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

Content is to be added here.

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Table. 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. 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 [3] was a trapped Michaelis complex of a thio oligosaccharide was observed spanning the avtive centre with the -1 subsite sugar in ²S_O conformation which suggestad a pathway around the B_2,5 conformation. Subsequent structural and modelling [4]support for these proposals comes from similarly distorted species on other GH6 enzymes and from the observation of a "cellobiosyl isofagomine" in B_2,5 conformation.

Family Firsts

First sterochemistry determination

Hypocrea jecorina cellobiohydrolase Cel6A by NMR [1].

First general acid/base residue identification

Cite some reference here, with a short (1-2 senetence) explanation.

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 [5]. The first endoglucanase in this family was the Thermomonospora fusca E2 enzyme (catalytic core) solved by the Wilson/Karplus groups[6]

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. 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 |
4. 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 |
5. 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 |
6. 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 |