DB code: T00245

RLCP classification	1.30.5050.993 : Hydrolysis
CATH domain	2.60.40.10 : Immunoglobulin-like
	1.50.10.10 : Glycosyltransferase	Catalytic domain
	1.10.1330.10 : Type 1 dockerin domain
E.C.	3.2.1.4
CSA
M-CSA
MACiE

CATH domain	Related DB codes (homologues)
1.10.1330.10 : Type 1 dockerin domain	D00167 D00503 T00246
1.50.10.10 : Glycosyltransferase	S00531 S00048 S00845 D00167 D00500 M00192 T00246
2.60.40.10 : Immunoglobulin-like	M00131 T00257 T00005 M00113 M00127 M00132 M00323 M00325 M00327 M00329 M00330 M00331 M00332 T00307 D00166 D00500 M00112 M00193 T00063 T00065 T00067

Uniprot Enzyme Name
UniprotKB	Protein name	Synonyms	Pfam
P0C2S4	Endoglucanase D	EGD EC 3.2.1.4 Endo-1,4-beta-glucanase Cellulase D	PF02927 (CelD_N) PF00404 (Dockerin_1) PF00759 (Glyco_hydro_9) [Graphical View]

KEGG enzyme name
cellulase endo-1,4-beta-D-glucanase beta-1,4-glucanase beta-1,4-endoglucan hydrolase celluase A cellulosin AP endoglucanase D alkali cellulase cellulase A 3 celludextrinase 9.5 cellulase avicelase pancellase SS 1,4-(1,3 1,4)-beta-D-glucan 4-glucanohydrolase

UniprotKB: Accession Number	Entry name	Activity	Subunit	Subcellular location	Cofactor
P0C2S4	GUND_CLOTM	Endohydrolysis of (1->4)-beta-D-glucosidic linkages in cellulose, lichenin and cereal beta-D-glucans.			Calcium.

KEGG Pathways
Map code	Pathways	E.C.
MAP00500	Starch and sucrose metabolism

Compound table
	Cofactors		Substrates				Products		Intermediates
KEGG-id	C00038	C00076	C00760	C00478	C00551	C00001	C00760	C00551
E.C.
Compound	Zinc	Calcium	Cellulose	Lichenin	beta-D-Glucan	H2O	Cellulose	beta-D-Glucan
Type	heavy metal	divalent metal (Ca2+, Mg2+)	polysaccharide	carbohydrate	polysaccharide	H2O	polysaccharide	polysaccharide
ChEBI	29105 29105	29108 29108				15377 15377
PubChem	32051 32051	271 271		439241 439241	46173706 46173706	22247451 962 22247451 962		46173706 46173706

1clcA01	Unbound	Unbound	Unbound	Unbound	Unbound		Unbound	Unbound
1clcA02	Bound:_ZN	Bound:3x_CA	Unbound	Unbound	Unbound		Unbound	Unbound

Reference for Active-site residues
resource	references	E.C.
PDB;1clc & literature [5], [6], [8], [14]

Active-site residues
PDB	Catalytic residues	Cofactor-binding residues	Modified residues	Main-chain involved in catalysis	Comment

1clcA01
1clcA02	ASP 198;ASP 201;TYR 205;TYR 354;GLU 555	CYS 155;CYS 173;HIS 174;HIS 197(Zinc binding);GLU 236;ASN 239;ILE 241;ASP 243;ASP 246(Calcium-1 binding);THR 356;SER 358;ASP 361;ASP 362;ASP 401(Calcium-2 binding);SER 520;ASP 523;ILE 525(Calcium-3 binding)

References for Catalytic Mechanism
References	Sections	No. of steps in catalysis
[5]	p.91
[8]	Fig.4, p.814815
[10]	p.657-658

References
[1]
Resource
Comments	CALCIUM-BINDING DATA.
Medline ID	90147577
PubMed ID	2302168
Journal	Biochem J
Year	1990
Volume	265
Pages	261-5
Authors	Chauvaux S, Beguin P, Aubert JP, Bhat KM, Gow LA, Wood TM, Bairoch A
Title	Calcium-binding affinity and calcium-enhanced activity of Clostridium thermocellum endoglucanase D.
Related PDB
Related UniProtKB	P04954
[2]
Resource
Comments	ACTIVE SITE HIS-516, AND MUTAGENESIS OF ALL HISTIDINE RESIDUES.
Medline ID	91244802
PubMed ID	2037583
Journal	J Biol Chem
Year	1991
Volume	266
Pages	10313-8
Authors	Tomme P, Chauvaux S, Beguin P, Millet J, Aubert JP, Claeyssens M
Title	Identification of a histidyl residue in the active center of endoglucanase D from Clostridium thermocellum.
Related PDB
Related UniProtKB	P04954
[3]
Resource
Comments	ACTIVE SITE ASP-546.
Medline ID	92344589
PubMed ID	1637316
Journal	Biochem J
Year	1992
Volume	285
Pages	319-24
Authors	Tomme P, van Beeumen J, Claeyssens M
Title	Modification of catalytically important carboxy residues in endoglucanase D from Clostridium thermocellum.
Related PDB
Related UniProtKB	P04954
[4]
Resource
Comments	ACTIVE SITE GLU-555, AND MUTAGENESIS OF ASPARTIC ACID AND GLUTAMIC ACID RESIDUES.
Medline ID	92165798
PubMed ID	1537833
Journal	J Biol Chem
Year	1992
Volume	267
Pages	4472-8
Authors	Chauvaux S, Beguin P, Aubert JP
Title	Site-directed mutagenesis of essential carboxylic residues in Clostridium thermocellum endoglucanase CelD.
Related PDB
Related UniProtKB	P04954
[5]
Resource
Comments	X-RAY CRYSTALLOGRAPHY (2.3 ANGSTROMS).
Medline ID
PubMed ID
Journal	Nature
Year	1992
Volume	357
Pages	89-91
Authors	Juy M, Amit AG, Alzari PM, Poljak RJ, Claeyssens M, Beguin P, Aubert J-P
Title	Three-dimensional structure of a thermostable bacterial cellulase.
Related PDB
Related UniProtKB	P04954
[6]
Resource
Comments
Medline ID
PubMed ID	7730353
Journal	J Biol Chem
Year	1995
Volume	270
Pages	9757-62
Authors	Chauvaux S, Souchon H, Alzari PM, Chariot P, Beguin P
Title	Structural and functional analysis of the metal-binding sites of Clostridium thermocellum endoglucanase CelD.
Related PDB
Related UniProtKB
[7]
Resource
Comments	X-RAY CRYSTALLOGRAPHY (1.9 ANGSTROMS).
Medline ID
PubMed ID	7739036
Journal	J Mol Biol
Year	1995
Volume	248
Pages	225-32
Authors	Chitarra V, Souchon H, Spinelli S, Juy M, Beguin P, Alzari PM
Title	Multiple crystal forms of endoglucanase CelD: signal peptide residues modulate lattice formation.
Related PDB
Related UniProtKB	P04954
[8]
Resource
Comments	X-RAY CRYSTALLOGRAPHY (1.9 ANGSTROMS) OF 47-651
Medline ID	9334746
PubMed ID	9334746
Journal	Nat Struct Biol
Year	1997
Volume	4
Pages	810-8
Authors	Sakon J, Irwin D, Wilson DB, Karplus PA
Title	Structure and mechanism of endo/exocellulase E4 from Thermomonospora fusca.
Related PDB
Related UniProtKB
[9]
Resource
Comments
Medline ID
PubMed ID	11273698
Journal	J Mol Biol
Year	2001
Volume	307
Pages	745-53
Authors	Lytle BL, Volkman BF, Westler WM, Heckman MP, Wu JH
Title	Solution structure of a type I dockerin domain, a novel prokaryotic, extracellular calcium-binding domain.
Related PDB
Related UniProtKB
[10]
Resource
Comments	X-ray crystallography (1.4 Angstroms)
Medline ID
PubMed ID	11914490
Journal	Acta Crystallogr D Biol Crystallogr
Year	2002
Volume	58
Pages	653-659
Authors	Khademi S, Guarino LA, Watanabe H, Tokuda G, Meyer EF
Title	Structure of an endoglucanase from termite, Nasutitermes takasagoensis.
Related PDB
Related UniProtKB
[11]
Resource
Comments
Medline ID
PubMed ID	11844767
Journal	J Bacteriol
Year	2002
Volume	184
Pages	1378-84
Authors	Belaich A, Parsiegla G, Gal L, Villard C, Haser R, Belaich JP
Title	Cel9M, a new family 9 cellulase of the Clostridium cellulolyticum cellulosome.
Related PDB
Related UniProtKB
[12]
Resource
Comments	X-ray crystallography
Medline ID
PubMed ID	12837787
Journal	J Bacteriol
Year	2003
Volume	185
Pages	4127-35
Authors	Mandelman D, Belaich A, Belaich JP, Aghajari N, Driguez H, Haser R
Title	X-Ray crystal structure of the multidomain endoglucanase Cel9G from Clostridium cellulolyticum complexed with natural and synthetic cello-oligosaccharides.
Related PDB
Related UniProtKB
[13]
Resource
Comments	X-ray crystallography
Medline ID
PubMed ID	14756552
Journal	Biochemistry
Year	2004
Volume	43
Pages	1163-70
Authors	Schubot FD, Kataeva IA, Chang J, Shah AK, Ljungdahl LG, Rose JP, Wang BC
Title	Structural basis for the exocellulase activity of the cellobiohydrolase CbhA from Clostridium thermocellum.
Related PDB
Related UniProtKB
[14]
Resource
Comments
Medline ID
PubMed ID	15274620
Journal	Biochemistry
Year	2004
Volume	43
Pages	9655-63
Authors	Zhou W, Irwin DC, Escovar-Kousen J, Wilson DB
Title	Kinetic studies of Thermobifida fusca Cel9A active site mutant enzymes.
Related PDB
Related UniProtKB

Comments
This enzyme belongs to the glycosidase family-9, with an inverting mechanism. Although the structure of the domain 3 (dockerin repeat region) has not been determined yet, it must have a similar structure to that of type-I dockerin domain (PDB;1daq). Althoug this enzyme binds a zinc ion and three calcium ions, they are not involved in catalysis. They must play structural roles (see [1] & [6]). Although a catalytic histdine residue is not conserved in this enzyme, the catalytic domain of this enzyme is homologous to those of the other glycosidase family-9 enzymes (S00531, D00167, T00245, M00192 in EzCatDB). Considering the active-site structure of this enzyme, instead of a catalytic histidine residue, Tyr205 must act as a modulator for Asp201. The reaction of this enzyme may proceeds as follows: (0) Tyr354 modulates the pKa of Asp198, whereas Tyr205 modulates the pKa of Asp201. (1) Glu555 acts as a general acid to protonate the leaving group, the glycosidic oxygen, forming an oxocarbonium ion in the transition state. (SN1-like reaction) (2) Both Asp198 and Asp201 act as general bases to deprotonate the nucleophilic water. (Here, Asp201 seems more likely to be the base.) (3) The activated water makes a nucleophilic attack on the anomeric carbon to complete the reaction.

Comments

This enzyme belongs to the glycosidase family-9, with an inverting mechanism.
Although the structure of the domain 3 (dockerin repeat region) has not been determined yet, it must have a similar structure to that of type-I dockerin domain (PDB;1daq).
Althoug this enzyme binds a zinc ion and three calcium ions, they are not involved in catalysis. They must play structural roles (see [1] & [6]).
Although a catalytic histdine residue is not conserved in this enzyme, the catalytic domain of this enzyme is homologous to those of the other glycosidase family-9 enzymes (S00531, D00167, T00245, M00192 in EzCatDB). Considering the active-site structure of this enzyme, instead of a catalytic histidine residue, Tyr205 must act as a modulator for Asp201.
The reaction of this enzyme may proceeds as follows:
(0) Tyr354 modulates the pKa of Asp198, whereas Tyr205 modulates the pKa of Asp201.
(1) Glu555 acts as a general acid to protonate the leaving group, the glycosidic oxygen, forming an oxocarbonium ion in the transition state. (SN1-like reaction)
(2) Both Asp198 and Asp201 act as general bases to deprotonate the nucleophilic water. (Here, Asp201 seems more likely to be the base.)
(3) The activated water makes a nucleophilic attack on the anomeric carbon to complete the reaction.

Created	Updated
2004-08-18	2009-02-26