Glycoside-Hydrolases overview

The IUBMB (International Union of Biochemistry and Molecular Biology) enzyme nomenclature is based on their substrate specificity and on the reaction catalyzed. In this classification, glycoside Hydrolases (GHs, EC 3.4…) and glycosyltransferases (GT EC 2.4..) belong to difference classes.

However, in the classification based on sequence alignment and molecular mechanism initially proposed by B. Henrissat and at the origin of the CAZy database (Carbohydrate Active Enzymes, http://www.cazy.org/), glycoside hydrolases (EC 3.2.) and some transferases (EC2.4..) showing a strong ability to catalyse glycosyl transfer were shown to be homologous, thus sharing related sequences and similar mechanism of glycosidic bond cleavage. Consequently, they were all classified in glycoside hydrolase family. In the CAZy classification, GHs are structurally divided into over 156 families (February 2019). In addition, these families are grouped in clans according to similarity in structure-function; this classification is showed in table 5 (Desmet et al. 2012; Cantarel et al. 2009).

Moreover, GHs have two different main mechanisms for the cleavage of the glycosidic bond. In 1953, Koshland for the first time described these mechanisms as retaining or inverting, which depends on the variation of the anomeric carbon configuration during the reaction (Koshland, 1953). Figure 4 shows the general mechanism of GHs. In the inverting mechanism, the catalytic acid residue donates a proton to the anomeric carbon while the catalytic base residue removes a proton from a water molecule, increasing its nucleophilicity, facilitating its attack on the anomeric center.

Table 5. The established Glycoside Hydrolases clans of related families in the CAZy database (adapted from http://www.cazy.org/).

In the retaining mechanism, a general acid/base catalyst works first as an acid and then as a base in two steps: glycosylation and deglycosylation, respectively. In the first step, it facilitates departure of the leaving group by donating a proton to the glycosyl oxygen atom while the nucleophile forms a glycosyl-enzyme intermediate of opposite anomeric configuration. In the second step, the deprotonated acid/base acts as a general base to activate a water molecule that carries out a nucleophilic attack on the glycosyl-enzyme intermediate; these two inversion steps resulted in the stereochemistry retention at the anomeric center (Davies and Henrissat, 1995; Vuong and Wilson, 2010).

GH clans Shared features Related GH Families

GH-A (β/α)8 1, 2, 5, 10, 17, 26, 30, 35, 39, 42, 50,51, 53, 59, 72, 79, 86, 113, 128, 147, 148

GH-B β-jelly roll 7, 16


GH-C β-jelly roll 11, 12

GH-D (β/α)8 27, 31, 36

GH-E 6-fold β-propeller 33,34 83,93

GH-F 5-fold β-propeller 43,62

GH-G (α/α)6 37, 63, 100, 125

GH-H (β/α)8 13, 70, 77

GH-I β+α 24, 80

GH-J 5-fold β-propeller 32, 68

GH-K (β/α)8 18, 20, 85

GH-L (α/α)6 15, 65

GH-M (α/α)6 8, 48

GH-N β-helix 28, 49

GH-O (α/α)6 52, 116

GH-P (α/α)6 127, 146

GH-Q (α/α)6 94, 149

GH-R (β/α)8 29,107

Figure 4. General glycoside hydrolases a) inverting and b) retaining mechanisms. AH: a catalytic acid residue, B-: a catalytic base residue, Nuc: a nucleophile, and R: a carbohydrate derivative. HOR: an exogenous nucleophile, often a water molecule (adapted from Vuong and Wilson, 2010).

In addition, GHs are able to perform hydrolysis and synthesis reactions (figure 5). The hydrolysis is the main reaction that these enzymes typically perform; thus they hydrolyze the glycosidic bond by transferring the cleaved glycosyl moiety to water as an acceptor substrate. For some enzymes, the glycosyl moiety can however be transferred on to a free hydroxylated group of an acceptor molecule different from water (e.g. simple sugar, oligosaccharides or aglycon). This process is called transglycosylation and allows the formation of a new glycosidic bond, instead of hydrolysis reaction.

Thus, transglycosylation is kinetically controlled, and during the reaction, it is assumed that there is a competition between the nucleophilic water and the acceptor substrate at the glycosyl-enzyme intermediate (Desmet et al. 2012). Moreover, when enzymatic synthesis is thermodynamically controlled this process is called reverse hydrolysis (van Rantwijk et al. 1999).

a) Inverting mechanism

b) Retaining mechanism

O O

R

O H H B

Catalytic base A

Catalytic acid

H

HOR

O

OH

BH A

O O

R

Nuc

Nucleophile A H Acid/base

HOR

O

Nuc

Intermediate A

O H

R*

O O

R*

Nuc AH

Figure 5. Synthetic and hydrolytic reactions catalyzed by Glycoside Hydrolases (Desmet et al. 2012).

Indeed, many GHs can perform hydrolysis or synthesis reactions using different kinds of acceptors such as glucose, fructose, lactose, xylose, sucrose, etc. The GHs that transfer fructosyl units are classified into clan GH-J according to the CAZy database (Carbohydrate Active Enzymes, http://

www.cazy.org/). The clan GH-J contains the GH32 and GH68 families; they share some similarities such as a retaining mechanism, a 5-fold β-propeller tertiary structure and conserved aspartate and glutamate residues that act as nucleophile and acid/base catalyst, respectively. Structurally, this clan has a common β-propeller catalytic domain with three conserved amino acids, located in the deep axial pocket of the active site. The propeller has a 5-fold repeat of blades, each consisting of four antiparallel β-strands with the classical ‘W’ topology around the central axis, enclosing the negatively charged cavity of the active site (Lammens et al. 2009).

Although, enzymes from GH-J clan have structural similarities, especially in the amino acid residues near to the active site, they have a broad variation in substrate specificities (Yuan et al. 2012). Mainly, these enzymes use sucrose or fructans as donor substrates and sucrose, fructans or water as acceptor substrates. Some examples are levansucrases and inulosucrases from the GH68 family, plant and microbial invertases, microbial endo- and exo-type inulinases as well as levanases and a wide variety of plant fructosyltransferases plant fructan and exo-hydrolases all of them from the GH32 family (Lammens et al. 2009). The next section is entirely devoted to the description of the enzymes belonging to the GH32 and GH68 families.

Glycosyl-O-R¹

transglycosylation R²OH R¹OH

Glycosyl-O-R²

hyd roly

sis H₂O

R¹OH

Glycosyl-O-OH

H₂O

R²OH reverse hydrolysis