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Glycoside Hydrolase Family 9

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Glycoside Hydrolase Family GH9
Clan GH-G
Mechanism inverting
Active site residues known/known
CAZy DB link

Substrate specificities

GH Family 9 is an inverting glycohydrolase family that mainly contains cellulases and is the second largest cellulase family. It contains mainly endoglucanases with a few processive endoglucanases. All of the processive endoglucanases contain a family 3c CBM rigidly attached to the C-terminus of the family 9 catalytic domain (cd) [1]. This domain is part of the active site and is essential for processivity [1]. CBM3c domains bind weakly to cellulose as they lack several of the conserved aromatic residues that are important for cellulose binding in family 3a and family 3b members [2]. All known plant cellulases belong to family 9, and most of the other members are eubacterial although there are two archael members and some fungal, earthworm, arthropod, chordate, echinoderma and molusk members. There are two subgroups in family 9, E1 which contains only cellulases from bacteria, including ones from both aerobes and anaeobes, and E2 which includes some bacterial and all nonbacterial cellulases [3]. An evolutionary study shows that the eucaryote members contain two monophyletic groups that are amcient; one including all animal members and the other including all plant members [4]. All known processive endoglucanase genes are in subgroup E1.

Kinetics and Mechanism

The processive endoglucanase,Cel9A from Thermobifda fusca, has high activity on bacterial cellulose and is the only cellulase tested that can degrade crystalline regions in bacterial cellulose by itself although it prefers amorphous regions [5]. A related cellulase in Clostridium phytofermentans, which is the only family 9 cellulase encoded in its genome, has been shown to be essential for cellulose degradation by this organism. This is the only case where a single cellulase has been shown to be essential for growth on cellulose [6].

Catalytic Residues

Content is to be added here. There is a conserved Glu residue that functions as the catalytic acid and two conserved Asp residues that bind the catalytic water, with one functioning as the catalytic base and mutation of the other also greatly reduces activity on all substrates [7].

Three-dimensional structures

Content is to be added here. All known family 9 cd structures have an ( a / a ) 6 barrel fold that contains an open active site cleft that contains at least six sugar binding subsites -4 to +2 [1, 8]. In processive endoglucanases the catalytic domain is joined to a family 3c CBM that is aligned with the active site cleft [1].

Family Firsts

First sterochemistry determination
The steriospecificity of three family 9 cellulases were all determined to be inverting by NMR [9].
First catalytic nucleophile identification
Asp 58 in T. fusca Cel9A was shown to be the catalytic nucleophile by site directed mutagenesis and azide rescue [10].
First general acid/base residue identification
Glu555 was shown to be the catalytic acid in C. thermocellum CelD by site directed mutagenesis [11].
First 3-D structure
The structure of endocellulase CelD from Clostridium thermocellum was determined by X-ray crystallography (PDB ID 1clc) [12].


  1. Sakon J, Irwin D, Wilson DB, and Karplus PA. (1997) Structure and mechanism of endo/exocellulase E4 from Thermomonospora fusca. Nat Struct Biol. 4, 810-8. PubMed ID:9334746 | HubMed [Sakon1997]
  2. Tormo J, Lamed R, Chirino AJ, Morag E, Bayer EA, Shoham Y, and Steitz TA. (1996) Crystal structure of a bacterial family-III cellulose-binding domain: a general mechanism for attachment to cellulose. EMBO J. 15, 5739-51. PubMed ID:8918451 | HubMed [Tormo1996]
  3. Tomme P, Warren RA, and Gilkes NR. (1995) Cellulose hydrolysis by bacteria and fungi. Adv Microb Physiol. 37, 1-81. PubMed ID:8540419 | HubMed [Tomme1995]
  4. Davison A and Blaxter M. (2005) Ancient origin of glycosyl hydrolase family 9 cellulase genes. Mol Biol Evol. 22, 1273-84. DOI:10.1093/molbev/msi107 | PubMed ID:15703240 | HubMed [Davison2005]
  5. Chen, Arthur J. Stipanovic, William T. Winter, David B. Wilson and Young-Jun Kim. Effect of digestion by pure cellulases on crystallinity and average chain length for bacterial and microcrystalline celluloses. Cellulose 2007: 14: 283-293.
  6. Tolonen AC, Chilaka AC, and Church GM. (2009) Targeted gene inactivation in Clostridium phytofermentans shows that cellulose degradation requires the family 9 hydrolase Cphy3367. Mol Microbiol. 74, 1300-13. DOI:10.1111/j.1365-2958.2009.06890.x | PubMed ID:19775243 | HubMed [Tolonen2009]
  7. Zhou W, Irwin DC, Escovar-Kousen J, and Wilson DB. (2004) Kinetic studies of Thermobifida fusca Cel9A active site mutant enzymes. Biochemistry. 43, 9655-63. DOI:10.1021/bi049394n | PubMed ID:15274620 | HubMed [Zhou2004]
  8. Guérin DM, Lascombe MB, Costabel M, Souchon H, Lamzin V, Béguin P, and Alzari PM. (2002) Atomic (0.94 A) resolution structure of an inverting glycosidase in complex with substrate. J Mol Biol. 316, 1061-9. DOI:10.1006/jmbi.2001.5404 | PubMed ID:11884144 | HubMed [Geurin2002]
  9. Gebler J, Gilkes NR, Claeyssens M, Wilson DB, Béguin P, Wakarchuk WW, Kilburn DG, Miller RC Jr, Warren RA, and Withers SG. (1992) Stereoselective hydrolysis catalyzed by related beta-1,4-glucanases and beta-1,4-xylanases. J Biol Chem. 267, 12559-61. PubMed ID:1618761 | HubMed [Gebler1992]
  10. Li Y, Irwin DC, and Wilson DB. (2007) Processivity, substrate binding, and mechanism of cellulose hydrolysis by Thermobifida fusca Cel9A. Appl Environ Microbiol. 73, 3165-72. DOI:10.1128/AEM.02960-06 | PubMed ID:17369336 | HubMed [Li2007]
  11. Lascombe, M.B., Souchon, H., Juy, M., Alzari, P.M. Three-Dimensional Structure of Endoglucanase D at 1.9 Angstroms Resolution. Deposited 1995, unpublished.
All Medline abstracts: PubMed | HubMed