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Glycoside Hydrolase Family 7
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|Glycoside Hydrolase Family 7|
|Active site residues||known|
|CAZy DB link|
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 22.214.171.124), [reducing end-acting] cellobiohydrolase (EC 3.2.1.-), chitosanase (EC 126.96.36.199) and endo-1,3-1,4-β-glucanase (EC 188.8.131.52). 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
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 , 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 , 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 , and with Fusarium oxysporum Endoglucanase I (EG I, Cel7B) the residue was identified by X-ray crystallography . 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 . 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 .
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) , and the first endoglucanase, Fusarium oxysporum EG I (Cel7B), in 1996 (PDB 1ovw) . 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) .
- 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 .
- Later studies of oligosaccharide binding in Melanocarpus albomyces Cel7B provide further insight into the flexibility of sugar binding within the tunnel of a cellobiohydrolase .
- First sterochemistry determination
- Hypocrea jecorina cellobiohydrolase Cel7A by NMR .
- First catalytic nucleophile identification
- Suggested in Hypocrea jecorina cellobiohydrolase Cel7A  and Fusarium oxysporum endoglucanase Cel7B  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 .
- 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 .
- First 3-D structure
- First cellobiohydrolase was Hypocrea jecorina Cel7A (CBH I; PDB 1cel) . First endo-1,4-β-glucanase was Endoglucanase I (EG I; Cel7B) from Fusarium oxysporum (PDB 1ovw) , both by X-ray crystallography.
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- 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
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