Glycoside Hydrolase Family 29

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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 VPI-5482, 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 k_cat/K_M 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 β-strands 4 (nucleophile) and 6 (acid/base). Crystallization experiments for the S. solfataricus α-L-fucosidase, Ssα-fuc, were not very fruitful, but a small angle scattering study has been reported [11], which suggests a nonameric assembly of the enzyme in solution. Two crystal structures, arising for Strucural Genomics initiatives, have been deposited in the Protein Data Bank for α-L-fucosidases from Bacteroides thetaiotaomicron VPI-5482, with accession numbers 3eyp and 3gza. The catalytic domain of Tmα-fuc does not adopt the canonical TIM-barrel (β/α)₈ fold, as it lacks helices α5 and α6. Helix α5 is missing as well in the structure of one of the B. thetaiotaomicron VPI-5482 α-L-fucosidases, BT3798 (3gza), whereas α-L-fucosidase BT2192 (3epy) from the same organism adopts the canonical TIM-barrel fold. The three structures differ furthermore by the insertion/deletion of a considerable number of additional α-helices, 3₁₀ helices, and extended surface loop regions. The closest structural homologues of GH29 enzymes within the CAZy classification can be found in families GH13 and GH27.

Family Firsts

First stereochemistry determination: Normal.dotm 0 0 1 33 189 AFMB 1 1 232 12.0 0 false 21 18 pt 18 pt 0 0 false false false

Retention of anomeric stereochemistry suggested for human liver α-fucosidase by the formation of methyl-α-L-fucoside using methanol as an alternative glycone acceptor [2]. Later confirmed by 1H NMR for α-L-fucosidase from Thermus sp. [3].

First catalytic nucleophile identification: Normal.dotm 0 0 1 11 67 AFMB 1 1 82 12.0 0 false 21 18 pt 18 pt 0 0 false false false

Sulfolobus solfataricus α-L-fucosidase by azide rescue of an inactivated mutant [6].

First general acid/base residue identification: Normal.dotm 0 0 1 11 65 AFMB 1 1 79 12.0 0 false 21 18 pt 18 pt 0 0 false false false Thermotoga maritima α-fucosidase by X-ray structural analysis and mutagenesis. [8].

First 3-D structure: Normal.dotm 0 0 1 17 101 AFMB 1 1 124 12.0 0 false 21 18 pt 18 pt 0 0 false false false Thermotoga maritima α-fucosidase, free enzyme, product complex and glycosyl-enzyme intermediate (PDB 1hl8, 1hl9, 1odu).

(PDB 1byh) [5].

References

All Medline abstracts: PubMed