EzCatDB: S00912
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DB codeS00912
RLCP classification1.30.35880.983 : Hydrolysis
CATH domainDomain 13.20.20.80 : TIM BarrelCatalytic domain
E.C.3.2.1.89

CATH domainRelated DB codes (homologues)
3.20.20.80 : TIM BarrelS00202,S00210,S00748,S00906,S00907,S00911,S00915,M00134,M00160,D00479,S00204,S00205,S00206,S00207,S00203,S00208,S00209,S00211,S00213,S00214,M00113,T00307,D00165,D00166,D00169,D00176,D00501,D00502,D00503,D00844,D00861,D00864,M00026,M00112,M00193,M00346,T00057,T00062,T00063,T00066,T00067

Enzyme Name
UniProtKBKEGG

Q65CX5
Protein name
Arabinogalactan endo-1,4-beta-galactosidase
Endo-1,4-beta-galactanase
Galactanase
Arabinogalactanase
SynonymsYvfO
RefSeqYP_006715543.1 (Protein)
NC_006322.1 (DNA/RNA sequence)
YP_081349.1 (Protein)
NC_006270.3 (DNA/RNA sequence)
PfamPF07745 (Glyco_hydro_53)
[Graphical view]
CAZyGH53 (Glycoside Hydrolase Family)


UniProtKB:Accession NumberQ65CX5
Entry nameGANA_BACLD
ActivityEndohydrolysis of 1,4-beta-D-galactosidic linkages in arabinogalactans.
Subunit
Subcellular location
CofactorBinds 1 calcium ion per subunit.

Compound table: links to PDB-related databases & PoSSuM

CofactorsSubstratesProductsintermediates
KEGG-idC00076L00048L00049C05796C00001L00048L00049C05796C03611I00121
CompoundCalciumRhamnogalacturonan IArabinogalactan type IGalactanH2ORhamnogalacturonan IArabinogalactanGalactanGalactose oligosaccharidePeptidyl-Glu-galactan
Typedivalent metal (Ca2+, Mg2+)carboxyl group,polysaccharidepolysaccharidepolysaccharideH2Ocarboxyl group,polysaccharidepolysaccharidepolysaccharidepolysaccharide
ChEBI29108



15377





PubChem271



962
22247451





                  
1r8lA00Bound:_CAUnboundUnboundUnbound UnboundUnboundUnboundUnboundUnbound
1r8lB00Bound:_CAUnboundUnboundUnbound UnboundUnboundUnboundUnboundUnbound
1ur0A00Bound:_CAUnboundUnboundUnbound UnboundUnboundBound:B4GUnboundUnbound
1ur0B00Bound:_CAUnboundUnboundUnbound UnboundUnboundBound:B4GUnboundUnbound
1ur4A00Bound:_CAUnboundUnboundUnbound UnboundUnboundBound:B2GUnboundUnbound
1ur4B00Bound:_CAUnboundUnboundUnbound UnboundUnboundBound:B2GUnboundUnbound
2ccrA00Bound:_CAUnboundUnboundUnbound UnboundUnboundBound:B4GBound:B2GUnbound
2ccrB00Bound:_CAUnboundUnboundUnbound UnboundUnboundBound:B4GBound:B2GUnbound
2gftA00Bound:_CAUnboundUnboundUnbound UnboundUnboundBound:GAL-GAL-GALUnboundUnbound
2gftB00Bound:_CAUnboundUnboundUnbound UnboundUnboundBound:GAL-GAL-GALUnboundUnbound
2j74A00Bound:_CAUnboundUnboundUnbound UnboundUnboundBound:B4GBound:B2GUnbound
2j74B00Bound:_CAUnboundUnboundUnbound UnboundUnboundBound:B4GBound:B2GUnbound

Active-site residues
resource
Literature [6], [8], [9], [11], Swiss-prot;Q65CX5, P48842, P83691 & P83692
pdbCatalytic residuesCofactor-binding residuescomment
           
1r8lA00ARG 66;ASN 164;GLU 165;TYR 234;GLU 263
ASP 272;ASP 274;HIS 276;ASN 278;SER 367;ASP 370 (Calcium binding)
 
1r8lB00ARG 66;ASN 164;GLU 165;TYR 234;GLU 263
ASP 272;ASP 274;HIS 276;ASN 278;SER 367;ASP 370 (Calcium binding)
 
1ur0A00ARG 66;ASN 164;GLU 165;TYR 234;GLU 263
ASP 272;ASP 274;HIS 276;ASN 278;SER 367;ASP 370 (Calcium binding)
 
1ur0B00ARG 66;ASN 164;GLU 165;TYR 234;GLU 263
ASP 272;ASP 274;HIS 276;ASN 278;SER 367;ASP 370 (Calcium binding)
 
1ur4A00ARG 66;ASN 164;GLU 165;TYR 234;GLU 263
ASP 272;ASP 274;HIS 276;ASN 278;SER 367;ASP 370 (Calcium binding)
 
1ur4B00ARG 66;ASN 164;GLU 165;TYR 234;GLU 263
ASP 272;ASP 274;HIS 276;ASN 278;SER 367;ASP 370 (Calcium binding)
 
2ccrA00ARG 66;ASN 164;GLU 165;TYR 234;GLU 263
ASP 272;ASP 274;HIS 276;ASN 278;SER 367;ASP 370 (Calcium binding)
 
2ccrB00ARG 66;ASN 164;GLU 165;TYR 234;GLU 263
ASP 272;ASP 274;HIS 276;ASN 278;SER 367;ASP 370 (Calcium binding)
 
2gftA00ARG 66;ASN 164;GLU 165;TYR 234;       
ASP 272;ASP 274;HIS 276;ASN 278;SER 367;ASP 370 (Calcium binding)
mutant E236A
2gftB00ARG 66;ASN 164;GLU 166;TYR 234;       
ASP 272;ASP 274;HIS 276;ASN 278;SER 367;ASP 370 (Calcium binding)
mutant E236A
2j74A00ARG 66;ASN 164;GLU 165;TYR 234;GLU 263
ASP 272;ASP 274;HIS 276;ASN 278;SER 367;ASP 370 (Calcium binding)
 
2j74B00ARG 66;ASN 164;GLU 165;TYR 234;GLU 263
ASP 272;ASP 274;HIS 276;ASN 278;SER 367;ASP 370 (Calcium binding)
 

References for Catalytic Mechanism
ReferencesSectionsNo. of steps in catalysis
[6]FIGURE 3, p.15138-15141
[8]Figure 5
[9]p.109
[11]p.978

references
[1]
PubMed ID823153
JournalJ Biol Chem
Year1976
Volume251
Pages5904-10
AuthorsLabavitch JM, Freeman LE, Albersheim P
TitleStructure of plant cell walls. Purification and characterization of a beta-1,4-galactanase which degrades a structural component of the primary cell walls of dicots.
[2]
JournalCarbohydr Polym
Year1991
Volume16
Pages167-87
Authorsvan de Visa JW, Searle-van Leeuwena MJF, Silihaa HA, Kormelinka FJM, Voragena AGJ
TitlePurification and characterization of Endo-1,4-ƒÀ-D-galactanases from Aspergillus niger and Aspergillus aculeatus: Use in combination with arabinanases from Aspergillus niger in enzymic conversion of potato arabinogalactan
[3]
PubMed ID7712292
JournalCurr Opin Struct Biol
Year1994
Volume4
Pages885-92
AuthorsMcCarter JD, Withers SG
TitleMechanisms of enzymatic glycoside hydrolysis.
[4]
PubMed ID8535779
JournalStructure
Year1995
Volume3
Pages853-9
AuthorsDavies G, Henrissat B
TitleStructures and mechanisms of glycosyl hydrolases.
[5]
PubMed ID9398278
JournalBiochemistry
Year1997
Volume36
Pages15489-500
AuthorsBraithwaite KL, Barna T, Spurway TD, Charnock SJ, Black GW, Hughes N, Lakey JH, Virden R, Hazlewood GP, Henrissat B, Gilbert HJ
TitleEvidence that galactanase A from Pseudomonas fluorescens subspecies cellulosa is a retaining family 53 glycosyl hydrolase in which E161 and E270 are the catalytic residues.
[6]
CommentsX-RAY CRYSTALLOGRAPHY (1.8 ANGSTROMS).
PubMed ID12484750
JournalBiochemistry
Year2002
Volume41
Pages15135-43
AuthorsRyttersgaard C, Lo Leggio L, Coutinho PM, Henrissat B, Larsen S
TitleAspergillus aculeatus beta-1,4-galactanase: substrate recognition and relations to other glycoside hydrolases in clan GH-A.
Related PDB1fhl,1fob
Related UniProtKBP48842
[7]
JournalCarbohydr Polym
Year2003
Volume53
Pages155-68
AuthorsLuonteria E, Laineb C, Uusitaloa S, Telemanc A, Siika-ahoa M, Tenkanen M
TitlePurification and characterization of Aspergillus ƒÀ-Image -galactanases acting on ƒÀ-1,4- and ƒÀ-1,3/6-linked arabinogalactans
[8]
CommentsX-RAY CRYSTALLOGRAPHY (2.6 ANGSTROMS), CATALYTIC ACTIVITY, GLYCOSYLATION AT ASN-111.
PubMed ID12761390
JournalProtein Sci
Year2003
Volume12
Pages1195-204
AuthorsLe Nours J, Ryttersgaard C, Lo Leggio L, Ostergaard PR, Borchert TV, Christensen LL, Larsen S
TitleStructure of two fungal beta-1,4-galactanases: searching for the basis for temperature and pH optimum.
Related PDB1hjq,1hjs,1hju
Related UniProtKBP83691,P83692
[9]
CommentsX-RAY CRYSTALLOGRAPHY (2.2 ANGSTROMS) OF 28-424 IN COMPLEX WITH CALCIUM IONS AND SUBSTRATE ANALOGS, COFACTOR.
PubMed ID15312766
JournalJ Mol Biol
Year2004
Volume341
Pages107-17
AuthorsRyttersgaard C, Le Nours J, Lo Leggio L, Jorgensen CT, Christensen LL, Bjornvad M, Larsen S
TitleThe structure of endo-beta-1,4-galactanase from Bacillus licheniformis in complex with two oligosaccharide products.
Related PDB1r8l,1ur0,1ur4
Related UniProtKBQ65CX5
[10]
PubMed ID16151143
JournalAppl Environ Microbiol
Year2005
Volume71
Pages5501-10
AuthorsHinz SW, Pastink MI, van den Broek LA, Vincken JP, Voragen AG
TitleBifidobacterium longum endogalactanase liberates galactotriose from type I galactans.
[11]
PubMed ID19089956
JournalProteins
Year2009
Volume75
Pages977-89
AuthorsLe Nours J, De Maria L, Welner D, Jorgensen CT, Christensen LL, Borchert TV, Larsen S, Lo Leggio L
TitleInvestigating the binding of beta-1,4-galactan to Bacillus licheniformis beta-1,4-galactanase by crystallography and computational modeling.
Related PDB2ccr,2j74
Related UniProtKBQ65CX5

comments
This enzyme belongs to glycosidase family-58, with a retaining mechanism.
This enzyme is homologous to a counterpart enzyme from fungi (S00748 in EzCatDB).
Although this enzyme binds calcium ion, it is not involved in catalysis. The reactiom mechanism must be similar to that of the homologous enzyme.
According to the literature [6], the reaction mechanism proceeds as follows:
(0) Arg66 and Tyr234 modulate the pKa of catalytic nucleophile, Glu263.
(1) Glu165 acts as a general acid to protonate the leaving oxygen, O4 atom at subsite +1, forming an oxocarbenium-ion-like transition-state. The transition-state is stabilized by Asn164 and Tyr234 through hydrogen bonding to the oxygen atoms at subsite -1. (SN1-like reaction)
(2) Glu263 makes a nucleophilic attack on C1 atom of substrate, galactan, to form a covalent intermediate.
(3) Glu165 acts as a general base to deprotonate a water molecule, to activate it.
(4) The activated water makes a nucleophilic attack on the C1 atom of the covalent intermediate. Thus, the substitution reaction completes.

createdupdated
2010-04-082012-01-26
Copyright: Nozomi Nagano, JST & CBRC-AIST
Funded by PRESTO/Japan Science and Technology Corporation (JST) (December 2001 - November 2004)
Funded by Grant-in-Aid for Publication of Scientific Research Results/Japan Society for the Promotion of Science (JSPS) (April 2005 - March 2006)
Funded by Grant-in-Aid for Scientific Research (B)/Japan Society for the Promotion of Science (JSPS) (April 2005 - March 2008)
Funded by BIRD/Japan Science and Technology Corporation (JST) (September 2005 - September 2008)
Funded by BIRD/Japan Science and Technology Corporation (JST) (October 2007 - September 2010)
Funded by Grant-in-Aid for Publication of Scientific Research Results/Japan Society for the Promotion of Science (JSPS) (April 2011 - March 2012)
Funded by Grant-in-Aid for Publication of Scientific Research Results/Japan Society for the Promotion of Science (JSPS) (April 2012 - March 2013)
Supported by the commission for the Development of Artificial Gene Synthesis Technology for Creating Innovative Biomaterial from the Ministry of Economy, Trade and Industry (METI) (October 2012 - )
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