Glycoside Hydrolase Family 6

This page has been approved by the Responsible Curator as essentially complete. CAZypedia is a living document, so further improvement of this page is still possible. If you would like to suggest an addition or correction, please contact the page's Responsible Curator directly by e-mail.

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

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

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. 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 [3] on cellobiohydrolase II (CBH II; Cel6A) from the fungus Trichoderma reesei (a clonal derivative of Hypocrea jecorina [4]).

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) [5] 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 [5], 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 [9]. 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 [10]. 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" [11]. 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 [14]).

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 [6] 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 suggested a pathway around the ^2,5B transition state conformation. Subsequent structural [7, 8] and modelling [9] 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 [13].

Family Firsts

First sterochemistry determination: Hypocrea jecorina cellobiohydrolase Cel6A by NMR [3].
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 [15]. Enzyme kinetics of variants, in conjunction with leaving groups requiring provided strong confirmation [5].
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 [15]. The first endoglucanase in this family was the Thermomonospora fusca E2 enzyme (catalytic core) solved by the Wilson/Karplus groups[16]

References

All Medline abstracts: PubMed