Glycoside Hydrolase Family 3

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Authors: ^^^Geoff Fincher^^^ and ^^^Brian Mark^^^
Responsible Curator: ^^^Bernard Henrissat^^^

Glycoside Hydrolase Family GH3
Clan	none
Mechanism	retaining
Active site residues	known
CAZy DB link
https://www.cazy.org/GH3.html

Substrate specificities

The GH3 glycoside hydrolase family currently group together β-D-glucosidases, α-L-arabinofuranosidases, β-D-xylopyranosidases and N-acetyl-β-D-glucosaminidases [1]. Widely distributed in bacteria, fungi and plants, GH3 enzymes carry out a range of functions including cellulosic biomass degradation, plant and bacterial cell wall remodeling, energy metabolism and pathogen defense. In many cases the enzymes have dual or broad substrate specificities with respect to monosaccharide residues, linkage position and chain length of the substrate. For example, there are several well-characterized ‘bifunctional’ enzymes in the family that have both α-L-arabinofuranosidase and β-D-xylopyranosidase activity [2], and one characterized example of an N-acetyl-β-D-glucosaminidase/β-glucosidase from Cellulomonas fimi (Nag3) [3]. GH3 β-D-glucosidases from barley, which are more precisely referred to as β-D-glucan glucohydrolases, are also broad specificity exo-hydrolases that remove single glucosyl residues from the non-reducing ends of a range of β-D-glucans, β-D-oligoglucosides and aryl b-D-glucosides, including (1,3)-β-D-glucans, (1,4)-β-D-glucans, (1,3;1,4)-β-D-glucans and (1,6)-β-D-glucans, 4-nitrophenyl β-D-glucoside, certain cyanogenic β-D-glucosides and some β-D-oligoxyloglucosides [4].

In contrast to the broad substrate specificities observed for the GH3 enzymes described above, GH3 N-acetyl-β-D-glucosaminidases are selective for N-acetyl-β-D-glucosamine (GlcNAc) [5] (though exceptions exist such as Cellulomonas fimi Nag3 [3]). A notable GH3 N-acetyl-β-D-glucosaminidase of prokaryotes is NagZ, which participates in bacterial cell wall recycling (for review see [6]) by removing GlcNAc from 1,6-anhydroMurNAc-peptides in Gram-negative bacteria [7], or GlcNAc from GlcNAc-MurNAc-peptides in Gram-positive bacteria[8], . The NagZ product 1,6-anhydroMurNAc-peptide is an important activator of AmpC β-lactamase hyper-production in several Gram-negative pathogens, making the enzyme of interest as a potential therapeutic target[9].

Kinetics and Mechanism

Family 3 glycoside hydrolases remove single glycosyl residues from the non-reducing ends of their substrates. Catalysis occurs via a classical Koshland double-displacement mechanism with the β-anomeric configuration of the released glycose being retained. The stereochemistry of the reaction has been determined experimentally for some family 3 enzymes. Detailed kinetic analyses are available for two purified barley β-D-glucan glucohydrolases and two barley ‘bifunctional’ α-L-arabinofuranosidase/β-D-xylopyranosidases [2, 4]. The reaction sequence and mechanism of a barley β-glucosidase was further outlined using a range of synthetic inhibitors and crystallographic analysis [10]. Detailed kinetic data are also available for a N-acetyl-β-D-glucosaminidase from Vibrio furnisii (ExoII) [11]

Catalytic Residues

The catalytic amino acid residues of GH3 members was first informed by the three-dimensional structure of a barley β-D-glucan glucohydrolase, solved in complex with glucose bound in the -1 subsite [12]. Whereas the catalytic nucleophile is well conserved among GH3 members from diverse species, the catalytic acid/base has been difficult to generally identify. In many cases, this residue is borne on a flexible loop structure, which exhibits significant sequence divergence and conformational flexibility. The following sections detail the definitive assignments of the catalytic residues in a growing number of GH3 enzymes.

Catalytic nucleophile

In plant family 3 β-D-glycosidases, typified by the barley enzyme, active site consists of two glucosyl-binding subsites (-1 and +1) and the catalytic amino acid residues are located between these two subsites [10, 12]. The catalytic nucleophile, as identified in the crystal structure of a trapped 2-fluoroglycosyl enzyme, is Asp285, which is located in a highly conserved GFVISDW motif [10].

The catalytic nucleophile for Vibrio furnisii ExoII (a NagZ) has been identified chemically as the corresponding Asp242, which is conserved throughout the family 3 NagZ enzymes [11].

Catalytic acid/base

The general acid, E491, is highly conserved in plant family 3 enzymes but is more difficult to locate in more distantly related members of the family [1].

A general acid residue has not been identified for family 3 NagZ enzymes.

Three-dimensional structures

Figure 1. Ribbon representation of barley β-glucan exohydrolase isoenzyme ExoI. Domain 1, domain 2, and the linker region of the enzyme are coloured in magenta, cyan, and yellow, respectively. Figure from [].

Figure 2. NagZ from Vibrio cholerae in complex with PUGNAc (PDB ID: 2OXN) [13]. NagZ enzymes are single domain proteins that adopt a TIM barrel fold. Active site residues are located within the loops that extend from the C-termini of the strands of the β-barrel.

The family 3 β-D-glycosidases are globular monomeric enzymes of molecular mass around 60-70 kDa. The 3D structure of the β-D-glucan glucohydrolase isoenzyme ExoI from barley, determined by X-ray crystallography to 2.2 Å resolution, shows a two-domain, globular protein of 605 amino acid residues that is N-glycosylated at three sites [12]. The two domains are connected by a 16-amino acid helix-like linker. The first 357 residues constitute a (β/α)₈ TIM barrel domain. The second domain consists of residues 374 to 559 arranged in a six-stranded β-sandwich, which contains a β-sheet of five parallel β-strands and one antiparallel β-strand, with 3 α-helices on either side of the sheet. A long antiparallel loop of 42 amino acid residues is found at the COOH-terminus of the enzyme. In some bacterial GH3 enzymes the order of the domains is reversed [1].

The active site of the barley β-D-glucan glucohydrolase consists of a relatively shallow substrate-binding pocket that is located at the interface of the two domains of the enzyme [12]. The active site pocket can accommodate the two glucosyl residues at the non-reducing terminus of the substrate and aligns the non-reducing terminal glycosidic linkage of the substrate with the catalytic amino acid residues Asp285 and Glu491. Thus, the catalytic amino acid residues are located on domains 1 and 2, respectively.

The broad specificity of the barley β-D-glucan glucohydrolase can be rationalized from the X-ray crystallographic data and from molecular modelling of enzyme-substrate complexes [12, 14]. The glucosyl residue occupying binding subsite –1 is tightly locked into a relatively fixed position through interactions with six amino acid residues near the bottom of the shallow active site pocket. In contrast, the glucosyl residue at subsite +1 is located between two tryptophan residues at the entrance of the pocket, where it is less tightly constrained. The flexibility of binding at subsite +1, coupled with the projection of the remainder of bound substrate away from the enzyme’s surface, means that the overall active site is largely independent of substrate conformation and will therefore accommodate a range of substrates in which the spatial dispositions of adjacent β-D-glucosyl residues vary as a result of glycosidic linkages between different C atoms of the adjacent β-D-glucosyl residues [14].

Family 3 NagZ enzymes are also globular, yet have a mass of ~ 36 kDa, which is a distinctive feature of NagZ enzymes from others within the family. NagZ from Vibrio cholerae has been determined in complex with GlcNAc (PDB ID: 1Y65) and with the N-acetyl-β-glucosaminidase inhibitor PUGNAc [13] and NagZ selective PUGNAc derivatives [15]. The enzyme is comprised of 340 amino acids and adopts a (β/α)₈ TIM barrel fold. The active site pocket is shallow and accommodates the 2-N-acetyl group of a terminal GlcNAc sugar in a solvent accessible groove alongside the binding site for the pyranose ring. This active site architecture is different from the active site architectures the functionally-related GH20 N-acetyl-β-hexosaminidases and GH84 O-GlcNAcases. These latter families, which also remove β-1,4-linked GlcNAc residues from glycoconjugates, use a mechanism involving neighboring group participation wherein the carbonyl oxygen of the 2-acetamido group of the terminal GlcNAc acts as a nucleophile, yielding an oxazolinium ion intermediate [16, 17, 18]. Thus, unlike family 3 NagZ enzymes, GH20 and GH84 do not possess an enzymic catalytic nucleophile; however, they do have an appropriately positioned catalytic acid residue. Together, these mechanistic differences have allowed for the development of 2-N-acyl derivatives of PUGNAc that are selective for family 3 NagZ over GH20 and GH84 enzymes [13, 15].

Family Firsts

First 3D Structure: Barley β-D-glucan glucohydrolase [12].
First Catalytic Residues: Barley β-D-glucan glucohydrolase [12].

References

All Medline abstracts: PubMed