EzCatDB: D00860
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DB codeD00860
RLCP classification3.903.137500.660 : Transfer
CATH domainDomain 1-.-.-.-
Domain 23.90.550.10 : Spore Coat Polysaccharide Biosynthesis Protein SpsA; Chain ACatalytic domain
E.C.2.4.1.223

CATH domainRelated DB codes (homologues)
3.90.550.10 : Spore Coat Polysaccharide Biosynthesis Protein SpsA; Chain AS00709,S00465,S00466,D00417,D00859,T00415

Enzyme Name
UniProtKBKEGG

Q9ES89
Protein nameExostosin-like 2Glucuronyl-galactosyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase
Alpha-N-acetylglucosaminyltransferase I
Alpha1,4-N-acetylglucosaminyltransferase
Glucuronosylgalactosyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase
SynonymsEC 2.4.1.223
Alpha-1,4-N-acetylhexosaminyltransferase EXTL2
Alpha-GalNAcT EXTL2
EXT-related protein 2
Glucuronyl-galactosyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase
RefSeqNP_001156986.1 (Protein)
NM_001163514.1 (DNA/RNA sequence)
NP_067363.3 (Protein)
NM_021388.4 (DNA/RNA sequence)
PfamPF09258 (Glyco_transf_64)
[Graphical view]
CAZyGT64 (Glycosyltransferase Family)

KEGG pathways
MAP codePathways
MAP00534Glycosaminoglycan biosynthesis - heparan sulfate

UniProtKB:Accession NumberQ9ES89
Entry nameEXTL2_MOUSE
ActivityUDP-N-acetyl-D-glucosamine + beta-D-glucuronosyl-(1->3)-beta-D-galactosyl-(1->3)-beta-D-galactosyl-(1->4)-beta-D-xylosyl-proteoglycan = UDP + alpha-N-acetyl-D-glucosaminyl-(1->4)-beta-D-glucuronosyl-(1->3)-beta-D-galactosyl-(1->3)-beta-D-galactosyl-(1->4)-beta-D-xylosyl-proteoglycan.
Subunit
Subcellular locationEndoplasmic reticulum membrane, Single-pass type II membrane protein (By similarity).
Cofactor

Compound table: links to PDB-related databases & PoSSuM

CofactorsSubstratesProducts
KEGG-idC00034C00043C04903C00015C15656
CompoundManganeseUDP-N-acetyl-D-glucosamine3-beta-D-Glucuronosyl-3-beta-D-galactosyl-4-beta-D-galactosyl-O-beta-D-xylosylproteinUDPalpha-N-acetyl-D-glucosaminyl-(1->4)-beta-D-glucuronosyl-(1->3)-beta-D-galactosyl-(1->3)-beta-D-galactosyl-(1->4)-beta-D-xylosyl-proteoglycan
Typeheavy metalamide group,carbohydrate,nucleotidecarboxyl group,peptide/protein,polysaccharideamide group,nucleotideamide group,carboxyl group,peptide/protein,polysaccharide
ChEBI18291
35154
16264

17659

PubChem23930
445675

6031

             
1omxA00UnboundUnboundUnboundUnboundUnbound
1omxB00UnboundUnboundUnboundUnboundUnbound
1omzA00Bound:_MNAnalogue:UD2UnboundUnboundUnbound
1omzB00Bound:_MNAnalogue:UD2UnboundUnboundUnbound
1on6A00Bound:_MNBound:UD1UnboundUnboundUnbound
1on6B00Bound:_MNBound:UD1UnboundUnboundUnbound
1on8A00Bound:_MNUnboundAnalogue:BDP-GALBound:UDPUnbound
1on8B00Bound:_MNUnboundAnalogue:BDP-GALBound:UDPUnbound

Active-site residues
resource
Literature [4]
pdbCatalytic residuesCofactor-binding residuesMain-chain involved in catalysiscomment
            
1omxA00ASN 243;ASP 246;ARG 293
ASP 153(Manganese binding)
TRP 284
invisible 274-286
1omxB00ASN 243;ASP 246;ARG 293
ASP 153(Manganese binding)
TRP 284
invisible 274-287
1omzA00ASN 243;ASP 246;ARG 293
ASP 153(Manganese binding)
TRP 284
invisible 275-286
1omzB00ASN 243;ASP 246;ARG 293
ASP 153(Manganese binding)
TRP 284
invisible 275-285
1on6A00ASN 243;ASP 246;ARG 293
ASP 153(Manganese binding)
TRP 284
invisible 275-285
1on6B00ASN 243;ASP 246;ARG 293
ASP 153(Manganese binding)
TRP 284
invisible 275-285
1on8A00ASN 243;ASP 246;ARG 293
ASP 153(Manganese binding)
TRP 284
 
1on8B00ASN 243;ASP 246;ARG 293
ASP 153(Manganese binding)
TRP 284
invisible 278-280

References for Catalytic Mechanism
ReferencesSectionsNo. of steps in catalysis
[4]FIG.7
[7]p.241
[8]Figure 10

references
[1]
PubMed ID10318803
JournalJ Biol Chem
Year1999
Volume274
Pages13933-7
AuthorsKitagawa H, Shimakawa H, Sugahara K
TitleThe tumor suppressor EXT-like gene EXTL2 encodes an alpha1, 4-N-acetylhexosaminyltransferase that transfers N-acetylgalactosamine and N-acetylglucosamine to the common glycosaminoglycan-protein linkage region. The key enzyme for the chain initiation of heparan sulfate.
[2]
PubMed ID11042448
JournalCurr Opin Struct Biol
Year2000
Volume10
Pages518-27
AuthorsSugahara K, Kitagawa H
TitleRecent advances in the study of the biosynthesis and functions of sulfated glycosaminoglycans.
[3]
PubMed ID12659829
JournalBiochem Biophys Res Commun
Year2003
Volume303
Pages393-8
AuthorsNegishi M, Dong J, Darden TA, Pedersen LG, Pedersen LC
TitleGlucosaminylglycan biosynthesis: what we can learn from the X-ray crystal structures of glycosyltransferases GlcAT1 and EXTL2.
[4]
CommentsX-RAY CRYSTALLOGRAPHY (2.1 ANGSTROMS) OF 38-330, METAL-BINDING, DISULFIDE BOND.
PubMed ID12562774
JournalJ Biol Chem
Year2003
Volume278
Pages14420-8
AuthorsPedersen LC, Dong J, Taniguchi F, Kitagawa H, Krahn JM, Pedersen LG, Sugahara K, Negishi M
TitleCrystal structure of an alpha 1,4-N-acetylhexosaminyltransferase (EXTL2), a member of the exostosin gene family involved in heparan sulfate biosynthesis.
Related PDB1omx,1omz,1on6,1on8
Related UniProtKBQ9ES89
[5]
PubMed ID15489968
JournalChem Commun (Camb)
Year2004
Volume(20)
Pages2243-8
AuthorsLairson LL, Withers SG
TitleMechanistic analogies amongst carbohydrate modifying enzymes.
[6]
PubMed ID15831490
JournalJ Biol Chem
Year2005
Volume280
Pages23441-5
AuthorsSobhany M, Dong J, Negishi M
TitleTwo-step mechanism that determines the donor binding specificity of human UDP-N-acetylhexosaminyltransferase.
[7]
JournalTop Curr Chem
Year2007
Volume272
Pages217-57
AuthorsSchuman B, Alfaro JA, Evans SV
TitleGlycosyltransferase structure and function.
[8]
PubMed ID18518825
JournalAnnu Rev Biochem
Year2008
Volume77
Pages521-55
AuthorsLairson LL, Henrissat B, Davies GJ, Withers SG
TitleGlycosyltransferases: structures, functions, and mechanisms.

comments
This enzyme belongs to glycosyltransferase family-64 (GT64 family), with a GT-A fold and a retaining mechanism (see [7]).
The structure of the N-terminal domain, which contains the N-terminal cytoplasmic region and a membrane-bound region, has not been determined yet.
According to the literature [4], [7] and [8], this enzyme catalyzes the following reaction (so-called substitution nucleophilic internal (SNi)-like mechanism):
(0) Manganese ion, bound to Asp153, and mainchain amide of Trp284 stabilize the negative charge on the leaving phosphate groups. Sidechains of Asn243 and Asp246 modulate the positive charge or pKa of sidechain of Arg293.
(1) The transferred group, N-acetyl-glucosamine, forms an oxocarbenium ion-like transition state, with a positive charge developed on C1' atom on the N-acetyl-glucosamine and a negative charge on the leaving phosphate oxygen atom (Dissociative reaction/SN1-like reaction). Here, Arg293 stabilizes the positive charge on C1' atom in the transition state.
(2) The leaving phosphate oxygen acts as a general base to deprotonate O4 atom of the acceptor, GlcA group.
(3) The activated O4 atom of the GlcA makes a nucleophilic attack on C1' atom of the N-acetyl-glucosamine from the same side as the leaving phosphate group, so that the N-acetyl-glucosamine group of the final product retains its configuration.

createdupdated
2010-08-192011-10-05


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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