Difference between revisions of "Glycoside Hydrolase Family 73"

Revision as of 17:45, 1 September 2010

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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 is comprised of bacterial and prokaryotic viral glycoside hydrolases. Most of the enzymes of this family are peptidoglycan hydrolases that cleave the β-1,4-glycosidic linkage between N-acetylglucosaminyl (NAG) and N-acetylmuramyl (NAM) moieties in the carbohydrate backbone of bacterial peptidoglycan. Because of their cleavage specificity, they are commonly described as N-acetylglucosaminidases. The activity of the GH73 is mainly focused in daughter cell separation during vegetative growth and it is very often involved in the hydrolysis of the septum after cell division (Acp from Clostridium Perfringens [1] AltA from Enterococcus faecalis [2]). Only Listeria monocytogene uses Auto as a virulence-associated peptidoglycan hydrolase for host-cell invasion [3]. The GH73 are mostly surface located and exhibit repeated sequences that could be involved in cell-wall binding and therefore reinforce the enzymes catalytic activity. 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 like CBM50 also known as LysM domain appended to AcmA for 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 challenging task.

Catalytic Residues

The catalytic general acid is a glutamate, strictly conserved in the GH73 family. Its catalytic role has been evidenced in FlgJ [9], Auto [3], AcmA [7] and AltWN [10]. Glu185 in FlgJ and Glu122 in Auto have also been identified through structural comparison with the actives sites from GH23, GH22 and GH19 enzymes [3, 11]. Nevertheless, both structures of FlgJ and Auto have in common the evident lack of a nearby second catalytic carboxylate, provided for instance by Asp52(53) in GH22 lysozymes (see figure 1). In FlgJ and Auto the catalytic nucleophile/general base, a Glu corresponding to Aps52, is strickly conserved in the GH73 family but is situated 13Å away from the Glu general acid in the active site.

Figure 1. Comparison of Auto (in yellow) and HEWL (in grey) active sites. Catalytic residues are in italic for HEWL (GH22)

Mutational analysis on the putative distant nucleophile (Glu156) in Auto, showed a drastic decrease of the catalytic activity [3]. Therefore, Bublitz et al proposed a single displacement mechanism involving a distant carboxylate that would serve as a base assisting a water molecule for the nucleophilic attack on the opposite side of the sugar ring (inverting mechanism). This mechanism also involves an important displacement on the β-lobe upon substrat binding that would bring the nucleophile/base closer to the active site.

On the other hand, mutational analyses on FlgJ , AcmA and AltWN revealed an uncertainty on the nucleophile/base residue and the putative existence of another key catalytic residue. It is noteworthy that only the mutational analysis on Auto revealed a decreased catalytic activity when the nucleophile Glu156 was mutated into glutamine. In FlgJ, AcmA and AltWN, an important residual activity upon mutation of this equivalent Glu into alanine, glutamine or asparagine (for Asp1275 in AltWN) ruled out this residue as a key catalytic residue.

In close proximity to the Glu proton donor is a Tyrosine highly conserved in the GH73 family (Fig1: Tyr220 in Auto). Amino acid substitution of this tyrosine on FlgJ, AcmA and AltWN exhibited reduced activity similar to the mutation of the Glu proton donor [7, 9, 10]. The substitution of this Tyr into a Phe or Trp, in AcmA and AltWN, retained substantial activity.

Inagaki and Murayama agreed on the fact that the Glu proton donor and this nearby Tyr are probably crucial for enzyme activities of FlgJ, AcmA, and AltWN. The role of the Tyr have already been discussed for Auto, they suggested the need for an hydrophobic residue in this position, to protonate the carboxylate group of the proton donor and maintain the stable conformation of the active site residues [3].

Finally, based on sequence analyses in the GH73 family and in comparison with families GH20, GH18, GH23 and GH56, enzymes that do not have a catalytic nucleophile residue, Inagaki et al suggested a neighboring group participation involving the Glu proton donor and the Tyr as essential catalytic residues. This mechanism implies that the 2-acetamido group of the NAG is acting as an intramolecular nucleophile [7].

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 coincidently reported, FlgJ from Sphingomonas sp. (SPH1045-C) [11] and Auto a virulence associated peptigoglycan hydrolase from Listeria monocytogenes [3]. A structure for a catalytic mutant (E185A) of FlgJ has been solved by Maruyama et al [9] but doesn’t show any conformational changes. 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 [12], the family GH19 chitinases and goose egg-white lysozyme (GEWL, GH23)[13]. FlgJ is structurally related to a peptidoglycan degrading enzyme from the bacteriophage phi 29 [14] and also to family GH22 and GH23 lysozymes.

Family Firsts

First stereochemistry determination
First catalytic nucleophile identification: Evidence for a putative nucleophile residue in Auto, a peptidoglycan hydrolase from Lytseria monocytogene [3]
First general acid/base/general acid residue identification
First 3-D structure: peptidoglycan hydrolase FlgJ from Sphingomonas sp. [11]

References

All Medline abstracts: PubMed

@@ Line 47: / Line 47: @@
 Inagaki and Murayama agreed on the fact that the Glu proton donor and this nearby Tyr  are probably crucial for enzyme activities of FlgJ, AcmA, and AltWN. The role of the Tyr have already been discussed for Auto, they suggested the need for an hydrophobic residue in this position, to protonate the carboxylate group of the proton donor and maintain the stable conformation of the active site residues <cite>Bublitz2009</cite>.
-Finally, based on sequence analyses in the GH73 family and in comparison with families [[GH20]], [[GH18]], [[GH23]] and [[GH56]], that don't have a catalytic nucleophile residue. Inagaki et al suggested a [[Neighboring group participation]] involving the Glu proton donor and the Tyr as essential catalytic residues. This mechanism implies that the 2-acetamido group of the NAG is acting as an intramolecular nucleophile <cite>Inagaki2009</cite>.
+Finally, based on sequence analyses in the GH73 family and in comparison with families [[GH20]], [[GH18]], [[GH23]] and [[GH56]], enzymes that do not have a catalytic nucleophile residue, Inagaki et al suggested a [[neighboring group participation]] involving the Glu proton donor and the Tyr as essential catalytic residues. This mechanism implies that the 2-acetamido group of the NAG is acting as an intramolecular nucleophile <cite>Inagaki2009</cite>.
 == Three-dimensional structures ==