Glycoside Hydrolase Family 35

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Authors: ^^^Anna Kulminskaya^^^, ^^^Mirko Maksimainen^^^, ^^^Juha Rouvinen^^^
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 [1, 2, 3], and are often most active under acidic conditions [4, 5, 6]. As with many other CAZy families [7, 8, 9], GH35 members tend to be represented by multi-gene families in plants [3, 10, 11, 12, 13]. 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 [14].

In addition to β-galactosidases, GH35 also contains a limited number of archeal exo-β-glucosaminidases (EC 3.2.1.165) [15, 16]. 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 [4] and has been subsequently confirmed by other investigators for microbial and plant enzymes [1, 5] .

Catalytic Residues

The catalytic residues for family 35 were first predicted on the basis of hydrophobic cluster analysis of proteins of similar protein fold [17]. 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 [18]. Subsequently, this approach was repeated for two bacterial β-galactosidases from Xanthomonas manihotis and Bacillus circulans [19]. The general acid/base catalyst was inferred to be Glu200 from structural studies of a Penicillium sp. β-galactosidase [20]. Recent structural studies [21] revealed two different conformations of the general acid/base catalyst in the β-galactosidase of Trichoderma reesei.

Three-dimensional structures

To date (March 2011), only three enzymes from GH35 have been structurally characterized. The first 3D-structures of a GH35 enzyme, those of a β-galactosidase from Pencillium sp. (Psp-β-gal) in native (PDB 1tg7) and product-complexed (PDB 1xc6) forms, were reported in 2004 at 1.90 Å and 2.10 Å resolution, respectively [20]. The structure of a β-galactosidase from Bacteriodes thetaiotamicron (Btm-β-gal) was subsequently reported by the New York Structural GenomiX Research Consortium in 2008 at 2.15 Å resolution (PDB 3d3a). In 2010, a high (1.2 Å) resolution crystal structure of a Trichoderma reesei (Hypocrea jecorina) β-galactosidase (Tr-β-gal, PDB 3og2) was reported, together with complex structures with galactose, IPTG and PETG at 1.5, 1.75 and 1.4 Å resolutions, respectively (PDB codes 3ogr, 3ogs, and 3ogv, respectively) [21].

GH35 enzymes belong to Clan GH-A, and thus have a (α/β)₈ TIM barrel comprising the catalytic domain. Comparison of the native structures of Psp-β-gal, Tr-β-gal and Btmβ-gal shows two remarkable things (Figure 1A): Firstly, Btm-β-gal consists of three different domains whereas Psp-β-gal and Tr-β-gal consist of five and six domains, respectively. The second and third domains of Btm-β-gal are quite similar with the fourth and fifth domains of Psp-β-gal and with the fifth and sixth of Tr-β-gal. Secondly, major structural differences between Psp-β-gal and Tr-β-gal are in the conformations of the loop regions. Although the crystal structures of Psp-β-gal and Tr-β-gal are similar, interpretation of the structure of Tr-β-gal is somewhat different from that presented earlier for Psp-β-gal: Rojas et al. considered Psp-β-gal to be composed of five distinct structural domains. The overall structure is built around the first, TIM barrel, domain. Domain 2 is an all β-sheet domain containing an immunoglobulin-like subdomain, domain 3 is based on a Greek-key β-sandwich and domains 4 and 5 are jelly rolls [20]. In contrast, Maksimainen et al. concluded that the Tr-β-gal and Psp-β-gal structures both form six similar domains [21].

A structural analysis of the galactose-binding active-site was based on the comparison of the crystallographic models of the native Psp-β-gal and Tr-β-gal enzymes and their respective complexes with galactose. A single galactose molecule is bound to the TIM barrel domain of the enzyme in the chair conformation in the β-anomeric configuration. Two glutamic acid residues act as the general acid-base and nucleophilic catalysts; these are presented on strands 4 and 7 of the barrel.

Additionally, Maksimainen et al. have described conformational changes in two loop regions of the active site of Tr-β-gal, thus implicating a conformational selection mechanism for the enzyme. Unlike, the induced fit theory which assumes that the initial interaction between a protein and its binding partner induces a conformational change in the protein through a stepwise process, the conformational selection theory is based on an assumption that the unbound protein exists as an ensemble of conformations in dynamic equilibrium. Interaction between a weakly populated, higher-energy conformation and a binding partner causes the equilibrium to move in favor of the selected conformation (Figure 2A) [22, 23]. This can be seen in the structures of Tr-β-gal: the open and closed conformation are both favorable in the native structure and the closed conformation becomes more favorable in the complex structures (Figure 2B,2C) [21]. Furthermore, The acid/base catalyst Glu200 has two different conformations in the IPTG and PETG complex structures. This clearly affects the pK_a value of this residue and thus the catalytic mechanism.

Structure images

Figure 1. A) Comparison of the native structures of the GH35 β-galactosidases. Psp-β-gal (PDB code 1tg7), Tr-β-gal (PDB code 3og2) and Btm-β-gal (PDB code 3d3a) are colored in green, brown and blue, respectively. B) Comparison of the active sites of the GH35 β-galactosidases. Psp-β-gal, Tr-β-gal and Btm-β-gal are colored as above-mentioned.

Figure 2. A) Molecular recognition mechanisms in proteins: Induced fit vs. conformational selection. The picture is taken from reference [] B) The native structure of Tr-β-gal. The two conformations (green and blue) of the loop region correspond to the colors of the energy states in Figure 2A. C) The galactose-bound structure of Tr-β-gal. The galactose molecule (yellow) corresponds to the substrate (triangle) and the purple conformation corresponds to the color of the substrate-bound energy state in Figure 2A.

Family Firsts

First stereochemistry determination

Human β-galactosidase precursor by NMR [4]

First catalytic nucleophile identification

Human β-galactosidase precursor by 2-fluorogalactose labeling [18].

First general acid/base residue identification

Penicillium sp. β-galactosidase by structural identification [20].

First 3-D structure

Penicillium sp. β-galactosidase [20].

References

All Medline abstracts: PubMed