Glycoside Hydrolase Family 9

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• Authors: David Wilson and Breeanna Urbanowicz
• Responsible Curator: David Wilson

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Glycoside Hydrolase Family GH9
Clan	not assigned
Mechanism	inverting
Active site residues	known/known
CAZy DB link
https://www.cazy.org/GH9.html

Substrate specificities

Members of family GH9 are mainly cellulases (EC 3.2.1.4), including primarily endo-glucanases and a few processive endo-glucanases. Indeed, as one of the first glycoside hydrolase families classified by hydrophobic cluster analysis, GH9 was previously known as "Cellulase Family E" [1, 2]. More recently, certain GH9 members from Clostridia [3] and Bacteroides [4, 5] have been shown to be endo-xyloglucanases (EC 3.2.1.151) or mixed-linkage endo-glucanases (EC 3.2.1.73). Exo-beta-glucosaminidases (EC 3.2.1.165) are also found in this family [6, 7].

All of the processive endoglucanases contain a family 3c CBM rigidly attached to the C-terminus of the GH9 catalytic domain (cd) [8]. This domain is part of the active site and is essential for processivity [8]. 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 [9]. All known plant cellulases belong to GH9, 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 GH9, E1 which contains only cellulases from bacteria, including ones from both aerobes and anaeobes, and E2 which includes some bacterial and all nonbacterial cellulases [10]. An evolutionary study shows that the eucaryote members contain two monophyletic groups that are ancient; one including all animal members and the other including all plant members [11]. All known processive endoglucanase genes are in subgroup E1. Most plant GH9 enzymes studied to date are endoglucanases ("cellulases", EC 3.2.1.4) with little or no activity on crystalline cellulose, but with discernible activity on soluble cellulose derivatives, including carboxymethyl cellulose (CMC), phosphoric acid swollen non-crystalline cellulose, and numerous plant polysaccharides including xylan, 1,3-1,4-ß-glucan, xyloglucan, and glucomannan [12, 13, 14, 15, 16]. Due to their ubiquity and large numbers, the phylogeny of plant GH9 enzymes has been further sub-divided into three classes [17], which are described in detail on the plant GH9 endoglucanase subpage.

Kinetics and Mechanism

GH9 enzymes operate with inversion of anomeric stereochemistry. 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 [18]. 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 [19].

Catalytic Residues

There is a conserved Glu residue that functions as a catalytic general acid and two conserved Asp residues that bind the catalytic water, with one functioning as the catalytic general base; mutation of the other also greatly reduces activity on all substrates [20]. Mutation of the conserved Glu to Ala, Gly or Gln reduced activity to less than >0.5% of WT on all forms of cellulose but the Ala and Gly mutant enzymes had higher than WT activity on dinitrophenyl-cellobioside which has a good leaving group, proving that this residue functions as the catalytic acid [20]. Mutation of either of two conserved Asp residues that bound the catalytic water to Ala or Asn reduced activity to less then 2% of WT on all cellulosic substrates. However, only one of the Ala mutant enzymes showed azide rescue proving that it was the actual catalytic base [21].

Three-dimensional structures

All reported GH9 catalytic domain 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 [5, 8, 22]. In processive endoglucanases the catalytic domain is joined to a family 3c carbohydrate-binding module that is aligned with the active site cleft [8].

Family Firsts

First stereochemistry determination

The stereospecificity of three family 9 cellulases were all determined to be inverting by NMR [23].

First general base identification

Asp 58 in T. fusca Cel9A was shown to be the general base by site directed mutagenesis and azide rescue [21].

First general acid residue identification

Glu555 was shown to be the catalytic acid in C. thermocellum CelD by site directed mutagenesis [24].

First 3-D structure

The structure of endocellulase CelD from Clostridium thermocellum was determined by X-ray crystallography (PDB ID 1clc) [25].

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 |
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 |
3. Ravachol J, de Philip P, Borne R, Mansuelle P, Maté MJ, Perret S, and Fierobe HP. (2016). Mechanisms involved in xyloglucan catabolism by the cellulosome-producing bacterium Ruminiclostridium cellulolyticum. Sci Rep. 2016;6:22770. DOI:10.1038/srep22770 |
4. Larsbrink J, Rogers TE, Hemsworth GR, McKee LS, Tauzin AS, Spadiut O, Klinter S, Pudlo NA, Urs K, Koropatkin NM, Creagh AL, Haynes CA, Kelly AG, Cederholm SN, Davies GJ, Martens EC, and Brumer H. (2014). A discrete genetic locus confers xyloglucan metabolism in select human gut Bacteroidetes. Nature. 2014;506(7489):498-502. DOI:10.1038/nature12907 |
5. Foley MH, Déjean G, Hemsworth GR, Davies GJ, Brumer H, and Koropatkin NM. (2019). A Cell-Surface GH9 Endo-Glucanase Coordinates with Surface Glycan-Binding Proteins to Mediate Xyloglucan Uptake in the Gut Symbiont Bacteroides ovatus. J Mol Biol. 2019;431(5):981-995. DOI:10.1016/j.jmb.2019.01.008 |
6. Honda Y, Arai S, Suzuki K, Kitaoka M, and Fushinobu S. (2016). The crystal structure of an inverting glycoside hydrolase family 9 exo-β-D-glucosaminidase and the design of glycosynthase. Biochem J. 2016;473(4):463-72. DOI:10.1042/BJ20150966 |
7. Wu L and Davies GJ. (2018). Structure of the GH9 glucosidase/glucosaminidase from Vibrio cholerae. Acta Crystallogr F Struct Biol Commun. 2018;74(Pt 8):512-523. DOI:10.1107/S2053230X18011019 |
8. Sakon J, Irwin D, Wilson DB, and Karplus PA. (1997). Structure and mechanism of endo/exocellulase E4 from Thermomonospora fusca. Nat Struct Biol. 1997;4(10):810-8. DOI:10.1038/nsb1097-810 |
9. 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. 1996;15(21):5739-51. | Google Books | Open Library
10. Tomme P, Warren RA, and Gilkes NR. (1995). Cellulose hydrolysis by bacteria and fungi. Adv Microb Physiol. 1995;37:1-81. DOI:10.1016/s0065-2911(08)60143-5 |
11. Davison A and Blaxter M. (2005). Ancient origin of glycosyl hydrolase family 9 cellulase genes. Mol Biol Evol. 2005;22(5):1273-84. DOI:10.1093/molbev/msi107 |
12. Master ER, Rudsander UJ, Zhou W, Henriksson H, Divne C, Denman S, Wilson DB, and Teeri TT. (2004). Recombinant expression and enzymatic characterization of PttCel9A, a KOR homologue from Populus tremula x tremuloides. Biochemistry. 2004;43(31):10080-9. DOI:10.1021/bi049453x |
13. Yoshida K and Komae K. (2006). A rice family 9 glycoside hydrolase isozyme with broad substrate specificity for hemicelluloses in type II cell walls. Plant Cell Physiol. 2006;47(11):1541-54. DOI:10.1093/pcp/pcl020 |
14. Ohmiya Y, Samejima M, Shiroishi M, Amano Y, Kanda T, Sakai F, and Hayashi T. (2000). Evidence that endo-1,4-beta-glucanases act on cellulose in suspension-cultured poplar cells. Plant J. 2000;24(2):147-58. DOI:10.1046/j.1365-313x.2000.00860.x |
15. Woolley LC, James DJ, and Manning K. (2001). Purification and properties of an endo-beta-1,4-glucanase from strawberry and down-regulation of the corresponding gene, cel1. Planta. 2001;214(1):11-21. DOI:10.1007/s004250100577 |
16. Urbanowicz BR, Catalá C, Irwin D, Wilson DB, Ripoll DR, and Rose JK. (2007). A tomato endo-beta-1,4-glucanase, SlCel9C1, represents a distinct subclass with a new family of carbohydrate binding modules (CBM49). J Biol Chem. 2007;282(16):12066-74. DOI:10.1074/jbc.M607925200 |
17. Urbanowicz BR, Bennett AB, Del Campillo E, Catalá C, Hayashi T, Henrissat B, Höfte H, McQueen-Mason SJ, Patterson SE, Shoseyov O, Teeri TT, and Rose JK. (2007). Structural organization and a standardized nomenclature for plant endo-1,4-beta-glucanases (cellulases) of glycosyl hydrolase family 9. Plant Physiol. 2007;144(4):1693-6. DOI:10.1104/pp.107.102574 |

18. 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.

19. 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. 2009;74(6):1300-13. DOI:10.1111/j.1365-2958.2009.06890.x |
20. Zhou W, Irwin DC, Escovar-Kousen J, and Wilson DB. (2004). Kinetic studies of Thermobifida fusca Cel9A active site mutant enzymes. Biochemistry. 2004;43(30):9655-63. DOI:10.1021/bi049394n |
21. Li Y, Irwin DC, and Wilson DB. (2007). Processivity, substrate binding, and mechanism of cellulose hydrolysis by Thermobifida fusca Cel9A. Appl Environ Microbiol. 2007;73(10):3165-72. DOI:10.1128/AEM.02960-06 |
22. 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. 2002;316(5):1061-9. DOI:10.1006/jmbi.2001.5404 |
23. 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. 1992;267(18):12559-61. | Google Books | Open Library
24. Chauvaux S, Béguin P, and Aubert JP. (1992). Site-directed mutagenesis of essential carboxylic residues in Clostridium thermocellum endoglucanase CelD. J Biol Chem. 1992;267(7):4472-8. | Google Books | Open Library

25. Lascombe, M.B., Souchon, H., Juy, M., Alzari, P.M. Three-Dimensional Structure of Endoglucanase D at 1.9 Angstroms Resolution. Deposited 1995, unpublished. PDB ID 1clc