Glycoside Hydrolase Family 25

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• Author: Ed Taylor
• Responsible Curator: Gideon Davies

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Glycoside Hydrolase Family GH25
Clan	GH-K
Mechanism	unknown, likely retaining by analogy with GH18,20,56,84,85 [1]
Active site residues	Asp/Glu
CAZy DB link
https://www.cazy.org/GH25.html

Substrate specificities

Glycoside hydrolases of family GH25 are lysozymes otherwise known as the Chalaropsis (CH) type of lysozymes (from their initial characterisation from Chalaropsis species of fungus [2]). They cleave the β-1,4-glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) in the carbohydrate backbone of bacterial peptidoglycan. The characterized lysozymes from this family exhibit both β-1,4-N-acetyl- and β-1,4-N,6-O-diacetylmuramidase activities and are able to degrade O-acetylated peptidoglycan present in Staphylococcus aureus and other pathogens [3]. The activity of GH25 enzymes appears to fulfil two main biological roles in bacteria. These roles are the re-modelling of peptidoglycan in cellular process such as division and the dissemination of phage progeny toward the end of the phage lytic cycle, which is achieved by lysis of the bacterial cell. For this reason many GH25 proteins are found to be chromosomal, phage or prophage encoded. The majority of the GH25 family is comprised of bacterial or prokaryotic viral (phage) members. There are however a few eukaryotic representatives, but these are so far restricted to the fungal kingdom. The roles of these fungal enzymes are less clear, many possess signal secretion peptides indicating a likely extracellular location, possibly for the purpose of obtaining or gaining assess to nutrients or even as a selective agent against bacteria.

Kinetics and Mechanism

The lack of activity on chitooligosaccharides and the difficulty of producing defined synthetic peptidoglycan substrates, has inevitably hampered the determination of kinetic parameters. Structural studies have shown the active-centre is closely similar to those from GH18, GH20, GH56, GH84 and GH85 implying that, in the absence of evidence to the contrary, GH25 enzymes are retaining glycoside hydrolases that use a neighboring group participation mechanism common to this “super-family” of enzymes [1].

Catalytic Residues

The super-family comprised of families GH18, GH20, GH56, GH84 and GH85 has been shown to use an active site DE or DXE sequence motif (or exceptionally in the case of GH85 a NXD motif [4, 5]). This carboxylate pair promotes a double displacement mechanism in which the catalytic nucleophile is not enzyme-derived but is provided by the substrate in an intramolecular “neighboring group” attack of the N-acetyl carbonyl group [6]. Thus catalysis occurs via the formation, and subsequent breakdown of a covalent oxazoline (or oxazolinium ion) intermediate.

Three-dimensional structures

So far four members of this family of enzymes have been structural characterized, that of the Streptomyces coelicolor enzyme “cellosyl” [7], the bacteriophage lysine PlyB [8], and Clp-1lysozyme from a Streptococcus pneumoniae phage [9, 10], whose structure was also obtained in complex with fragments of peptidoglycan analogues 10 and BaGh25c from Bacillus anthracis str. Ames [1]. The GH25 lysozymes, are structurally unrelated to the GH22, GH23, GH24, GH73 and GH108 lysozyme folds and instead these enzymes display a modified α -barrel-like fold that, like the classical “TIM-barrel” is composed of a eight-stranded β-barrel, but which is flanked by just three (as opposed to the normal eight) α-helices [7]. There is a prominent, long groove, very likely the substrate binding site, located on the C-terminal face. This groove culminates in a deep hole of a highly negative electrostatic potential forming the catalytic site [11].

Family Firsts

First sterochemistry determination

Has yet to be experimentally determined.

First catalytic nucleophile identification

This remains to be experimentally proven. Inverting mechanisms have been proposed [9], but it is perhaps more likely that the enzyme is retaining and that the nucleophile is not enzyme-derived, but is provided by the substrate in a neighboring group participation mechanism [1]. This is based upon the considerable active centre structural similarity with other neighboring group enzymes in GH18, 20, 56, 84 and also 85.

First general acid/base residue identification

Has yet to be fully experimentally determined but again structural similarity strongly implicates the second carboxylate in the DXE motif described previously. In the case of the Bacillus anthracis enzyme this is Glu107 [1].

First 3-D structure

The Streptomyces coelicolor enzyme "cellosyl" [7] was the first GH25 structure to be described in 2001.

References

1. Martinez-Fleites C, Korczynska JE, Davies GJ, Cope MJ, Turkenburg JP, and Taylor EJ. (2009). The crystal structure of a family GH25 lysozyme from Bacillus anthracis implies a neighboring-group catalytic mechanism with retention of anomeric configuration. Carbohydr Res. 2009;344(13):1753-7. DOI:10.1016/j.carres.2009.06.001 |
2. Hash JH and Rothlauf MV. (1967). The N,O-diacetylmuramidase of Chalaropsis species. I. Purification and crystallization. J Biol Chem. 1967;242(23):5586-90. | Google Books | Open Library
3. Vollmer W (2008). Structural variation in the glycan strands of bacterial peptidoglycan. FEMS Microbiol Rev. 2008;32(2):287-306. DOI:10.1111/j.1574-6976.2007.00088.x |
4. Ling Z, Suits MD, Bingham RJ, Bruce NC, Davies GJ, Fairbanks AJ, Moir JW, and Taylor EJ. (2009). The X-ray crystal structure of an Arthrobacter protophormiae endo-beta-N-acetylglucosaminidase reveals a (beta/alpha)(8) catalytic domain, two ancillary domains and active site residues key for transglycosylation activity. J Mol Biol. 2009;389(1):1-9. DOI:10.1016/j.jmb.2009.03.050 |
5. Abbott DW, Macauley MS, Vocadlo DJ, and Boraston AB. (2009). Streptococcus pneumoniae endohexosaminidase D, structural and mechanistic insight into substrate-assisted catalysis in family 85 glycoside hydrolases. J Biol Chem. 2009;284(17):11676-89. DOI:10.1074/jbc.M809663200 |
6. Vocadlo DJ and Davies GJ. (2008). Mechanistic insights into glycosidase chemistry. Curr Opin Chem Biol. 2008;12(5):539-55. DOI:10.1016/j.cbpa.2008.05.010 |
7. Rau A, Hogg T, Marquardt R, and Hilgenfeld R. (2001). A new lysozyme fold. Crystal structure of the muramidase from Streptomyces coelicolor at 1.65 A resolution. J Biol Chem. 2001;276(34):31994-9. DOI:10.1074/jbc.M102591200 |
8. Porter CJ, Schuch R, Pelzek AJ, Buckle AM, McGowan S, Wilce MC, Rossjohn J, Russell R, Nelson D, Fischetti VA, and Whisstock JC. (2007). The 1.6 A crystal structure of the catalytic domain of PlyB, a bacteriophage lysin active against Bacillus anthracis. J Mol Biol. 2007;366(2):540-50. DOI:10.1016/j.jmb.2006.11.056 |
9. Hermoso JA, Monterroso B, Albert A, Galán B, Ahrazem O, García P, Martínez-Ripoll M, García JL, and Menéndez M. (2003). Structural basis for selective recognition of pneumococcal cell wall by modular endolysin from phage Cp-1. Structure. 2003;11(10):1239-49. DOI:10.1016/j.str.2003.09.005 |
10. Pérez-Dorado I, Campillo NE, Monterroso B, Hesek D, Lee M, Páez JA, García P, Martínez-Ripoll M, García JL, Mobashery S, Menéndez M, and Hermoso JA. (2007). Elucidation of the molecular recognition of bacterial cell wall by modular pneumococcal phage endolysin CPL-1. J Biol Chem. 2007;282(34):24990-9. DOI:10.1074/jbc.M704317200 |
11. Vollmer W, Joris B, Charlier P, and Foster S. (2008). Bacterial peptidoglycan (murein) hydrolases. FEMS Microbiol Rev. 2008;32(2):259-86. DOI:10.1111/j.1574-6976.2007.00099.x |