Glycoside Hydrolase Family 7
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, or using this form.
|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).
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 [5, 6]. 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-parallell 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 [4, 11].
- 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 [3, 4, 11]. 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.
- 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
- Kuhls K, Lieckfeldt E, Samuels GJ, Kovacs W, Meyer W, Petrini O, Gams W, Börner T, and Kubicek CP. Molecular evidence that the asexual industrial fungus Trichoderma reesei is a clonal derivative of the ascomycete Hypocrea jecorina. Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7755-60.
- Divne C, Ståhlberg J, Reinikainen T, Ruohonen L, Pettersson G, Knowles JK, Teeri TT, and Jones TA. The three-dimensional crystal structure of the catalytic core of cellobiohydrolase I from Trichoderma reesei. Science. 1994 Jul 22;265(5171):524-8.
- Ståhlberg J, Divne C, Koivula A, Piens K, Claeyssens M, Teeri TT, and Jones TA. Activity studies and crystal structures of catalytically deficient mutants of cellobiohydrolase I from Trichoderma reesei. J Mol Biol. 1996 Nov 29;264(2):337-49. DOI:10.1006/jmbi.1996.0644 |
- MacKenzie LF, Sulzenbacher G, Divne C, Jones TA, Wöldike HF, Schülein M, Withers SG, and Davies GJ. Crystal structure of the family 7 endoglucanase I (Cel7B) from Humicola insolens at 2.2 A resolution and identification of the catalytic nucleophile by trapping of the covalent glycosyl-enzyme intermediate. Biochem J. 1998 Oct 15;335 ( Pt 2):409-16.
- Ducros VM, Tarling CA, Zechel DL, Brzozowski AM, Frandsen TP, von Ossowski I, Schülein M, Withers SG, and Davies GJ. Anatomy of glycosynthesis: structure and kinetics of the Humicola insolens Cel7B E197A and E197S glycosynthase mutants. Chem Biol. 2003 Jul;10(7):619-28.
- Klarskov K, Piens K, Ståhlberg J, Høj PB, Beeumen JV, and Claeyssens M. Cellobiohydrolase I from Trichoderma reesei: identification of an active-site nucleophile and additional information on sequence including the glycosylation pattern of the core protein. Carbohydr Res. 1997 Nov 10;304(2):143-54.
- Sulzenbacher G, Schülein M, and Davies GJ. Structure of the endoglucanase I from Fusarium oxysporum: native, cellobiose, and 3,4-epoxybutyl beta-D-cellobioside-inhibited forms, at 2.3 A resolution. Biochemistry. 1997 May 13;36(19):5902-11. DOI:10.1021/bi962963+ |
- Viladot JL, de Ramon E, Durany O, and Planas A. Probing the mechanism of Bacillus 1,3-1,4-beta-D-glucan 4-glucanohydrolases by chemical rescue of inactive mutants at catalytically essential residues. Biochemistry. 1998 Aug 11;37(32):11332-42. DOI:10.1021/bi980586q |
- Sulzenbacher G, Driguez H, Henrissat B, Schülein M, and Davies GJ. Structure of the Fusarium oxysporum endoglucanase I with a nonhydrolyzable substrate analogue: substrate distortion gives rise to the preferred axial orientation for the leaving group. Biochemistry. 1996 Dec 3;35(48):15280-7. DOI:10.1021/bi961946h |
- Divne C, Ståhlberg J, Teeri TT, and Jones TA. High-resolution crystal structures reveal how a cellulose chain is bound in the 50 A long tunnel of cellobiohydrolase I from Trichoderma reesei. J Mol Biol. 1998 Jan 16;275(2):309-25. DOI:10.1006/jmbi.1997.1437 |
- Ubhayasekera W, Muñoz IG, Vasella A, Ståhlberg J, and Mowbray SL. Structures of Phanerochaete chrysosporium Cel7D in complex with product and inhibitors. FEBS J. 2005 Apr;272(8):1952-64. DOI:10.1111/j.1742-4658.2005.04625.x |
- Parkkinen T, Koivula A, Vehmaanperä J, and Rouvinen J. Crystal structures of Melanocarpus albomyces cellobiohydrolase Cel7B in complex with cello-oligomers show high flexibility in the substrate binding. Protein Sci. 2008 Aug;17(8):1383-94. DOI:10.1110/ps.034488.108 |