Glycoside Hydrolase Family 73

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Author: ^^^Florence Vincent^^^
Responsible Curator: ^^^Bernard Henrissat^^^

Glycoside Hydrolase Family GH73
Clan	none, α+β "lysozyme fold"
Mechanism	not known
Active site residues	partially known
CAZy DB link
https://www.cazy.org/GH73.html

Substrate specificities

Family GH73 contains bacterial and viral glycoside hydrolases. Most of the enzymes of this family cleave the β-1,4-glycosidic linkage between N-acetylglucosaminyl (NAG) and N-acetylmuramyl (NAM) moieties in the carbohydrate backbone of bacterial peptidoglycans. Because of their cleavage specificity, they are commonly described as β-N-acetylglucosaminidases. The enzymes from family GH73 are mainly involved in daughter cell separation during vegetative growth, and they often hydrolyze the septum after cell division (Acp from Clostridium perfringens [1] AltA from Enterococcus faecalis [2]). Occasionally GH73 enzymes are used during host-cell invasion such as the virulence-associated peptidoglycan hydrolase Auto from Listeria monocytogene [3]. GH73 enzymes are mostly surface-located and often exhibit repeated sequences that could be involved in bacterial cell-wall binding (figure 1)[[Image:Diapositive1recadree.jpg|left|Figure 1. Examples of modular GH73 enzymes: SLH, S‐layer homology domains; CBM50, carbohydrate binding module of family 50, in purple, signal peptide; in grey and red, unknown repaeted domains. Unknown repeated domains are appended for instance to LytD and LytG from Bacillus subtilis [4, 5], AcmB from Lactococcus lactis [6] and Auto from L. monocytogene [3]. Some of these repeated domains have been identified such as the carbohydrate-binding modules of family CBM50 (also known as LysM domains) appended to AcmA of Lactococcus lactis [7], AltA from Enterococcus faecalis [2] and Mur2-Mur2 from Enterococcus hirae [8].

Kinetics and Mechanism

No kinetic parameters have been determined for any enzyme of the GH73 family, as the production of synthetic peptidoglycan substrates remains a challenge.

Three-dimensional structures

Figure 2. Ribbon diagram of Auto structure (orange) and its surface, superimposed on FlgJ structure (green).

Crystal structures of GH73 are available and have been reported simultaneously, namely FlgJ from Sphingomonas sp. (SPH1045-C) [9] and Auto a virulence associated peptigoglycan hydrolase from Listeria monocytogenes [3]. The two GH73 show the same fold, with two subdomains consisting of a β-lobe and an α-lobe that together create an extended substrate binding groove (Figure 2). With a typical lysozyme (α+β) fold, the catalytic domain of Auto is structurally related to the catalytic domain of Slt70 from E. coli [10], the family GH19 chitinases and goose egg-white lysozyme (GEWL, GH23)[11]. FlgJ is structurally related to a peptidoglycan degrading enzyme from the bacteriophage phi 29 [12] and also to family GH22 and GH23 lysozymes.

Catalytic Residues

The catalytic general acid is a glutamate, strictly conserved in the GH73 family. Its catalytic role has been evidenced in FlgJ [13], Auto [3], AcmA [7] and AltWN [14]. Glu185 in FlgJ and Glu122 in Auto have also been identified through structural comparison with the actives sites of GH19, GH22 and GH23 enzymes [3, 9]. However, in contrast to GH22 lysozymes, the structures of FlgJ and Auto both lack a nearby second catalytic carboxylate such as Asp52, which is the catalytic nucleophile/general base in hen egg white lysozyme (HEWL) (see figure 3). Interestingly this amino acid is present and strictly conserved in the sequences of GH73 enzymes but it is situated 13Å away from the Glu general acid in the active site.[[Image:GH73activesite.jpg|thumb|left|Figure 3. Comparison of Auto (in yellow) and HEWL (in grey) active sites. Catalytic residues are in italic for HEWL (GH22)]]

The identification of the catalytic nucleophile/base is not conclusive. On one hand, Bublitz et al found that a mutation of the putative distant nucleophile Glu156 was accompanied of a large decrease in the catalytic activity, compatible with the role of a base activating a water molecule for the nucleophilic attack on the opposite side of the sugar ring (inverting mechanism). On the other hand, significant residual activity was found when the putative nucleophile/base residue of FlgJ , AcmA and AltWN was converted to alanine, glutamine or asparagine (for Asp1275 in AltWN), which is more compatible with a substrate-assisted catalysis involving anchimeric assistance by the acetamido group of the GlcNAc moiety. In such a mechanism a neighbouring aromatic residue is frequently involved. A Tyr residue is highly conserved in family GH73 (Fig2: Tyr220 in Auto), in close proximity to the catalytic general acid Glu. Substitution of this Tyr residue in FlgJ, AcmA and AltWN was associated with a reduced activity similar to that resulting from the mutation of the general acid Glu [7, 13, 14]. The neighboring group participation mechanism involving the general acid Glu and the Tyr as essential catalytic residues found support from the sequence comparison of family GH73 with families GH20, GH18, GH23 and GH56, which do not have a catalytic nucleophile residue [7].

Family Firsts

First general acid/base/general acid residue identification
First 3-D structure: peptidoglycan hydrolase FlgJ from Sphingomonas sp. [9]

References

All Medline abstracts: PubMed