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.

Fucosynthases

Transglycosylation activity had been observed in 1987 for human liver α-fucosidase [2]. The first successful transformation of an α-fucosidase into an α-transfucosidase by directed evolution has been reported for Thermotoga maritima α-fucosidase [12]. α-fucosidases mutated in the catalytic nucleophile from both Sulfolobus solfataricus and Thermotoga maritima could be successfully transformed into fucosynthases by the use of β-L-fucopyranosyl azide as donor substrate [13]

Family Firsts

First stereochemistry determination: 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: Sulfolobus solfataricus α-L-fucosidase by azide rescue of an inactivated mutant [6].
First general acid/base residue identification: Thermotoga maritima α-fucosidase by X-ray structural analysis and mutagenesis [8].
First 3-D structure: Thermotoga maritima α-fucosidase, free enzyme (PDB 1hl8), product complex (PDB 1odu) and glycosyl-enzyme intermediate (PDB 1hl9) [8].

References

All Medline abstracts: PubMed