Glycoside Hydrolase Family 7

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• Author: Jerry Stahlberg
• Responsible Curator: Jerry Stahlberg

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Glycoside Hydrolase Family 7
Clan	GH-B
Mechanism	retaining
Active site residues	known
CAZy DB link
https://www.cazy.org/GH7.html

Substrate specificities

Most glycoside hydrolases of family 7 cleave β-1,4 glycosidic bonds in cellulose/β-1,4-glucans. Several members also show activity on xylan. The substrate specificities found in GH7 are: endo-1,4-β-glucanase (EC 3.2.1.4), [reducing end-acting] cellobiohydrolase (EC 3.2.1.-), chitosanase (EC 3.2.1.132) and endo-1,3-1,4-β-glucanase (EC 3.2.1.73). GH7 was one of the first glycoside hydrolase families classified by hydrophobic cluster analysis, and was previously known as "Cellulase Family C" [1, 2].

Kinetics and Mechanism

Family 7 enzymes are retaining enzymes, as first shown by NMR analysis [3] on cellobiohydrolase I (CBH I; Cel7A) from the fungus Trichoderma reesei (a clonal derivative of Hypocrea jecorina [4]).

Catalytic Residues

In GH7 enzymes the catalytic residues are positioned close to each other in sequence in the consensus motif -Glu-X-Asp-X-X-Glu-, where the first Glu acts as catalytic nucleophile and the other Glu as general acid/base. This was proposed in the first 3-D structure publication, of Hypocrea jecorina Cel7A [5], based on the position of the residues relative to an o-iodo-benzyl-cellobioside molecule bound at the active site. It was supported by mutational studies with the same enzyme [6], which also showed that the Aspartate residue in the consensus motif is important for catalysis, and with Endoglucanase I (EG I, Cel7B) from Humicola insolens [7, 8]. The catalytic nucleophile was further supported by affinity labelling with 3,4-epoxybutyl-β-cellobioside; with Hypocrea jecorina Cel7A the identification was done by ESI-MS peptide mapping and sequencing [9], and with Fusarium oxysporum Endoglucanase I (EG I, Cel7B) the residue was identified by X-ray crystallography [10]. This was subsequently verified by trapping of a 2-deoxy-2-fluorocellotriosyl covalent enzyme intermediate in Humicola insolens Cel7B and identification of the labelled peptide by tandem MS [7]. The general acid/base has been inferred by homology to GH16, the other family in clan GH-B, where it has been verified by azide rescue of inactivated mutants of a Bacillus licheniformis 1,3-1,4-β-D-glucan 4-glucanohydrolase [11].

Three-dimensional structures

Three-dimensional structures are available for both endoglucanases and cellobiohydrolases of GH7. The first cellobiohydrolase structure, the catalytic module of Hypocrea jecorina Cel7A, was published in 1994 (CBH I; PDB 1cel) [5], and the first endoglucanase, Fusarium oxysporum EG I (Cel7B), in 1996 (PDB 1ovw) [12]. The proteins are built up around a β-jellyroll folded framework, in which two large anti-parallel β-sheets pack face-to-face to form a highly curved β-sandwich. The β-sandwich is further extended along both edges by several of the loops that connect the β-strands, resulting in a long (~50 Å) substrate-binding surface that runs perpendicular to the β-strands of the inner, concave β-sheet. A few short α-helical segments occur in some of the loops at the perifery of the structure. Endoglucanases have an open substrate binding cleft/groove, while in cellobiohydrolases some loops are further elongated and bend around the active site so that a more or less closed tunnel is formed through the enzyme. Further structural studies have provided detailed knowledge about catalytic mechanism and substrate binding in family 7. Some key studies include:

• A complex of Fusarium oxysporum EG1 (Cel7B) with a non-hydrolysable substrate analog (thio-cellopentaose) indicated that transition of the glucose residue at site -1 from a ⁴C₁ chair to a distorted ^1,4B boat conformation is reqiured prior to hydrolysis (PDB 1ovw) [12].
• Cellooligosaccharides bound in catalytically deficient mutants of Hypocrea jecorina Cel7A revealed 10 discrete glucosyl-binding subsites, -7 to +3, and allowed modelling of a productively bound cellulose chain along the entire tunnel of the enzyme [6, 13].
• The discovery of two discrete binding modes for cellobiose in the product sites +1/+2 in Hypocrea jecorina Cel7A and Phanerochaete chrysosporium Cel7D, indicated that hydrolysis of the glycosyl-enzyme intermediate may proceed without prior release of the cellobiose product, and suggests a product ejection mechanism during processive hydrolysis of cellulose [14].
• Later studies of oligosaccharide binding in Melanocarpus albomyces Cel7B provide further insight into the flexibility of sugar binding within the tunnel of a cellobiohydrolase [15].

Family Firsts

First sterochemistry determination

Hypocrea jecorina cellobiohydrolase Cel7A by NMR [3].

First catalytic nucleophile identification

Suggested in Hypocrea jecorina cellobiohydrolase Cel7A [9] and Fusarium oxysporum endoglucanase Cel7B [10] via affinity labelling with 3,4-epoxybutyl-β-cellobioside. Verified in Humicola insolens Cel7B by trapping of a covalent 2-deoxy-2-fluorocellotriosyl enzyme intermediate [7].

First general acid/base residue identification

Suggested by structural studies and mutation in Hypocrea jecorina Cel7A [5, 6, 13]. Verified in Bacillus licheniformis 1,3-1,4-β-D-glucan 4-glucanohydrolase of GH16 by azide rescue of inactivated mutants [11].

First 3-D structure

First cellobiohydrolase was Hypocrea jecorina Cel7A (CBH I; PDB 1cel) [5]. First endo-1,4-β-glucanase was Endoglucanase I (EG I; Cel7B) from Fusarium oxysporum (PDB 1ovw) [12], both by X-ray crystallography.

References

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3. 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