Glycoside Hydrolase Family 35

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• Authors: ^^^Anna Kulminskaya^^^, ^^^Mirko Maksimainen^^^, Juha Rouvinen
• Responsible Curator: ^^^Anna Kulminskaya^^^

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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

As of February 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 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. 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.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 us 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 and [21]. The most of the structural differences between them are in the conformations of the loop regions.

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 [22]. 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 2) [21]. The acid/base catalyst Glu200 exhibited two different conformations which affect 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 [23] 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

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17. Henrissat B, Callebaut I, Fabrega S, Lehn P, Mornon JP, and Davies G. (1995). Conserved catalytic machinery and the prediction of a common fold for several families of glycosyl hydrolases. Proc Natl Acad Sci U S A. 1995;92(15):7090-4. DOI:10.1073/pnas.92.15.7090 |
20. Rojas AL, Nagem RA, Neustroev KN, Arand M, Adamska M, Eneyskaya EV, Kulminskaya AA, Garratt RC, Golubev AM, and Polikarpov I. (2004). Crystal structures of beta-galactosidase from Penicillium sp. and its complex with galactose. J Mol Biol. 2004;343(5):1281-92. DOI:10.1016/j.jmb.2004.09.012 |

21. Maksimainen M, Hakulinen N, Kallio JM, Timoharju T, Turunen O, Rouvinen J. Crystal structures of Trichoderma reesei beta-galactosidase reveal conformational changes in the active site. J Struct Biol. 2010, in press.

    Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out: DOI:10.1016/j.jsb.2010.11.024 PMID:21130883

23. Boehr DD, Wright PE How do proteins interact? Science 2008, 320 1429-1430.