CAZypedia needs your help!
We have many unassigned pages in need of Authors and Responsible Curators. See a page that's out-of-date and just needs a touch-up? - You are also welcome to become a CAZypedian. Here's how.
Scientists at all career stages, including students, are welcome to contribute.
Learn more about CAZypedia's misson here and in this article.
Totally new to the CAZy classification? Read this first.

Glycoside Hydrolase Family 7

From CAZypedia
Jump to navigation Jump to search
Approve icon-50px.png

This page has been approved by the 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.


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

  1. Henrissat B, Claeyssens M, Tomme P, Lemesle L, and Mornon JP. (1989). Cellulase families revealed by hydrophobic cluster analysis. Gene. 1989;81(1):83-95. DOI:10.1016/0378-1119(89)90339-9 | PubMed ID:2806912 [Henrissat1989]
  2. Gilkes NR, Henrissat B, Kilburn DG, Miller RC Jr, and Warren RA. (1991). Domains in microbial beta-1, 4-glycanases: sequence conservation, function, and enzyme families. Microbiol Rev. 1991;55(2):303-15. DOI:10.1128/mr.55.2.303-315.1991 | PubMed ID:1886523 [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. (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 | PubMed ID:8755548 [Kuhls1996]
  5. 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 | PubMed ID:8036495 [Divne1994]
  6. 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 | PubMed ID:8951380 [Stahlberg1996]
  7. 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 | PubMed ID:9761741 [Mackenzie1998]
  8. Ducros VM, Tarling CA, Zechel DL, Brzozowski AM, Frandsen TP, von Ossowski I, Schülein M, Withers SG, and Davies GJ. (2003). Anatomy of glycosynthesis: structure and kinetics of the Humicola insolens Cel7B E197A and E197S glycosynthase mutants. Chem Biol. 2003;10(7):619-28. DOI:10.1016/s1074-5521(03)00143-1 | PubMed ID:12890535 [Ducros2003]
  9. 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 | PubMed ID:9449766 [Klarskov1997]
  10. 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+ | PubMed ID:9153432 [Sulzenbacher1997]
  11. Viladot JL, de Ramon E, Durany O, and Planas A. (1998). 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;37(32):11332-42. DOI:10.1021/bi980586q | PubMed ID:9698381 [Viladot1998]
  12. 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 | PubMed ID:8952478 [Sulzenbacher1996]
  13. 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 | PubMed ID:9466911 [Divne1998]
  14. 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 | PubMed ID:15819888 [Ubhayasekera2005]
  15. 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 | PubMed ID:18499583 [Parkkinen2008]

All Medline abstracts: PubMed