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
http://www.cazy.org/fam/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).

Kinetics and Mechanism

Family 7 enzymes are retaining enzymes, as first shown by NMR [1] on Cellobiohydrolase I (CBH I; Cel7A) from the fungus Trichoderma reesei (a clonal derivative of Hypocrea jecorina [2]).

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 [3], based on the position of the residues relative to a o-iodo-benzyl-cellobioside molecule bound at the active site. It was supported by mutational studies with the same enzyme [4], 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]. 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 [6], and with Fusarium oxysporum Endoglucanase I (EG I, Cel7B) the residue was identified by X-ray crystallography [7]. 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 [5].

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) [3], and the first endoglucanase, Fusarium oxysporum EG I (Cel7B), in 1996 (PDB 1ovw) [8]. 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 bends 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:

i) 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) [8].

ii) Cellooligosaccharides bound in catalytically deficient mutants of Hypocrea jecorina Cel7A [4, 9], 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.

iii) 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 [10].

iv) Later studies of oligosaccharide binding in Melanocarpus albomyces Cel7B provide further insight into the flexibility of sugar binding within the tunnel of a cellobiohydrolase [11].

Family Firsts

First sterochemistry determination

Hypocrea jecorina cellobiohydrolase Cel7A by NMR [1].

First catalytic nucleophile identification

Suggested in Hypocrea jecorina cellobiohydrolase Cel7A [6] and Fusarium oxysporum endoglucanase Cel7B [7] via affinity labelling with 3,4-epoxybutyl-β-cellobioside. Verified in Humicola insolens Cel7B by trapping of a 2-deoxy-2-fluorocellotriosyl covalent enzyme intermediate [5].

First general acid/base residue identification

Cite some reference here, with a short (1-2 sentence) explanation.

First 3-D structure

First cellobiohydrolase was Hypocrea jecorina Cel7A (CBH I; PDB 1cel) [3]. First endo-1,4-β-glucanase was Endoglucanase I (EG I, Cel7B) from Fusarium oxysporum (PDB 1ovw) [8], both by X-ray crystallography.

References

1. 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

2. Kuhls K, Lieckfeldt E, Samuels GJ, Kovacs W, Meyer W, Petrini O, Gams W, Börner T, and Kubicek CP. (1996). 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;93(15):7755-60. DOI:10.1073/pnas.93.15.7755 |
3. Divne C, Ståhlberg J, Reinikainen T, Ruohonen L, Pettersson G, Knowles JK, Teeri TT, and Jones TA. (1994). The three-dimensional crystal structure of the catalytic core of cellobiohydrolase I from Trichoderma reesei. Science. 1994;265(5171):524-8. DOI:10.1126/science.8036495 |
4. Ståhlberg J, Divne C, Koivula A, Piens K, Claeyssens M, Teeri TT, and Jones TA. (1996). Activity studies and crystal structures of catalytically deficient mutants of cellobiohydrolase I from Trichoderma reesei. J Mol Biol. 1996;264(2):337-49. DOI:10.1006/jmbi.1996.0644 |
5. MacKenzie LF, Sulzenbacher G, Divne C, Jones TA, Wöldike HF, Schülein M, Withers SG, and Davies GJ. (1998). 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;335 ( Pt 2)(Pt 2):409-16. DOI:10.1042/bj3350409 |
6. Klarskov K, Piens K, Ståhlberg J, Høj PB, Beeumen JV, and Claeyssens M. (1997). 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;304(2):143-54. DOI:10.1016/s0008-6215(97)00215-2 |
7. Sulzenbacher G, Schülein M, and Davies GJ. (1997). 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;36(19):5902-11. DOI:10.1021/bi962963+ |
8. Sulzenbacher G, Driguez H, Henrissat B, Schülein M, and Davies GJ. (1996). 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;35(48):15280-7. DOI:10.1021/bi961946h |
9. Divne C, Ståhlberg J, Teeri TT, and Jones TA. (1998). 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;275(2):309-25. DOI:10.1006/jmbi.1997.1437 |
10. Ubhayasekera W, Muñoz IG, Vasella A, Ståhlberg J, and Mowbray SL. (2005). Structures of Phanerochaete chrysosporium Cel7D in complex with product and inhibitors. FEBS J. 2005;272(8):1952-64. DOI:10.1111/j.1742-4658.2005.04625.x |
11. Parkkinen T, Koivula A, Vehmaanperä J, and Rouvinen J. (2008). Crystal structures of Melanocarpus albomyces cellobiohydrolase Cel7B in complex with cello-oligomers show high flexibility in the substrate binding. Protein Sci. 2008;17(8):1383-94. DOI:10.1110/ps.034488.108 |