Glycoside Hydrolase Family 6

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• Author: ^^^Kathleen Piens^^^ and ^^^Gideon Davies^^^
• Responsible Curator: ^^^Gideon Davies^^^

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Glycoside Hydrolase Family GH6
Clan	none
Mechanism	inverting
Active site residues	acid known, base debated
CAZy DB link
http://www.cazy.org/fam/GH6.html

Substrate specificities

Glycoside hydrolases of family 6 cleave β-1,4 glycosidic bonds in cellulose/β-1,4-glucans. Only endoglucanase (EC 3.2.1.4) and cellobiohydrolase (EC 3.2.1.91) activity has been reported for both bacterial and eukaryotic members of this family.

Kinetics and Mechanism

Family 6 enzymes are inverting enzymes, as first shown by NMR [1] on Cellobiohydrolase II (CBH II; Cel6A) from the fungus Trichoderma reesei (a clonal derivative of Hypocrea jecorina [2]).

Catalytic Residues

The first 3-D structure of CBHII provided a strong clue as to the identification of the catalytic acid in the inverting mechanism (Asp 221 in the case of this Trichoderma reesei Cel6A enzyme. This assigment has withstood the tests of time with strong kinetic support (from kinetics as a function of leaving group ability for a series of enzyme variants) [3] as well as from all subsequent 3-D analyses of enzyme-ligand complexes (for example [4][5][6] ). The identification of the base is, however, far less clear. Simply put this is because there is no clear potential base within hydrogen-bonding distance of a water molecule that could act as the nucleophile in the inversion mechanism. Thus, although there are mutagenesis / kinetic proposals for a base [3], the current Zeitgeist is that the attacking water is deprotonated via a string of water molecules in what Sinnott has descibed as a "Grotthuss" mechanism; for which there is solvent kinetic isotope effect support [7]. On the basis of structure the residue most likely to act as the base is Asp175 on the Trichoderma reesei Cel6A although Asp392 may also play a role (see Table 1).

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Table 1. Putative catalytic residues of some representatives in GH family 6
(with biochemical characterization of wt and mutant enzymes).

Proposed role	CfCel6A (endo)	HiCel6A (exo)	HjCel6A (exo)	TfCel6A (endo)	TfCel6B (exo)
Substrate distortion	Tyr210	Tyr174	Tyr169	Tyr73	Tyr220
Increase in pKa acid/Catalytic base	Asp216	Asp180	Asp175	Asp79	Asp226
Proton network	Gly222?	Ser186	Ser181	Ser85	Ser232
Catalytic acid	Asp252	Asp226	Asp221	Asp117	Asp274
Catalytic base/substrate binding	Asp392	Asp405	Asp401	Asp265	Asp497

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Three-dimensional structures

The first crystal structures of cellobiohydrolases and endoglucanases from family GH7 revealed modified a/b barrel folds which, unlike the classical (b/a)₈ "TIM" barrel has just seven b-strands forming the central b-barrel. The CBHII structure revealed an active centre (see above) enclosed in a tunnel formed primarily by two surface loops. When, subsequently, the first endoglucanase from this family was solved the active center was observed in a long open groove. The comparison of these two structures thus provided the first insight into how endo or processive activity was modulated, through display of the active centre in a in an open grove, or loop-enclosed tunnel, respectively. In 1995 the UBC group were able to truncate the extended loops of a cellobiohydrolase resulting in an enzyme with more endo-activity [8]

The nature of how catalysis was achived, and the conformational itinerary of catalysis was first provided by the Uppsala, Grenoble and Gent groups in 1999 [4] was a trapped Michaelis complex of a thio oligosaccharide was observed spanning the active centre with the -1 subsite sugar in ²S_O conformation which suggestad a pathway around the ^2,5B conformation. Subsequent structural [5][6] and modelling [7]support for these proposals comes from similarly distorted species on other GH6 enzymes and from the observation of a "cellobiosyl isofagomine" in ^2,5B conformation [9].

Family Firsts

First sterochemistry determination

Hypocrea jecorina cellobiohydrolase Cel6A by NMR [1].

First general acid/base residue identification

The role of Asp221 as the potenbtial caralytic acid was first proposed on the basis of 3-D structure of the Hypocrea jecorina cellobiohydrolase CBHII / Cel6A [10]. Enzyme kinetics of variants, in conjunction with leaving groups requiring provided strong confirmation [3]. The existance / identification of the base is less clear and current beliefs are that the water is deprotonated via a solvent wire through to one of teh conserved aspratates near the active centre.

First 3-D structure

The catalytic core domain of the Trichoderma reesei (the organism now known as Hypocrea jecorina) cellobiohydrolase II by the Jones group [10]. The first endoglucanase in this family was the Thermomonospora fusca E2 enzyme (catalytic core) solved by the Wilson/Karplus groups[9]

References

1. Knowles, J.K.C., Lehtovaara, P., Murray, M. and Sinnott, M.L. (1988) Stereochemical course of the action of the cellobioside hydrolases I and II of Trichoderma reesei. J. Chem. Soc., Chem. Commun., 1988, 1401-1402. DOI: 10.1039/C39880001401

2. Kuhls K, Lieckfeldt E, Samuels GJ, Kovacs W, Meyer W, Petrini O, Gams W, Börner T, and Kubicek CP. (1996). Molecular evidence that the asexual industrial fungus Trichoderma reesei is a clonal derivative of the ascomycete Hypocrea jecorina. Proc Natl Acad Sci U S A. 1996;93(15):7755-60. DOI:10.1073/pnas.93.15.7755 |
3. Damude HG, Withers SG, Kilburn DG, Miller RC Jr, and Warren RA. (1995). Site-directed mutation of the putative catalytic residues of endoglucanase CenA from Cellulomonas fimi. Biochemistry. 1995;34(7):2220-4. DOI:10.1021/bi00007a016 |
4. Zou Jy, Kleywegt GJ, Ståhlberg J, Driguez H, Nerinckx W, Claeyssens M, Koivula A, Teeri TT, and Jones TA. (1999). Crystallographic evidence for substrate ring distortion and protein conformational changes during catalysis in cellobiohydrolase Ce16A from trichoderma reesei. Structure. 1999;7(9):1035-45. DOI:10.1016/s0969-2126(99)80171-3 |
5. Varrot A, Leydier S, Pell G, Macdonald JM, Stick RV, Henrissat B, Gilbert HJ, and Davies GJ. (2005). Mycobacterium tuberculosis strains possess functional cellulases. J Biol Chem. 2005;280(21):20181-4. DOI:10.1074/jbc.C500142200 |
6. Varrot A, Frandsen TP, Driguez H, and Davies GJ. (2002). Structure of the Humicola insolens cellobiohydrolase Cel6A D416A mutant in complex with a non-hydrolysable substrate analogue, methyl cellobiosyl-4-thio-beta-cellobioside, at 1.9 A. Acta Crystallogr D Biol Crystallogr. 2002;58(Pt 12):2201-4. DOI:10.1107/s0907444902017006 |
7. Koivula A, Ruohonen L, Wohlfahrt G, Reinikainen T, Teeri TT, Piens K, Claeyssens M, Weber M, Vasella A, Becker D, Sinnott ML, Zou JY, Kleywegt GJ, Szardenings M, Ståhlberg J, and Jones TA. (2002). The active site of cellobiohydrolase Cel6A from Trichoderma reesei: the roles of aspartic acids D221 and D175. J Am Chem Soc. 2002;124(34):10015-24. DOI:10.1021/ja012659q |
8. Meinke A, Damude HG, Tomme P, Kwan E, Kilburn DG, Miller RC Jr, Warren RA, and Gilkes NR. (1995). Enhancement of the endo-beta-1,4-glucanase activity of an exocellobiohydrolase by deletion of a surface loop. J Biol Chem. 1995;270(9):4383-6. DOI:10.1074/jbc.270.9.4383 |
9. Spezio M, Wilson DB, and Karplus PA. (1993). Crystal structure of the catalytic domain of a thermophilic endocellulase. Biochemistry. 1993;32(38):9906-16. DOI:10.1021/bi00089a006 |
10. Rouvinen J, Bergfors T, Teeri T, Knowles JK, and Jones TA. (1990). Three-dimensional structure of cellobiohydrolase II from Trichoderma reesei. Science. 1990;249(4967):380-6. DOI:10.1126/science.2377893 |
11. Varrot A, Macdonald J, Stick RV, Pell G, Gilbert HJ, and Davies GJ. (2003). Distortion of a cellobio-derived isofagomine highlights the potential conformational itinerary of inverting beta-glucosidases. Chem Commun (Camb). 2003(8):946-7. DOI:10.1039/b301592k |