7</cite>. The trapping of the 2-deoxy-2-fluorofucosyl-enzyme intermediate in Tmα-fuc was corroborated by crystallographic studies <cite>8</cite>. The [[catalytic nucleophile]] of the human enzyme FucA1 has recently been identified as being Asp225 <cite>9</cite>. | The [[catalytic nucleophile]] in GH29 was first identified in the ''Sulfolobus solfataricus'' α-L-fucosidase, Ssα-fuc, as Asp242 in the sequence VYF<u>'''D'''</u>WWI via chemical rescue of an inactive mutant with sodium azide <cite>6</cite>. Concomitantly the [[catalytic nucleophile]] of ''Thermotoga maritima'' α-L-fucosidase, Tmα-fuc, was confirmed to be Asp224 in the sequence LWN<u>'''D'''</u>MGW 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 <cite>7</cite>. The trapping of the 2-deoxy-2-fluorofucosyl-enzyme intermediate in Tmα-fuc was corroborated by crystallographic studies <cite>8</cite>. The [[catalytic nucleophile]] of the human enzyme FucA1 has recently been identified as being Asp225 <cite>9</cite>. | ||
− | 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]] <cite>8</cite>. 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 [http://www.pdb.org/ 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/K<sub>M</sub>'' and could be chemically rescued <cite>10</cite>. In the crystal structure of Tmα-fuc in complex with fucose <cite>8</cite>, the residue corresponding to Ssa-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]]. | + | 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]] <cite>8</cite>. 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 [http://www.pdb.org/ 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/K<sub>M</sub>'' and could be chemically rescued <cite>10</cite>. In the crystal structure of Tmα-fuc in complex with fucose <cite>8</cite>, the residue corresponding to Ssa-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]]. Normal.dotm 0 0 1 32 187 AFMB 1 1 229 12.0 0 false 21 18 pt 18 pt 0 0 false false false A recent study of the human α-L-fucosidase FucA1, carefully done combining bioinformatics, structural modeling, mutagenesis, chemical rescue with azide and 1H NMR spectral analysis, identified Glu289 as the general acid/base. |
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Revision as of 11:05, 6 January 2010
This page is currently under construction. This means that the Responsible Curator has deemed that the page's content is not quite up to CAZypedia's standards for full public consumption. All information should be considered to be under revision and may be subject to major changes.
- 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 Ssa-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. Normal.dotm 0 0 1 32 187 AFMB 1 1 229 12.0 0 false 21 18 pt 18 pt 0 0 false false false A recent study of the human α-L-fucosidase FucA1, carefully done combining bioinformatics, structural modeling, mutagenesis, chemical rescue with azide and 1H NMR spectral analysis, identified Glu289 as the general acid/base.
Three-dimensional structures
Content is to be added here.
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 |