Glycoside Hydrolase Family 5

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• Author: ^^^Gideon Davies^^^ and ^^^Mohamed Attia^^^
• 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/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]. Likewise, the GH5 predominant endo-xyloglucanases can be only observed in the subfamily GH_4 [4, 7].

Kinetics and Mechanism

Family GH5 enzymes are retaining enzymes, as first shown by NMR [8] 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) [9, 10].

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 [11]. 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) [9, 10].

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 [12] but also as a testbed for glycosidase inhibitor design as crystals often diffract to atomic resolution (for example [13]). 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 [14] and family GH2 β-mannosidases [15] 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 [16]. 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 [17, 18, 19]).

The strict GH5_4 endo-xyloglucanases possess a wide active-site cleft that uniquely recognize the xylosyl substitutions of the polymeric substrate via discrete aromatic and hydrogen bond interactions. This is indeed contrary to the strict GH5 endo-glucanases which display a tight constriction in their active-site clefts leading to the apparent incapability of accommodating the highly branched xyloglucan substrate [20]. Notably, most of the GH5_4 endo-xyloglucanases cleave at the unbranched glucosyl units of the backbone due to the displayed constricted subsite -1 adjacent to the catalytic residues. Widening of that subsite, as observed in one of bovine rumen GH5_4 endo-xyloglucanase, can clearly confer the ability to cleave at the substituted X unit leading to a different cleavage pattern [21]. Although GH5_4 endo-xyloglucanases share amino acid identity as low as 30%, they display high substrate specificity towards xyloglucan which can be ultimately attributed to the high conservation of the amino acid residues interacting with the xyloglucan substrate in the active site cleft [22].

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, 23]. Very limited interactions with the xylan backbone is observed out with the -1 active site of the GH5_34 enzymes [23]. 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 [24]. In 2007 the first 3-D structure of a highly specific GH5 xyloglucanase was reported [25]; 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 [8]. GH5 enzymes were also in the comprehensive Gebler study [26].

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 [27].

First general acid/base identification

Several mutagenesis papers has alluded to the importance of a conserved glutamate- one that both Dominguez [28] and Ducros [29] 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) [28]. Subsequently, Ducros and colleagues reported the Clostridium cellulolyticum Cel5A also in 1995 [29].

References

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 |
3. St John FJ, González JM, and Pozharski E. (2010). Consolidation of glycosyl hydrolase family 30: a dual domain 4/7 hydrolase family consisting of two structurally distinct groups. FEBS Lett. 2010;584(21):4435-41. DOI:10.1016/j.febslet.2010.09.051 |
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 |
5. Dodd D, Moon YH, Swaminathan K, Mackie RI, and Cann IK. (2010). Transcriptomic analyses of xylan degradation by Prevotella bryantii and insights into energy acquisition by xylanolytic bacteroidetes. J Biol Chem. 2010;285(39):30261-73. DOI:10.1074/jbc.M110.141788 |
6. Correia MA, Mazumder K, Brás JL, Firbank SJ, Zhu Y, Lewis RJ, York WS, Fontes CM, and Gilbert HJ. (2011). Structure and function of an arabinoxylan-specific xylanase. J Biol Chem. 2011;286(25):22510-20. DOI:10.1074/jbc.M110.217315 |
7. Attia MA and Brumer H. (2016). Recent structural insights into the enzymology of the ubiquitous plant cell wall glycan xyloglucan. Curr Opin Struct Biol. 2016;40:43-53. DOI:10.1016/j.sbi.2016.07.005 |
8. 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 |
9. 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 |
10. 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 |
12. 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. 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 |
14. 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 |
15. 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 |
16. 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 |
17. Hogg D, Pell G, Dupree P, Goubet F, Martín-Orúe SM, Armand S, and Gilbert HJ. (2003). The modular architecture of Cellvibrio japonicus mannanases in glycoside hydrolase families 5 and 26 points to differences in their role in mannan degradation. Biochem J. 2003;371(Pt 3):1027-43. DOI:10.1042/BJ20021860 |
18. Tailford LE, Ducros VM, Flint JE, Roberts SM, Morland C, Zechel DL, Smith N, Bjørnvad ME, Borchert TV, Wilson KS, Davies GJ, and Gilbert HJ. (2009). Understanding how diverse beta-mannanases recognize heterogeneous substrates. Biochemistry. 2009;48(29):7009-18. DOI:10.1021/bi900515d |
19. 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 |
20. Naas AE, MacKenzie AK, Dalhus B, Eijsink VGH, and Pope PB. (2015). Structural Features of a Bacteroidetes-Affiliated Cellulase Linked with a Polysaccharide Utilization Locus. Sci Rep. 2015;5:11666. DOI:10.1038/srep11666 |
21. Dos Santos CR, Cordeiro RL, Wong DW, and Murakami MT. (2015). Structural basis for xyloglucan specificity and α-d-Xylp(1 → 6)-D-Glcp recognition at the -1 subsite within the GH5 family. Biochemistry. 2015;54(10):1930-42. DOI:10.1021/acs.biochem.5b00011 |
22. Attia MA, Nelson CE, Offen WA, Jain N, Davies GJ, Gardner JG, and Brumer H. (2018). In vitro and in vivo characterization of three Cellvibrio japonicus glycoside hydrolase family 5 members reveals potent xyloglucan backbone-cleaving functions. Biotechnol Biofuels. 2018;11:45. DOI:10.1186/s13068-018-1039-6 |
23. Labourel A, Crouch LI, Brás JL, Jackson A, Rogowski A, Gray J, Yadav MP, Henrissat B, Fontes CM, Gilbert HJ, Najmudin S, Baslé A, and Cuskin F. (2016). The Mechanism by Which Arabinoxylanases Can Recognize Highly Decorated Xylans. J Biol Chem. 2016;291(42):22149-22159. DOI:10.1074/jbc.M116.743948 |
24. 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 |
25. 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 |
26. 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
27. 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
28. 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 |
29. 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 |