Difference between revisions of "Glycoside Hydrolase Family 35"

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Authors: ^^^Alexander Golubev^^^ and ^^^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 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, 2, 3, 4, 5] 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) [6], [7]. 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 [8] 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 [9]. 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 [10]. Further, the same work was done for two bacterial β-galactosidases, from Xanthomonas manihotis and Bacillus circulans [11]. The general acid/base catalyst was inferred by structural studies of Penicillium β-galactosidase as Glu200 [12]. Recent structural studies of Maksimainen et al. [13] revealed two different conformations of the general acid/base catalyst Glu200 in the β-galactosidase of Trichoderma reeesei, which influence the catalytic machinery of the enzyme.

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 by Rojas et al. (2004). The crystallographic structures of Psp-β-gal and its complex with galactose 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) 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. 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 [8]

First catalytic nucleophile identification

Human β-galactosidase precursor by 2-fluorogalactose labeling [14].

First general acid/base residue identification

Penicillium sp. β-galactosidase by structural identification [12].

First 3-D structure

Penicillium β-galactosidase [12].

References

All Medline abstracts: PubMed

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 == Three-dimensional structures ==
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+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  by Rojas et al. (2004). The crystallographic structures of Psp-β-gal and its complex with galactose 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) 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. 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.