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;First 3-D structure: The structure of endocellulase CelD from ''Clostridium thermocellum'' was determined by X-ray crystallography (PDB ID [{{PDBlink}}1clc 1clc]) <cite>Lascombe1995</cite>.
 
;First 3-D structure: The structure of endocellulase CelD from ''Clostridium thermocellum'' was determined by X-ray crystallography (PDB ID [{{PDBlink}}1clc 1clc]) <cite>Lascombe1995</cite>.
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== GH9 Enzymes Found in Plants ==
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  12.00    Normal  0          false  false  false    EN-US  X-NONE  X-NONE                                                                                                                                                                                                                                                                                                                                                                      12.00    Normal  0          false  false  false    EN-US  X-NONE  X-NONE                                                                                                                                                                                                                                                                                                                                                                 
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Early reports described the existence of plant ‘cellulases’ or Egases (e.g. Hall, 1963).  Subsequently, cellulases have been shown to be associated with plant cell wall restructuring during cell expansion, the wall disassembly that accompanies processes such as fruit ripening and abscission (reviewed in del Campillo, 1999; Rose and Bennett, 1999; '''Mølhøj et al., 2002''') and cellulose biosynthesis (Nicol et al., 1998; Lane et al., 2001; Sato et al., 2001). The amino acid sequences of the first plant ‘cellulases’/endo-b-1,4-glucanases revealed that these enzymes belong to the CAZy family GH9 glycoside hydrolases (Henrissat 1991). Plant GH9 enzymes are expected to affect cell wall strength and extendibility.
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Most plant ‘cellulases’ studied to date are  endoglucanases (EC 3.2.1.4) with low 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-b-glucan, xyloglucan, and glucomannan (Master et al., 2004; Yoshida and Komae 2006; Hayashi et al., 1984; Ohmiya et al., 2000; Woolley et al., 2001; Urbanowicz et al., 2007). The inability of plant “cellulases” to hydrolyze crystaline cellulose is distinct from microbial cellulases, whose modular structure and synergistic action with  other enzymes facilitates effective degradation of crystalline cellulose.  ''In muro'', the substrates of microbial cellulases likely include xyloglucan, glucomannans, and non-crystalline cellulose, especially amorphous regions of cellulose where the microfibrils may be interwoven with xyloglucan.
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In the model plant'' Arabidopsis thaliana'', 25 different GH9 coding regions have been identified. Phylogenic analysis of the deduced amino acid sequences group the proteins into nine classes or three subfamilies ('''Mølhøj''' et al., 2002; Libertini et al., 2004; Urbanowicz et al., 2007a). Three distinct types of GH9 proteins are present in plants. Class A proteins are membrane-anchored, Class B proteins are secreted, and Class C proteins are also secreted but contain a family 49 carbohydrate binding module (CBM49) (Urbanowicz et al., 2007a). Class A plant EGases have been reported to lack tryptophans corresponding to substrate binding at subsites -4, -3, and -2 in ''T. fusca ''Cel9A (Master et al., 2004). Class C EGases are the only plant EGases to date, that contain a tryptophan residue corresponding to the one in subsite -2 in TfCel9A (Urbanowicz et al., 2007b).  This tryptophan has been shown to be important for hydrolysis in TfCel9A. The enzyme retains less than 10% of its normal activity on polymeric cellulose substrates, and less than 1% of wild type activity on cellohexaose when the Trp is replaced by another amino acid (Li et al., 2007a; Master et al 2004).
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The Class A EGases are integral type II membrane proteins with a GH9 catalytic core that lack a canonical secretion signal sequence. These enzymes are predicted to have a high degree of N-glycosylation and a long amino-terminal extension with a membrane-spanning domain that anchors the protein to the plasma membrane and/or to intracellular organelles ('''Mølhøj''' et al., 2002; Brummell et al., 1997). Membrane anchored EGases were first described in studies of the ''KORRIGAN'' (''KOR'') genes in<sup> </sup>''Arabidopsis thaliana'', which showed that they encode EGases that are required for normal cellulose synthesis or assembly.  Plants with mutant alleles of the ''KOR1'' gene are cellulose-deficient and dwarfed ('''Mølhøj''' et al., 2002; Szyjanowicz et al 2004).  The role of the Class A EGases is not known.  However, the KOR proteins have been proposed to cleave sitosterol-b-glucoside primers from the growing cellulose polymer, or may have a role in editing incorrectly formed growing microfibrils (Peng et al. 2002). Recently, it has been shown that during cell expansion KOR1 is cycled from the plasma membrane through intracellular compartments, comprising both<sup> </sup>the Golgi apparatus and early endosomes; however the role of KOR1 in cellulose biosynthesis remains to be determined (Robert et al., 2005).  The catalytic domain of PttCel9A, a Class A GH9 enzyme that is upregulated during secondary cell wall synthesis in ''Populus tremula x tremuloides'', has been biochemically characterized and shown to hydrolyse a narrow range of substrates ''in vitro'' including CMC, phosphoric acid swollen cellulose and cellulose oligosaccharides (DP≥5).
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Class B proteins are the most common form of plant Egases and are associated with virtually all stages of plant growth and development.  These enzymes have a GH9 catalytic domain and a signal sequence for ER targeting and secretion.  Different isoforms are expressed during fruit ripening, in abscission zones, in reproductive organ development, and in expanding cells (Brummel et al., 1999; Brummel et al., 1997; Kalaitzis et al., 1999; Shani et al., 1997).  Numerous studies, especially in tomato, have also shown that many class B EGases are under hormonal control (Catalá''' '''et al., 1997; Brummell et al., 1997; Bonghi et al., 1998).
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Plant Class C EGases are the least studied. These proteins have a signal sequence followed by a GH9 catalytic domain and a long carboxyl-terminal extension, which contains a CBM49 that has been shown to bind to crystalline cellulose ''in vitro'' (Urbanowicz et al., 2007a; Urbanowicz et al., 2007b).  CBMs are necessary for activity on crystalline substrates and may promote hydrolysis by increasing the local enzyme concentration at the substrate surface as well as modifying cellulose microfibril structure (for review see Boraston et al., 2004). The catalytic domain (CD) SlGH9C1 is promiscuous and can effectively hydrolyze artificial cellulosic polymers, cellulose oligosaccharides, and several plant cell wall polysaccharides (Urbanowicz et al., 2007b).  Nevertheless, the activity of the full length, modular enzyme has still not been characterized.  A Class C EGase from rice, OsCel9A, has been shown to be post-translationaly modified at the linker region to yield a 51 kDa GH9 CD and a CBM49, and it was suggested that the cleavage is necessary for function (Yoshida et al., 2006a).  The OsCel9A CD also displays a broad substrate range and was able to hydrolyze CMC, phosphoric acid-swollen cellulose, mixed linkage 1,3-1,4-ß-glucan,<sup> </sup>xylan, glucomannan, cellooligosaccharides (DP≥3) and 1,4-ß-xylohexaose (Yoshida et al., 2006b).
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== References ==
 
== References ==

Revision as of 14:23, 12 August 2010

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Glycoside Hydrolase Family GH9
Clan GH-G
Mechanism inverting
Active site residues known/known
CAZy DB link
http://www.cazy.org/fam/GH9.html


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].

GH9 Enzymes Found in Plants

  12.00     Normal  0          false  false  false    EN-US  X-NONE  X-NONE                                                                                                                                                                                                                                                                                                                                                                       12.00     Normal  0          false  false  false    EN-US  X-NONE  X-NONE                                                                                                                                                                                                                                                                                                                                                                  

Early reports described the existence of plant ‘cellulases’ or Egases (e.g. Hall, 1963). Subsequently, cellulases have been shown to be associated with plant cell wall restructuring during cell expansion, the wall disassembly that accompanies processes such as fruit ripening and abscission (reviewed in del Campillo, 1999; Rose and Bennett, 1999; Mølhøj et al., 2002) and cellulose biosynthesis (Nicol et al., 1998; Lane et al., 2001; Sato et al., 2001). The amino acid sequences of the first plant ‘cellulases’/endo-b-1,4-glucanases revealed that these enzymes belong to the CAZy family GH9 glycoside hydrolases (Henrissat 1991). Plant GH9 enzymes are expected to affect cell wall strength and extendibility.


Most plant ‘cellulases’ studied to date are endoglucanases (EC 3.2.1.4) with low 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-b-glucan, xyloglucan, and glucomannan (Master et al., 2004; Yoshida and Komae 2006; Hayashi et al., 1984; Ohmiya et al., 2000; Woolley et al., 2001; Urbanowicz et al., 2007). The inability of plant “cellulases” to hydrolyze crystaline cellulose is distinct from microbial cellulases, whose modular structure and synergistic action with other enzymes facilitates effective degradation of crystalline cellulose. In muro, the substrates of microbial cellulases likely include xyloglucan, glucomannans, and non-crystalline cellulose, especially amorphous regions of cellulose where the microfibrils may be interwoven with xyloglucan.


In the model plant Arabidopsis thaliana, 25 different GH9 coding regions have been identified. Phylogenic analysis of the deduced amino acid sequences group the proteins into nine classes or three subfamilies (Mølhøj et al., 2002; Libertini et al., 2004; Urbanowicz et al., 2007a). Three distinct types of GH9 proteins are present in plants. Class A proteins are membrane-anchored, Class B proteins are secreted, and Class C proteins are also secreted but contain a family 49 carbohydrate binding module (CBM49) (Urbanowicz et al., 2007a). Class A plant EGases have been reported to lack tryptophans corresponding to substrate binding at subsites -4, -3, and -2 in T. fusca Cel9A (Master et al., 2004). Class C EGases are the only plant EGases to date, that contain a tryptophan residue corresponding to the one in subsite -2 in TfCel9A (Urbanowicz et al., 2007b). This tryptophan has been shown to be important for hydrolysis in TfCel9A. The enzyme retains less than 10% of its normal activity on polymeric cellulose substrates, and less than 1% of wild type activity on cellohexaose when the Trp is replaced by another amino acid (Li et al., 2007a; Master et al 2004).


The Class A EGases are integral type II membrane proteins with a GH9 catalytic core that lack a canonical secretion signal sequence. These enzymes are predicted to have a high degree of N-glycosylation and a long amino-terminal extension with a membrane-spanning domain that anchors the protein to the plasma membrane and/or to intracellular organelles (Mølhøj et al., 2002; Brummell et al., 1997). Membrane anchored EGases were first described in studies of the KORRIGAN (KOR) genes in Arabidopsis thaliana, which showed that they encode EGases that are required for normal cellulose synthesis or assembly. Plants with mutant alleles of the KOR1 gene are cellulose-deficient and dwarfed (Mølhøj et al., 2002; Szyjanowicz et al 2004). The role of the Class A EGases is not known. However, the KOR proteins have been proposed to cleave sitosterol-b-glucoside primers from the growing cellulose polymer, or may have a role in editing incorrectly formed growing microfibrils (Peng et al. 2002). Recently, it has been shown that during cell expansion KOR1 is cycled from the plasma membrane through intracellular compartments, comprising both the Golgi apparatus and early endosomes; however the role of KOR1 in cellulose biosynthesis remains to be determined (Robert et al., 2005). The catalytic domain of PttCel9A, a Class A GH9 enzyme that is upregulated during secondary cell wall synthesis in Populus tremula x tremuloides, has been biochemically characterized and shown to hydrolyse a narrow range of substrates in vitro including CMC, phosphoric acid swollen cellulose and cellulose oligosaccharides (DP≥5).


Class B proteins are the most common form of plant Egases and are associated with virtually all stages of plant growth and development. These enzymes have a GH9 catalytic domain and a signal sequence for ER targeting and secretion. Different isoforms are expressed during fruit ripening, in abscission zones, in reproductive organ development, and in expanding cells (Brummel et al., 1999; Brummel et al., 1997; Kalaitzis et al., 1999; Shani et al., 1997). Numerous studies, especially in tomato, have also shown that many class B EGases are under hormonal control (Catalá et al., 1997; Brummell et al., 1997; Bonghi et al., 1998).


Plant Class C EGases are the least studied. These proteins have a signal sequence followed by a GH9 catalytic domain and a long carboxyl-terminal extension, which contains a CBM49 that has been shown to bind to crystalline cellulose in vitro (Urbanowicz et al., 2007a; Urbanowicz et al., 2007b). CBMs are necessary for activity on crystalline substrates and may promote hydrolysis by increasing the local enzyme concentration at the substrate surface as well as modifying cellulose microfibril structure (for review see Boraston et al., 2004). The catalytic domain (CD) SlGH9C1 is promiscuous and can effectively hydrolyze artificial cellulosic polymers, cellulose oligosaccharides, and several plant cell wall polysaccharides (Urbanowicz et al., 2007b). Nevertheless, the activity of the full length, modular enzyme has still not been characterized. A Class C EGase from rice, OsCel9A, has been shown to be post-translationaly modified at the linker region to yield a 51 kDa GH9 CD and a CBM49, and it was suggested that the cleavage is necessary for function (Yoshida et al., 2006a). The OsCel9A CD also displays a broad substrate range and was able to hydrolyze CMC, phosphoric acid-swollen cellulose, mixed linkage 1,3-1,4-ß-glucan, xylan, glucomannan, cellooligosaccharides (DP≥3) and 1,4-ß-xylohexaose (Yoshida et al., 2006b).


References

  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.
    [Chen2007]
  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.
    [Lascombe1995]
All Medline abstracts: PubMed | HubMed