Glycoside Hydrolase Family 5

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

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

Substrate specificities

GH5 is one of the largest of all CAZy glycoside hydrolase families. Previously known as "cellulase family A" [1, 2], a variety of specificities are now known in 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. Following the reclassification of a number of GH5 members into GH30 [3], a GH5 subfamily classification has been presented that delineates members into a number of monospecific and polyspecific clades [4]. It should be noted that enzymes specifically targeting xylans are exclusively arabinoxylanases, and are found in subfamilies GH_21 [5] and GH_34 [6]. Likewise, the GH5 predominant endo-xyloglucanases can be only observed in the subfamily GH_4 [4, 7].

Kinetics and Mechanism

Family GH5 enzymes are retaining enzymes, as first shown by NMR [8] and follow a classical Koshland double-displacement mechanism.

Catalytic Residues

GH5 enzymes use the classical Koshland double-displacement mechanism and the two catalytic residues (catalytic nucleophile and general acid/base) are known to be glutamates found at the C-terminal ends of β-strands 4 (acid/base) and 7 (nucleophile) [9, 10].

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 [11]. 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) [9, 10].

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 [12] but also as a testbed for glycosidase inhibitor design as crystals often diffract to atomic resolution (for example [13]). The reaction coordinate work on the endoglucanases (thus working on gluco-configured substrates) shows that the substrate binds in ¹S₃ conformation with the glycosyl enzyme intermediate in ⁴C₁ chair conformation implying catalysis via a near ⁴H₃ half-chair transition state.

By analogy with family GH26 mannnanases [14] and family GH2 β-mannosidases [15] 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 [16]. An interesting dissection of mannan-degrading enzyme systems has been provided by work in the Gilbert group on the diverse GH5 and GH26 mannanases in Cellvibrio japonicus(see for example [17, 18, 19]).

The strict GH5_4 endo-xyloglucanases possess a wide active-site cleft that uniquely recognize the xylosyl substitutions of the polymeric substrate via discrete aromatic and hydrogen bond interactions. This is indeed contrary to the strict GH5 endo-glucanases which display a tight constriction in their active-site clefts leading to the apparent incapability of accommodating the highly branched xyloglucan substrate [20]. Notably, most of the GH5_4 endo-xyloglucanases cleave at the unbranched glucosyl units of the backbone due to the displayed constricted subsite -1 adjacent to the catalytic residues. Widening of that subsite, as observed in one of bovine rumen Gh5_4 endoxyloglucanase, can definitely confer the capability of cleavage at the substituted X unit leading to a different cleavage pattern [21, 22].

The GH5_34 enzymes target arabinoxylan through essential interactions with single arabinose substituents linked O3 to the xylose positioned in the active site -1 subsite [6, 23]. Very limited interactions with the xylan backbone is observed out with the -1 active site of the GH5_34 enzymes [23]. This explains why these glycoside hydrolases cleave highly decorated glucuronoarabinoxylans that are recalcitrant to cleavage by classical xylanases found in GH10 and GH11.

The Rhodococcal endoglycoceramidase II (EGC) in this family has found application in the chemoenzymatic synthesis of ceramide derivatives [24]. In 2007 the first 3-D structure of a highly specific GH5 xyloglucanase was reported [25]; 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 [8]. GH5 enzymes were also in the comprehensive Gebler study [26].
First catalytic nucleophile identification: Trapped using the classical Withers 2-fluoro method, here with 2',4'-dinitrophenyl 2-deoxy-2-fluoro-β-D-cellobioside, reported in Wang and Withers in 1993 [27].

First general acid/base identification: Several mutagenesis papers has alluded to the importance of a conserved glutamate- one that both Dominguez [28] and Ducros [29] 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) [28]. Subsequently, Ducros and colleagues reported the Clostridium cellulolyticum Cel5A also in 1995 [29].

References

All Medline abstracts: PubMed