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Glycoside Hydrolase Family 6
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|Glycoside Hydrolase Family GH6|
|Active site residues||acid known, base debated|
|CAZy DB link|
Glycoside hydrolases of family GH6 cleave β-1,4 glycosidic bonds in cellulose / β-1,4-glucans. Only endoglucanase (EC 220.127.116.11) and cellobiohydrolase (EC 18.104.22.168) activity has been reported for the bacterial and eukaryotic members of this family. GH6 was one of the first glycoside hydrolase families classified by hydrophobic cluster analysis, and was previously known as "Cellulase Family B" [1, 2].
Kinetics and Mechanism
GH6 enzymes perform catalysis with inversion of anomeric stereochemistry, as first shown by NMR  on cellobiohydrolase II (CBH II; Cel6A) from the fungus Trichoderma reesei (a clonal derivative of Hypocrea jecorina ).
The first 3D structure of CBHII provided a strong clue as to the identification of the catalytic general 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)  as well as from all subsequent 3-D analyses of enzyme-ligand complexes (for example [6, 7, 8] ). The identification of the catalytic general 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 inverting mechanism. Thus, although there are mutagenesis / kinetic proposals for a base , 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 . On the basis of structure the residue most likely to act as the general base is Asp175 on the Trichoderma reesei Cel6A, although Asp401 may also play a role (see Table 1).
|Proposed role||CfCel6A (endo)||HiCel6A (exo)||HjCel6A (exo)||TfCel6A (endo)||TfCel6B (exo)|
|Increase in pKa acid/Catalytic base||Asp216||Asp180||Asp175||Asp79||Asp226|
|Catalytic base/substrate binding||Asp392||Asp405||Asp401||Asp265||Asp497|
The first crystal structures of cellobiohydrolases and endoglucanases from family GH6 revealed modified α/β barrel folds which, unlike the classical (β/α)8 "TIM" barrel has just seven β-strands forming the central β-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 GH6 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 . To this day the debate continues about the possibilities of loop conformational change in moderating the activity of cellobiohydrolases between exo and endo. Ståhlberg was perhaps the first to explicitly state that T. reesei "has no true exo-cellulases" . It is clear that there is no absolute steric demand for the exo activity of cellobiohydrolases; the enzymes have a viable "-3" subsite [12, 13], the loops of the cellobiohydrolases are clearly mobile and show multiple conformations (consistent with occasional opening to support endo-activity, and are also able to act on artificial substrates in which both ends have large appended groups (for example ).
The nature of how catalysis was achieved, and the conformational itinerary of catalysis was first provided by the Uppsala, Grenoble and Gent groups in 1999  was a trapped Michaelis complex of a thio-oligosaccharide was observed spanning the active centre with the -1 subsite sugar in 2SO conformation, which suggested a pathway around the 2,5B transition state conformation. Subsequent structural [7, 8] and modelling  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 .
- First sterochemistry determination
- Hypocrea jecorina cellobiohydrolase Cel6A by NMR .
- First general acid residue identification
- The role of Asp221 as the potential catalytic acid was first proposed on the basis of 3-D structure of the Hypocrea jecorina cellobiohydrolase CBHII / Cel6A . Enzyme kinetics of variants, in conjunction with leaving groups requiring provided strong confirmation .
- First general base residue identification
- The existence / identification of the catalytic base is less clear and current beliefs are that the water is deprotonated through a "solvent wire" through to one of the conserved aspartates 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 . The first endoglucanase in this family was the Thermomonospora fusca E2 enzyme (catalytic core) solved by the Wilson/Karplus groups
- Henrissat B, Claeyssens M, Tomme P, Lemesle L, and Mornon JP. (1989) Cellulase families revealed by hydrophobic cluster analysis. Gene. 81, 83-95. DOI:10.1016/0378-1119(89)90339-9 |
- 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. 55, 303-15.
- 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
- 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. 93, 7755-60. DOI:10.1073/pnas.93.15.7755 |
- 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. 34, 2220-4. DOI:10.1021/bi00007a016 |
- 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. 7, 1035-45. DOI:10.1016/s0969-2126(99)80171-3 |
- 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. 280, 20181-4. DOI:10.1074/jbc.C500142200 |
- 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. 58, 2201-4. DOI:10.1107/s0907444902017006 |
- 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. 124, 10015-24. DOI:10.1021/ja012659q |
- 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. 270, 4383-6. DOI:10.1074/jbc.270.9.4383 |
- Ståhlberg J, Johansson G, and Pettersson G. (1993) Trichoderma reesei has no true exo-cellulase: all intact and truncated cellulases produce new reducing end groups on cellulose. Biochim Biophys Acta. 1157, 107-13. DOI:10.1016/0304-4165(93)90085-m |
- Varrot A, Schülein M, and Davies GJ. (1999) Structural changes of the active site tunnel of Humicola insolens cellobiohydrolase, Cel6A, upon oligosaccharide binding. Biochemistry. 38, 8884-91. DOI:10.1021/bi9903998 |
- 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)., 946-7. DOI:10.1039/b301592k |
- Armand S, Drouillard S, Schülein M, Henrissat B, and Driguez H. (1997) A bifunctionalized fluorogenic tetrasaccharide as a substrate to study cellulases. J Biol Chem. 272, 2709-13. DOI:10.1074/jbc.272.5.2709 |
- Rouvinen J, Bergfors T, Teeri T, Knowles JK, and Jones TA. (1990) Three-dimensional structure of cellobiohydrolase II from Trichoderma reesei. Science. 249, 380-6. DOI:10.1126/science.2377893 |
- Spezio M, Wilson DB, and Karplus PA. (1993) Crystal structure of the catalytic domain of a thermophilic endocellulase. Biochemistry. 32, 9906-16. DOI:10.1021/bi00089a006 |