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Glycoside Hydrolase Family 6

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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/GH6.html

Contents

Substrate specificities

Glycoside hydrolases of family GH6 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 the bacterial and eukaryotic members of this family.

Kinetics and Mechanism

GH6 enzymes perform catalysis with inversion of anomeric stereochemistry, 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 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) [3] as well as from all subsequent 3-D analyses of enzyme-ligand complexes (for example [4, 5, 6] ). 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 [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 general base is Asp175 on the Trichoderma reesei Cel6A, although Asp401 may also play a role (see Table 1).



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


Three-dimensional structures

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 [8]. 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" [9]. It is clear that there is no absolute steric demand for the exo activity of cellobiohydrolases; the enzymes have a viable "-3" subsite [10, 11], 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 [12]).

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 2SO conformation, which suggested a pathway around the 2,5B transition state 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 [11].

Family Firsts

First sterochemistry determination
Hypocrea jecorina cellobiohydrolase Cel6A by NMR [1].
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 [13]. Enzyme kinetics of variants, in conjunction with leaving groups requiring provided strong confirmation [3].
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 [13]. The first endoglucanase in this family was the Thermomonospora fusca E2 enzyme (catalytic core) solved by the Wilson/Karplus groups[14]

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 [Knowles1988]
  2. Kuhls K, Lieckfeldt E, Samuels GJ, Kovacs W, Meyer W, Petrini O, Gams W, Börner T, and Kubicek CP. 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 Jul 23;93(15):7755-60. PubMed ID:8755548 | HubMed [Kuhls1996]
  3. Damude HG, Withers SG, Kilburn DG, Miller RC Jr, and Warren RA. Site-directed mutation of the putative catalytic residues of endoglucanase CenA from Cellulomonas fimi. Biochemistry. 1995 Feb 21;34(7):2220-4. PubMed ID:7857933 | HubMed [Damude1995]
  4. Zou Jy, Kleywegt GJ, Ståhlberg J, Driguez H, Nerinckx W, Claeyssens M, Koivula A, Teeri TT, and Jones TA. Crystallographic evidence for substrate ring distortion and protein conformational changes during catalysis in cellobiohydrolase Ce16A from trichoderma reesei. Structure. 1999 Sep 15;7(9):1035-45. PubMed ID:10508787 | HubMed [Zou1999]
  5. Varrot A, Leydier S, Pell G, Macdonald JM, Stick RV, Henrissat B, Gilbert HJ, and Davies GJ. Mycobacterium tuberculosis strains possess functional cellulases. J Biol Chem. 2005 May 27;280(21):20181-4. DOI:10.1074/jbc.C500142200 | PubMed ID:15824123 | HubMed [Varrot2005]
  6. Varrot A, Frandsen TP, Driguez H, and Davies GJ. 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 Dec;58(Pt 12):2201-4. PubMed ID:12454501 | HubMed [Varrot2002]
  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. The active site of cellobiohydrolase Cel6A from Trichoderma reesei: the roles of aspartic acids D221 and D175. J Am Chem Soc. 2002 Aug 28;124(34):10015-24. PubMed ID:12188666 | HubMed [Koivula2002]
  8. Meinke A, Damude HG, Tomme P, Kwan E, Kilburn DG, Miller RC Jr, Warren RA, and Gilkes NR. Enhancement of the endo-beta-1,4-glucanase activity of an exocellobiohydrolase by deletion of a surface loop. J Biol Chem. 1995 Mar 3;270(9):4383-6. PubMed ID:7876202 | HubMed [Meinke1995]
  9. Ståhlberg J, Johansson G, and Pettersson G. Trichoderma reesei has no true exo-cellulase: all intact and truncated cellulases produce new reducing end groups on cellulose. Biochim Biophys Acta. 1993 May 7;1157(1):107-13. PubMed ID:8499476 | HubMed [Stberg1993]
  10. Varrot A, Schülein M, and Davies GJ. Structural changes of the active site tunnel of Humicola insolens cellobiohydrolase, Cel6A, upon oligosaccharide binding. Biochemistry. 1999 Jul 13;38(28):8884-91. DOI:10.1021/bi9903998 | PubMed ID:10413461 | HubMed [Varrot1999]
  11. Varrot A, Macdonald J, Stick RV, Pell G, Gilbert HJ, and Davies GJ. Distortion of a cellobio-derived isofagomine highlights the potential conformational itinerary of inverting beta-glucosidases. Chem Commun (Camb). 2003 Apr 21(8):946-7. PubMed ID:12744312 | HubMed [Varrot2003]
  12. Armand S, Drouillard S, Schülein M, Henrissat B, and Driguez H. A bifunctionalized fluorogenic tetrasaccharide as a substrate to study cellulases. J Biol Chem. 1997 Jan 31;272(5):2709-13. PubMed ID:9006908 | HubMed [Armand1997]
  13. Rouvinen J, Bergfors T, Teeri T, Knowles JK, and Jones TA. Three-dimensional structure of cellobiohydrolase II from Trichoderma reesei. Science. 1990 Jul 27;249(4967):380-6. PubMed ID:2377893 | HubMed [Rouvinen1990]
  14. Spezio M, Wilson DB, and Karplus PA. Crystal structure of the catalytic domain of a thermophilic endocellulase. Biochemistry. 1993 Sep 28;32(38):9906-16. PubMed ID:8399160 | HubMed [Spezio1993]
All Medline abstracts: PubMed | HubMed
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