Glycoside Hydrolase Family 5

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• Author: ^^^Gideon Davies^^^
• Responsible Curator: ^^^Gideon Davies^^^

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Glycoside Hydrolase Family GH5
Clan	GH-A
Mechanism	retaining
Active site residues	known
CAZy DB link
http://www.cazy.org/fam/GH5.html

Substrate specificities

GH5 is one of the largest of all CAZy glycoside hydrolase families. A variety of specificties are annotated to this family notably endoglucanase (cellulase) and endomannanase as well as exoglucanases, exomannanases and β-glucosidase and β-mannosidase. Other activities include 1,6 galactanase, 1,3 mannanase, 1,4 xylanase, endoglycoceramidase, as well as high specificity xyloglucanases. Family GH5 enzymes are found widely distributed across Archae, bacteria and eukaryotes, notably fungi and plants. There are no known human enzymes in GH5.

Kinetics and Mechanism

Family GH5 enzymes are retaining enzymes, as first shown by NMR [1] and follow a classical Koshland double-displacement mechanism.

Catalytic Residues

GH5 enzymes use the classical Koshland double-displacement mechanism and the two catalytic residues are known to be glutamates found at the C-terminal ends of β-strands 4 (acid/base) and 7 (nucleophile) [2, 3].

Three-dimensional structures

Three-dimensional structures are available for a very large number of Family GH5 enzymes, the first solved being that of the Clostridium thermocellum endoglucanase CelC [4]. As members of Clan GH-A they have a classical (α/β)₈ TIM barrel fold with the two key active site glutamic acids being approximately 200 residues apart in sequence and located at the C-terminal ends of β-strands 4 (acid/base) and 7 (nucleophile) [2, 3].

With so many 3D structures in this family, covering many specificities it is clearly hard to pick out notable structural papers. The Bacillus agaradhaerens Cel5A has been extensively studied, notably in the trapping of enzymatic snapshots along the reaction coordinate [5] but also as a testbed for glycosidase inhibitor design as crystals often diffract to atomic resolution (for example [6]). The reaction coordinate work on the endoglucanases (thus working on gluco-configured substrates) shows that the substrate binds in ¹S₃ conformation with the covalent intermediate in ⁴C₁ chair conformation implying catalysis via a ⁴H₃ half-chair (near) transition-state.

By analogy with family GH26 mannnanases [7] and family GH2 β-mannosidases [8] it would seem likely that GH5 mannanases use a different conformational itinerary to their glucosidase relatives, likely via a ¹S₅-^OS₂ glycosylation pathway and thus via a B_2,5 (near) transition-state although direct evidence in this family is limited [9]. An interesting dissection of mannan degrading enzyme systems has been provided by work in the Gilbert group on the diverse GH5 and 26 mannanases in Cellvibrio japonicus(see for example [10, 11, 12]).

The Rhodococcal endoglycoceramidase II (EGC) in this family has found application in the chemoenzymatic synthesis of ceramide derivatives [13]. In 2007 the first 3-D structure of a highly specific GH5 xyloglucanase was reported [14]; this enzyme makes kinetically productive interactions with both xylose and galactose substituents, as reflected in both a high specific activity on xyloglucan and the kinetics of a series of aryl glycosides.

Family Firsts

First sterochemistry determination

The curator believes this to be the ¹H NMR stereochemical determination for EGZ from Erwinia chrysanthemi [1]. GH5 enzymes were also in the comprehensive Gebler study [15].

First catalytic nucleophile identification

Trapped using the classical Withers 2-fluoro method, here with 2',4'-dinitrophenyl-2-deoxy-2-fluoro-beta-D-cellobioside, reported in Wang and Withers in 1993 [16].

First general acid/base residue identification

Several mutagenesis papers has alluded to the importance of a conserved glutamate- one that both Dominguez [17] and Ducros [18] correctly postulated as the catalytic acid when the 3-D structures were determined.

First 3-D structure

The first 3D structures in family GH5 was an endoglucanase (cellulase) from Clostridium thermocellum reported by the Alzari in 1995 (in a paper which also reported a family GH10 xylanase structure and the similarities between them) [17]. Subsequently, Ducros and colleagues reported the Clostridium cellulolyticum Cel5A also in 1995 [18].

References

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2. Henrissat B, Callebaut I, Fabrega S, Lehn P, Mornon JP, and Davies G. (1996). Conserved catalytic machinery and the prediction of a common fold for several families of glycosyl hydrolases. Proc Natl Acad Sci U S A. 1996;93(11):5674. DOI:10.1073/pnas.93.11.5674 |
3. Jenkins J, Lo Leggio L, Harris G, and Pickersgill R. (1995). Beta-glucosidase, beta-galactosidase, family A cellulases, family F xylanases and two barley glycanases form a superfamily of enzymes with 8-fold beta/alpha architecture and with two conserved glutamates near the carboxy-terminal ends of beta-strands four and seven. FEBS Lett. 1995;362(3):281-5. DOI:10.1016/0014-5793(95)00252-5 |
5. Davies GJ, Mackenzie L, Varrot A, Dauter M, Brzozowski AM, Schülein M, and Withers SG. (1998). Snapshots along an enzymatic reaction coordinate: analysis of a retaining beta-glycoside hydrolase. Biochemistry. 1998;37(34):11707-13. DOI:10.1021/bi981315i |