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
https://www.cazy.org/GH5.html

Substrate specificities

GH5 is one of the largest of all CAZy glycoside hydrolase families. Previously known as "cellulase family A" [1, 2], a variety of specificities are now known in 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. Following the reclassification of a number of GH5 members into GH30 [3], a GH5 subfamily classification has been presented that delineates members into a number of monospecific and polyspecific clades [4]. It should be noted that enzymes specifically targeting xylans are exclusively arabinoxylanases, and are found in subfamilies GH_21 [5] and GH_34 [6].

Kinetics and Mechanism

Family GH5 enzymes are retaining enzymes, as first shown by NMR [7] and follow a classical Koshland double-displacement mechanism.

Catalytic Residues

GH5 enzymes use the classical Koshland double-displacement mechanism and the two catalytic residues (catalytic nucleophile and general acid/base) are known to be glutamates found at the C-terminal ends of β-strands 4 (acid/base) and 7 (nucleophile) [8, 9].

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 [10]. 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) [8, 9].

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 [11] but also as a testbed for glycosidase inhibitor design as crystals often diffract to atomic resolution (for example [12]). The reaction coordinate work on the endoglucanases (thus working on gluco-configured substrates) shows that the substrate binds in ¹S₃ conformation with the glycosyl enzyme intermediate in ⁴C₁ chair conformation implying catalysis via a near ⁴H₃ half-chair transition state.

By analogy with family GH26 mannnanases [13] and family GH2 β-mannosidases [14] it would seem likely that GH5 mannanases use a different conformational itinerary to their glucosidase relatives, likely via a ¹S₅-^OS₂ glycosylation pathway and thus via a B_2,5 (near) transition-state although direct evidence in this family is limited [15]. An interesting dissection of mannan-degrading enzyme systems has been provided by work in the Gilbert group on the diverse GH5 and GH26 mannanases in Cellvibrio japonicus(see for example [16, 17, 18]).

Within the huge functional diversity of the GH5 family, the GH5 subfamily 4 (GH5_4) is the only subfamily which was found to contain predominant endo-xyloglucanases [4, 19]. Although the GH5_4 endo-xyloglucanases share amino acid identity as low as 30%, the exhibited strict substrate specificity is ultimately attributed to the high conservation of the amino acid residues interacting with the xyloglucan substrate in the active site cleft [20].

The GH5_34 enzymes target arabinoxylan through essential interactions with single arabinose substituents linked O3 to the xylose positioned in the active site -1 subsite [6, 21]. Very limited interactions with the xylan backbone is observed out with the -1 active site of the GH5_34 enzymes [21]. This explains why these glycoside hydrolases cleave highly decorated glucuronoarabinoxylans that are recalcitrant to cleavage by classical xylanases found in GH10 and GH11.

The Rhodococcal endoglycoceramidase II (EGC) in this family has found application in the chemoenzymatic synthesis of ceramide derivatives [22]. In 2007 the first 3-D structure of a highly specific GH5 xyloglucanase was reported [23]; 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 [7]. GH5 enzymes were also in the comprehensive Gebler study [24].

First catalytic nucleophile identification

Trapped using the classical Withers 2-fluoro method, here with 2',4'-dinitrophenyl 2-deoxy-2-fluoro-β-D-cellobioside, reported in Wang and Withers in 1993 [25].

First general acid/base identification

Several mutagenesis papers has alluded to the importance of a conserved glutamate- one that both Dominguez [26] and Ducros [27] 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) [26]. Subsequently, Ducros and colleagues reported the Clostridium cellulolyticum Cel5A also in 1995 [27].

References

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1. Henrissat B, Claeyssens M, Tomme P, Lemesle L, and Mornon JP. (1989). Cellulase families revealed by hydrophobic cluster analysis. Gene. 1989;81(1):83-95. DOI:10.1016/0378-1119(89)90339-9 |
2. Gilkes NR, Henrissat B, Kilburn DG, Miller RC Jr, and Warren RA. (1991). Domains in microbial beta-1, 4-glycanases: sequence conservation, function, and enzyme families. Microbiol Rev. 1991;55(2):303-15. DOI:10.1128/mr.55.2.303-315.1991 |
4. Aspeborg H, Coutinho PM, Wang Y, Brumer H 3rd, and Henrissat B. (2012). Evolution, substrate specificity and subfamily classification of glycoside hydrolase family 5 (GH5). BMC Evol Biol. 2012;12:186. DOI:10.1186/1471-2148-12-186 |
8. Henrissat B, Callebaut I, Fabrega S, Lehn P, Mornon JP, and Davies G. (1995). Conserved catalytic machinery and the prediction of a common fold for several families of glycosyl hydrolases. Proc Natl Acad Sci U S A. 1995;92(15):7090-4. DOI:10.1073/pnas.92.15.7090 |
11. 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 |
13. 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 |
14. 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 |
18. Cartmell A, Topakas E, Ducros VM, Suits MD, Davies GJ, and Gilbert HJ. (2008). The Cellvibrio japonicus mannanase CjMan26C displays a unique exo-mode of action that is conferred by subtle changes to the distal region of the active site. J Biol Chem. 2008;283(49):34403-13. DOI:10.1074/jbc.M804053200 |
24. 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