Glycoside Hydrolase Family 35

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Author: ^^^Anna Kulminskaya^^^
Responsible Curator: ^^^Anna Kulminskaya^^^

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

Substrate specificities

The majority of GH35 members are β-galactosidases (EC 3.2.1.23). GH35 enzymes have been isolated from microorganisms such as fungi, bacteria and yeasts, as well as higher organisms such as plants, animals, and human cells. These β-galactosidases catalyse the hydrolysis of terminal non-reducing β-D-galactose residues in, for example, lactose (1,4-O-β-D-galactopyranosyl-D-glucose), oligosaccharides, glycolipids, and glycoproteins. Various GH35 β-galactosidases demonstrate specificity towards β1,3-, β1,6- or β1,4-galactosidic linkages (REFS NEEDED), and are often most active under acidic conditions (REFS NEEDED). As with many other CAZy families [1, 2, 3], GH35 members tend to be represented by multi-gene families in plants [4, 5, 6, 7, 8]. Moreover, plant GH35 β-galactosidases have be divided into two classes: members of the first are capable of hydrolyzing pectic β-1,4-galactans, while those of the second can specifically cleave β-1,3- and β1,6-galactosyl linkages of arabinogalactan proteins (REFS NEEDED).

In addition to β-galactosidases, GH35 also contains a limited number of archeal exo-β-glucosaminidases (EC 3.2.1.165) [9, 10] (NEED ORIGINAL BIOCHEMICAL REF FOR PYROCCOCCUS ENZYME; NOT GENOME PAPER). Such enzymes hydrolyze chitosan or chitosan oligosaccharides to remove successive D-glucosamine residues from non-reducing termini.

Kinetics and Mechanism

Beta-galactosidases of GH35 catalyze the hydrolysis of terminal β-galactosyl residues via a double-displacement mechanism, which leads to net retention of the β-anomeric configuration of the released galactose molecule. The stereochemistry of the reaction was first shown by NMR for the human β-galactosidase precursor [11] and has been subsequently confirmed by other investigators for microbial and plant enzymes (REFS NEEDED).

Catalytic Residues

The catalytic residues for family 35 were first predicted on the basis of hydrophobic cluster analysis of proteins of similar protein fold [12]. Experimentally, the glutamic acid residue 268 was first identified as the catalytic nucleophile in human lysosomal β-galactosidase precursor using a slow substrate, 2,4-dinitrophenyl-2-deoxy-2-fluoro- β-D-galactopyranoside, that allowed trapping of a covalent glycosyl-enzyme intermediate. This allowed subsequent peptide mapping to exactly identify the catalytic nucleophile [13]. Subsequently, this approach was repeated for two bacterial β-galactosidases from Xanthomonas manihotis and Bacillus circulans [14]. The general acid/base catalyst was inferred to be Glu200 from structural studies of a Penicillium sp. β-galactosidase [15]. Recent structural studies [16] revealed two different conformations of the general acid/base catalyst in the β-galactosidase of Trichoderma reesei.

Three-dimensional structures

To date, there are only three enzymes from GH family 35 are structurally characterized. First 3D-structure has appeared available at PDB for the β-galactosidase from Pencillium sp. (Psp-β-gal, PDB code1tg7) by Rojas et al. [15]. The crystallographic structures of Psp-β-gal and its complex with galactose (PDB code 1xc6) were solved at 1.90 Å and 2.10 Å, respectively. The structure of β-galactosidase from Bacteriodes thetaiotamicron was reported by the New York Structural GenomiX Research Consortium in 2008. In 2010, the crystal structure of Trichoderma reesei (Hypocrea jecorina) β-galactosidase (Tr-β-gal, PDB code 3OG2) at a 1.20 Å resolution and its complex structures with galactose, IPTG and PETG at 1.5, 1.75 and 1.4 Å resolutions, respectively, were reported (PDB codes 3OGR, 3OGS, and 3OGV) by Maksimainen et al. [16]. Like β-galactosidases from other families, they belong to GH-A super-family, which usually have an (α/β)8 TIM barrel as a catalytic domain. The structural analysis of the galactose-binding site was based on the comparison of the crystallographic models of the native Psp-β-gal and Tr-β-gal and their complexes with galactose. A single galactose molecule is bound to the TIM barrel domain of the enzyme in the chair conformation with its O1 in the β-anomer configuration. Two glutamic acid residues act as proton donor and nucleophile and emanate from strands 4 and 7 of the barrel. Both crystal structures, Psp-β-gal and Tr-β-gal, are similar. However, interpretation of Maksimainen et al. of the structure of Tr-β-gal is a bit different from that presented earlier for Psp-β-gal. Rojas et al considered Psp-β-gal to be divided into five domains combining the second and the third domain, although they form separate sub-units in the structure. So, it was concluded that Tr-β-gal structure contains a central catalytic α/β-barrel surrounded by a horseshoe consisting of five ant-parallel β-sandwich structures.

Additionally, Maksimainen et al. described conformational changes in the two loop regions in the active site of Tr-β-gal, implicating a conformational selection-mechanism for the enzyme. An acid/base catalyst Glu200 showed two different conformations which affect pKa value of this residue and the catalytic mechanism.

Family Firsts

First stereochemistry determination

Human β-galactosidase precursor by NMR [11]

First catalytic nucleophile identification

Human β-galactosidase precursor by 2-fluorogalactose labeling [17].

First general acid/base residue identification

Penicillium sp. β-galactosidase by structural identification [15].

First 3-D structure

Penicillium β-galactosidase [15].

References

All Medline abstracts: PubMed