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Glycoside Hydrolase Family 7

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Glycoside Hydrolase Family 7
Clan GH-B
Mechanism retaining
Active site residues known
CAZy DB link

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, [reducing end-acting] cellobiohydrolase (EC 3.2.1.-), chitosanase (EC and endo-1,3-1,4-β-glucanase (EC 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-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) [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.


  1. Henrissat B, Claeyssens M, Tomme P, Lemesle L, and Mornon JP. Cellulase families revealed by hydrophobic cluster analysis. Gene. 1989 Sep 1;81(1):83-95. PubMed ID:2806912 | HubMed [Henrissat1989]
  2. Gilkes NR, Henrissat B, Kilburn DG, Miller RC Jr, and Warren RA. Domains in microbial beta-1, 4-glycanases: sequence conservation, function, and enzyme families. Microbiol Rev. 1991 Jun;55(2):303-15. PubMed ID:1886523 | HubMed [Gilkes1991]
  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 [Knowles1988]
  4. 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. PubMed ID:8755548 | HubMed [Kuhls1996]
  5. 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. PubMed ID:8036495 | HubMed [Divne1994]
  6. 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 | PubMed ID:8951380 | HubMed [Stahlberg1996]
  7. 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. PubMed ID:9761741 | HubMed [Mackenzie1998]
  8. 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. PubMed ID:12890535 | HubMed [Ducros2003]
  9. 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. PubMed ID:9449766 | HubMed [Klarskov1997]
  10. 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+ | PubMed ID:9153432 | HubMed [Sulzenbacher1997]
  11. 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 | PubMed ID:9698381 | HubMed [Viladot1998]
  12. 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 | PubMed ID:8952478 | HubMed [Sulzenbacher1996]
  13. 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 | PubMed ID:9466911 | HubMed [Divne1998]
  14. 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 | PubMed ID:15819888 | HubMed [Ubhayasekera2005]
  15. 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 | PubMed ID:18499583 | HubMed [Parkkinen2008]
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