Glycoside Hydrolase Family 29

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• Author: ^^^Gerlind Suzlenbacher^^^
• Responsible Curator: ^^^Steve Withers^^^

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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 [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 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 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 Thermotoga maritima α-L-fucosidase 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 Thermotoga maritima α-L-fucosidase 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). Normal.dotm 0 0 1 45 259 AFMB 2 1 318 12.0 0 false 21 18 pt 18 pt 0 0 false false false Studies of Sulfolobus solfataricus α-L-fucosidase demonstrated that mutation of the Glu residue corresponding in sequence to T. maritima α-L-fucosidase 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.

Three-dimensional structures

Content is to be added here.

Family Firsts

First sterochemistry determination

Cite some reference here, with a short (1-2 sentence) explanation [10].

First catalytic nucleophile identification

Cite some reference here, with a short (1-2 sentence) explanation [11].

First general acid/base residue identification

Cite some reference here, with a short (1-2 sentence) explanation [12].

First 3-D structure

Cite some reference here, with a short (1-2 sentence) explanation [3].

References

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