Glycoside Hydrolase Family 5

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• Author: ^^^Gideon Davies^^^
• Responsible Curator: ^^^Gideon Davies^^^

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Glycoside Hydrolase Family GH5
Clan	GH-A
Mechanism	retaining
Active site residues	known
CAZy DB link
http://www.cazy.org/fam/GH5.html

Substrate specificities

GH5 is one of the largest of all CAZy glycoside hydrolase families. A variety of specificties are annotated to this family notably endoglucanase (cellulase) and endomannanase as well as exoglucanases, exomannanases and β-glucosidase and β-mannosidase. Other activities include 1,6 galactanase, 1,3 mannanase, 1,4 xylanase, endoglycoceramidase, as well as high specificity xyloglucanases. Family GH5 enzymes are found widely distributed across Archae, bacteria and eukaryotes, notably fungi and plants. There are no known human enzymes in GH5.

Kinetics and Mechanism

Family GH5 enzymes are retaining enzymes, as first shown by NMR [1] and follow a classical Koshland double-displacement mechanism.

Catalytic Residues

GH5 enzymes use the classical Koshland double-displacement mechanism and the two catalytic residues are known to be glutamates found at the C-terminal ends of β-strands 4 (acid/base) and 7 (nucleophile) [2, 3].

Three-dimensional structures

Three-dimensional structures are available for a very large number of Family GH5 enzymes, the first solved being that of the Clostridium thermocellum endoglucanase CelC [4]. As members of Clan GH-A they have a classical (α/β)₈ TIM barrel fold with the two key active site glutamic acids being approximately 200 residues apart in sequence and located at the C-terminal ends of β-strands 4 (acid/base) and 7 (nucleophile) [2, 3].

With so many 3D structures in this family, covering many specificities it is clearly hard to pick out notable structural papers. The Bacillus agaradhaerens Cel5A has been extensively studied, notably in the trapping of enzymatic snapshots along the reaction coordinate [5] but also as a testbed for glycosidase inhibitor design as crystals often diffract to atomic resolution (for example [6]). The reaction coordinate work on the endoglucanases (thus working on gluco-configured substrates) shows that the substrate binds in ¹S₃ conformation with the covalent intermediate in ⁴C₁ chair conformation implying catalysis via a ⁴H₃ half-chair (near) transition-state. By analogy with family GH26 mannnanases [7] and family GH2 β-mannosidases [8] it would seem likely that GH5 mannanases use a different conformational itinerary to their glucosidase relatives, likely via a ¹S₅-^OS₂ glycosylation pathway although direct evidence in this family is limited [9].

The Rhodococcal endoglycoceramidase II (EGC) in this family has found application in the chemoenzymatic synthesis of ceramide derivatives [10]. In 2007 the first 3-D structure of a highly specific GH5 xyloglucanase was reported [11]; this enzyme makes kinetically productive interactions with both xylose and galactose substituents, as reflected in both a high specific activity on xyloglucan and the kinetics of a series of aryl glycosides.

Family Firsts

First sterochemistry determination

The curator believes this to be the ¹H NMR stereochemical determination for EGZ from Erwinia chrysanthemi [1]. GH5 enzymes were also in the comprehensive Gebler study [12].

First catalytic nucleophile identification

Trapped using the classical Withers 2-fluoro method, here with 2',4'-dinitrophenyl-2-deoxy-2-fluoro-beta-D-cellobioside, reported in Wang and Withers in 1993 [13].

First general acid/base residue identification

Several mutagenesis papers has alluded to the importance of a conserved glutamate- one that both Dominguez [14] and Ducros [15] correctly postulated as the catalytic acid when the 3-D structures were determined.

First 3-D structure

The first 3D structures in family GH5 was an endoglucanase (cellulase) from Clostridium thermocellum reported by the Alzari in 1995 (in a paper which also reported a family GH10 xylanase structure and the similarities between them) [14]. Subsequently, Ducros and colleagues reported the Clostridium cellulolyticum Cel5A also in 1995 [15].

References

1. Barras F, Bortoli-German I, Bauzan M, Rouvier J, Gey C, Heyraud A, and Henrissat B. (1992). Stereochemistry of the hydrolysis reaction catalyzed by endoglucanase Z from Erwinia chrysanthemi. FEBS Lett. 1992;300(2):145-8. DOI:10.1016/0014-5793(92)80183-h |
2. Henrissat B, Callebaut I, Fabrega S, Lehn P, Mornon JP, and Davies G. (1996). Conserved catalytic machinery and the prediction of a common fold for several families of glycosyl hydrolases. Proc Natl Acad Sci U S A. 1996;93(11):5674. DOI:10.1073/pnas.93.11.5674 |
3. Jenkins J, Lo Leggio L, Harris G, and Pickersgill R. (1995). Beta-glucosidase, beta-galactosidase, family A cellulases, family F xylanases and two barley glycanases form a superfamily of enzymes with 8-fold beta/alpha architecture and with two conserved glutamates near the carboxy-terminal ends of beta-strands four and seven. FEBS Lett. 1995;362(3):281-5. DOI:10.1016/0014-5793(95)00252-5 |
5. Davies GJ, Mackenzie L, Varrot A, Dauter M, Brzozowski AM, Schülein M, and Withers SG. (1998). Snapshots along an enzymatic reaction coordinate: analysis of a retaining beta-glycoside hydrolase. Biochemistry. 1998;37(34):11707-13. DOI:10.1021/bi981315i |
6. Varrot A, Tarling CA, Macdonald JM, Stick RV, Zechel DL, Withers SG, and Davies GJ. (2003). Direct observation of the protonation state of an imino sugar glycosidase inhibitor upon binding. J Am Chem Soc. 2003;125(25):7496-7. DOI:10.1021/ja034917k |
7. Ducros VM, Zechel DL, Murshudov GN, Gilbert HJ, Szabó L, Stoll D, Withers SG, and Davies GJ. (2002). Substrate distortion by a beta-mannanase: snapshots of the Michaelis and covalent-intermediate complexes suggest a B(2,5) conformation for the transition state. Angew Chem Int Ed Engl. 2002;41(15):2824-7. DOI:10.1002/1521-3773(20020802)41:15<2824::AID-ANIE2824>3.0.CO;2-G |
8. Tailford LE, Offen WA, Smith NL, Dumon C, Morland C, Gratien J, Heck MP, Stick RV, Blériot Y, Vasella A, Gilbert HJ, and Davies GJ. (2008). Structural and biochemical evidence for a boat-like transition state in beta-mannosidases. Nat Chem Biol. 2008;4(5):306-12. DOI:10.1038/nchembio.81 |
9. Vincent F, Gloster TM, Macdonald J, Morland C, Stick RV, Dias FM, Prates JA, Fontes CM, Gilbert HJ, and Davies GJ. (2004). Common inhibition of both beta-glucosidases and beta-mannosidases by isofagomine lactam reflects different conformational itineraries for pyranoside hydrolysis. Chembiochem. 2004;5(11):1596-9. DOI:10.1002/cbic.200400169 |
10. Caines ME, Vaughan MD, Tarling CA, Hancock SM, Warren RA, Withers SG, and Strynadka NC. (2007). Structural and mechanistic analyses of endo-glycoceramidase II, a membrane-associated family 5 glycosidase in the Apo and GM3 ganglioside-bound forms. J Biol Chem. 2007;282(19):14300-8. DOI:10.1074/jbc.M611455200 |
11. Gloster TM, Ibatullin FM, Macauley K, Eklöf JM, Roberts S, Turkenburg JP, Bjørnvad ME, Jørgensen PL, Danielsen S, Johansen KS, Borchert TV, Wilson KS, Brumer H, and Davies GJ. (2007). Characterization and three-dimensional structures of two distinct bacterial xyloglucanases from families GH5 and GH12. J Biol Chem. 2007;282(26):19177-89. DOI:10.1074/jbc.M700224200 |
12. Gebler J, Gilkes NR, Claeyssens M, Wilson DB, Béguin P, Wakarchuk WW, Kilburn DG, Miller RC Jr, Warren RA, and Withers SG. (1992). Stereoselective hydrolysis catalyzed by related beta-1,4-glucanases and beta-1,4-xylanases. J Biol Chem. 1992;267(18):12559-61. | Google Books | Open Library
13. Wang Q, Tull D, Meinke A, Gilkes NR, Warren RA, Aebersold R, and Withers SG. (1993). Glu280 is the nucleophile in the active site of Clostridium thermocellum CelC, a family A endo-beta-1,4-glucanase. J Biol Chem. 1993;268(19):14096-102. | Google Books | Open Library
14. Dominguez R, Souchon H, Spinelli S, Dauter Z, Wilson KS, Chauvaux S, Béguin P, and Alzari PM. (1995). A common protein fold and similar active site in two distinct families of beta-glycanases. Nat Struct Biol. 1995;2(7):569-76. DOI:10.1038/nsb0795-569 |
15. Ducros V, Czjzek M, Belaich A, Gaudin C, Fierobe HP, Belaich JP, Davies GJ, and Haser R. (1995). Crystal structure of the catalytic domain of a bacterial cellulase belonging to family 5. Structure. 1995;3(9):939-49. DOI:10.1016/S0969-2126(01)00228-3 |