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 β-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].

As more plant genome sequences are published, it is becoming clear that the GH3 glycoside hydrolases in plants are encoded by multi-gene families. In both monocot and dicot species, there are 15-18 genes for the GH3 enzymes, while in lower plants such as the Physcomitrella and Selaginella mosses 6-7 genes encoding GH3 glycoside hydrolases have been identified. Furthermore, phylogenetic analyses of the plant GH3 enzymes reveal their separation into two major groups (Fig. 1). Existing annotations suggest that one plant group might include β-D-glucosidases and the other α-L-arabinofuranosidases, β-D-xylopyranosidase and other enzymes. However, this suggestion is limited to plant GH3 enzymes and must be viewed with caution until further confirmation is obtained, because relatively few members of the enzyme family have been purified for detailed characterization of their substrate specificities. There do not appear to be any confirmed plant β-N-acetylglucosaminidases in either clade.

Kinetics and Mechanism

GH3 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 retention of anomeric configuration has been established experimentally for several enzymes. The active site of GH3 enzymes consists of two glucosyl-binding subsites (-1 and +1) with an enzymic nucleophile and general acid/base residue flanking the junction of these two subsites (see [10] for subsite nomenclature). This arrangement of catalytic residues was first visualized in 1999 when the crystal structure of barley β-D-glucan glucohydrolase was determined in complex with glucose [11] (Fig. 2).

Several studies have contributed to the understanding of the kinetics and mechanism of GH3 enzymes, including detailed analyses of β-glucosidases from fungi (Aspergillus wentii [12] and Aspergillus niger [13, 14]) and Gram-negative bacteria (Flavobacterium meningosepticum [15, 16], Thermotoga neapolitana [17]), as well as a GH3 glucosylceramidase from the Gram-positive microbe Paenibacillus sp. TS12 [18]. Kinetic and mechanistic analyses of β-D-glucan glucohydrolases and two ‘bifunctional’ α-L-arabinofuranosidase/β-D-xylopyranosidases from plants (barley) have also been carried out [2, 4]. These studies, combined with kinetic and mechanistic analyses of N-acetyl-β-D-glucosaminidases from the Gram-positive microbe Bacilus subtillus [19, 20], and Gram-negative microbes Vibrio furnisii [21, 22], Vibrio cholerea [23] and Salmonella typhimerium [20] confirm that while the catalytic nucleophile of GH3 enzymes is well conserved, the location and identity of the general acid/base residue is not.

Catalytic Residues

Catalytic nucleophile

Early labeling experiments of a GH3 β-glucosidases from Aspergillus wentii using conduritol B-epoxide by Bause and Legler in 1974 [12] identified an aspartate residue within the sequence VMSDW as the putative catalytic nucleophile. This was later supported by the crystal structure of barley β-D-glucan glucohydrolase, which identified Asp285 within the sequence GFVISDW as being appropriately positioned to act as a nucleophile during catalysis [11]. Direct identification of this aspartate as the catalytic nucleophile via active-site labeling using 2-deoxy-2-fluoro-β-D-glycosides of GH3 β-glucosidases from A. niger [13] and F. meningosepticum [15], a glucosylceramidase from Paenibacillus sp [18], as well as the barley β-D-glucan glucohydrolase [24], and labeling of NagZ enzymes from V. furnisii [21] and S. typhimerium [20] using 2-acetamido-2-Deoxy-5-Fluoro-β-D-glucopyranosyl fluoride, have confirmed that this catalytic residue is well conserved in GH3 enzymes from diverse species. The conservation of this aspartate becomes clear when known crystal structures of a number of GH3 enzymes are superposed (Fig. 3).

Catalytic acid/base

Though sequence alignments can now be used to easily identify the catalytic nucleophile of GH3 members, the position of type of general acid/base residue is phylogenetically variable and less readily identifiable. An increase in 3D structural information for GH3 enzymes has recently provided insight into identity of the elusive general acid-base residue. The barley β-D-glucan glucohydrolase structure is a two-domain enzyme consisting of an N-terminal (b/a)₈ barrel domain housing the active site pocket and catalytic aspartate nucleophile (Asp285), and a C-terminal domain with a glutamate residue (Glu 491) that projects into the active site of (b/a)₈ barrel domain to act as the catalytic acid/base [11]. Structural studies of additional GH3 enzymes have since shown that while insertion of additional domains is possible (which shifts the location of the glutamic acid general acid-base within the primary sequence with respect to the barely enzyme), the unusual two-domain active site architecture observed for the barley enzyme appears to be a core feature of multidomain GH3 b-glucanases [17, 25, 26]. Superposition of available GH3 β-D-glucan glucohydrolase structures clearly reveals the conservation of this architecture (Fig. 3).

GH3 NagZ enzymes depart significantly from the above two-domain active site architecture. A crystal structure of NagZ from B. subtilis recently revealed that the catalytic acid-base is an unusual histidine/aspartate dyad that resides within a flexible loop that is present on the catalytic (b/a)₈ barrel and not on a separate domain [19]. Though the enzyme adopts a two-domain fold similar to barely β-D-glucan glucohydrolase, the C-terminal domain does not participate in catalysis (Fig. 4). In fact, most NagZ enzymes from Gram-negative bacteria are single domain enzymes comprised solely of a catalytic (b/a)₈ barrel that contains the conserved aspartate nucleophile and a catalytic histidine/aspartate dyad on flexible loop as seen for B. subtilis NagZ [20] (Fig. 5). The flexible loop containing the His/Asp dyad is unique to GH3 NagZ enzymes and can be identified by the consensus motif [KH(F/I)PG(H/L)GXXXXD(S/T)H] (catalytic dyad highlighted in boldface) [21].

Three-dimensional structures

Figure 2. 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) [23]. 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.

Crystal structures are now available for a number multidomain GH3 members, including barley β-D-glucan glucohydrolase [11] and a mechanistically related β-glucanase from the marine bacterium Pseudoalteromonas sp. [25], as well as β-glucosidases from Kluyveromyces marxianus (KmBglI) [26] , Trichoderma reesei (Cel3A) (PDB: 4I8D (unpublished)), Thermotoga neapolitana [17] and a macrolide β-glycosidase / β-glucosidase (DesR) from Streptomyces venezuelae [27]. These structures have revealed considerable diversity in architecture and arrangement of domains in GH3 enzymes that are present in addition to the core catalytic domain, and how these domains affect function. Crystal structures of a number of NagZ enzymes are also available, including the two-domain NagZ from B. subtilis [19] and single domain NagZ enzymes from Vibrio cholera (PDB: 1TR9 (unpublished)) & [23], S. typhimurium [20], Deinococcus radiodurans (PDB 3TEV (unpublished)) and Burkholderia cenocepacia (PDB: 4GNV (unpublished)). Given the role of NagZ in the activation of AmpC-mediated β-lactam resistance in Gram-negative bacteria [9]., a number of crystallographic and kinetic studies have focused on the development of inhibitors that are selective for NagZ over functionally related human enzymes from families GH20 and GH84 [23, 28, 29].

Insight into the broad specificity observed for some GH3 enzymes has been provided from X-ray crystallographic data and from molecular modeling of enzyme-substrate complexes of the barley β-D-glucan glucohydrolase [11, 30]. Recent structural studies of NagZ enzymes from S. typhimurium and B. subtilis have shown the flexible catalytic loop participates in distorting the bound terminal GlcNAc sugar toward a ¹S₃ conformation during catalysis [20] (Fig. .5). Distortion of the substrate toward a ¹S₃ conformation is in agreement with other retaining β-glycosidases; however, this has not been observed for other GH3 enzymes.

Family Firsts

First 3D Structure: Barley β-D-glucan glucohydrolase [11].
First Catalytic Residues: Barley β-D-glucan glucohydrolase [11] and B. subtilis NagZ [19].

References

All Medline abstracts: PubMed