Glycoside Hydrolase Family 35

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• Authors: ^^^Alexander Golubev^^^ and ^^^Anna Kulminskaya^^^
• Responsible Curator: ^^^Anna Kulminskaya^^^

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Glycoside Hydrolase Family GH35
Clan	GH-A
Mechanism	retaining (inferred)
Active site residues	known
CAZy DB link
https://www.cazy.org/GH35.html

Substrate specificities

The major activity of enzymes of this GH family is β-galactosidase (EC 3.2.1.23). Reported enzymes were isolated from microorganisms such as fungi, bacteria and yeasts; plants, animals and human cells, and from recombinant sources and act in acidic conditions. The β-galactosidase (EC 3.2.1.23) catalyses the hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides as, for example, lactose (1,4-O-β-D-galactopyranosyl-D-glucose) and structurally related compounds. GH35 includes multiple genes in various plant species [1-6], suggesting ubiquity of GH35 gene multiplicity in plants. Family 35 β-galactosidases demonstrate specificity towards β1,3-, β1,6- or β1,4-galactosidic linkages. Plant β-galactosidases can be divided into two classes: members of the first are capable of hydrolyzing pectic β-1,4-galactans; another ones can specifically cleave β-1,3- and β1,6-galactosyl linkages of arabinogalactan proteins.

Besides β-galactosidases, GHF35 contains two exo-β-glucosaminidases (EC 3.2.1.165) [7,8]. This enzyme hydrolyze chitosan or chitosan oligosaccharides to remove successive D-glucosamine residues from the non-reducing termini.

Kinetics and Mechanism

Beta-galactosidases of GH35 family catalyze hydrolysis of β-galactosyl linkages between terminal galactosyl residues of oligosaccharides, glycolipids, and glycoproteins acting via a double-displacement mechanism and retaining β-anomeric configuration of the released galactose molecule. The stereochemistry of the reaction has been first shown by NMR for human β-galactosidase precursor [Zhang et al. 1997] and then confirmed by other investigators for microbial and plant enzymes.

Catalytic Residues

The catalytic residues for family 35 were first predicted on the basis of hydrophobic cluster analysis of proteins of similar protein fold [ ]. Experimentally, the glutamic acid residue 268 was first identified as the catalytic nucleophile in human lysosomal β-galactosidase precursor using the slow substrate 2,4-dinitrophenyl-2-deoxy-2-fluoro- β-D-galactopyranoside that trapped a glycosyl enzyme intermediate. It allowed subsequent peptide mapping and exact nulceophile ID. Further, the same work was done for two bacterial β-galactosidases. Recent structural studies of Maksimainen et al. [ ] revealed the general acid/base catalyst as Glu200 in the β-galactosidase of Trichoderma reeesei which showed two different conformations influencing the catalytic machinery of the enzyme.

Three-dimensional structures

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Family Firsts

First stereochemistry determination

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

First catalytic nucleophile identification

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

First general acid/base residue identification

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

First 3-D structure

Cite some reference here, with a short (1-2 sentence) explanation [4].

References

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2. Sinnott, M.L. (1990) Catalytic mechanisms of enzymic glycosyl transfer. Chem. Rev. 90, 1171-1202. DOI: 10.1021/cr00105a006