DB code: D00860

RLCP classification 3.903.137500.660 : Transfer
CATH domain -.-.-.- :
3.90.550.10 : Spore Coat Polysaccharide Biosynthesis Protein SpsA; Chain A Catalytic domain
E.C. 2.4.1.223
CSA
M-CSA
MACiE

CATH domain Related DB codes (homologues)
3.90.550.10 : Spore Coat Polysaccharide Biosynthesis Protein SpsA; Chain A S00709 S00465 S00466 D00417 D00859 T00415

Uniprot Enzyme Name
UniprotKB Protein name Synonyms RefSeq CAZy Pfam
Q9ES89 Exostosin-like 2
EC 2.4.1.223
Alpha-1,4-N-acetylhexosaminyltransferase EXTL2
Alpha-GalNAcT EXTL2
EXT-related protein 2
Glucuronyl-galactosyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase
NP_001156986.1 (Protein)
NM_001163514.1 (DNA/RNA sequence)
NP_067363.3 (Protein)
NM_021388.4 (DNA/RNA sequence)
GT64 (Glycosyltransferase Family 64)
PF09258 (Glyco_transf_64)
[Graphical View]

KEGG enzyme name
Glucuronyl-galactosyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase
Alpha-N-acetylglucosaminyltransferase I
Alpha1,4-N-acetylglucosaminyltransferase
Glucuronosylgalactosyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase

UniprotKB: Accession Number Entry name Activity Subunit Subcellular location Cofactor
Q9ES89 EXTL2_MOUSE UDP-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. Endoplasmic reticulum membrane, Single-pass type II membrane protein (By similarity).

KEGG Pathways
Map code Pathways E.C.
MAP00534 Glycosaminoglycan biosynthesis - heparan sulfate

Compound table
Cofactors Substrates Products Intermediates
KEGG-id C00034 C00043 C04903 C00015 C15656
E.C.
Compound Manganese UDP-N-acetyl-D-glucosamine 3-beta-D-Glucuronosyl-3-beta-D-galactosyl-4-beta-D-galactosyl-O-beta-D-xylosylprotein 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
Type heavy metal amide group,carbohydrate,nucleotide carboxyl group,peptide/protein,polysaccharide amide group,nucleotide amide group,carboxyl group,peptide/protein,polysaccharide
ChEBI 18291
35154
16264
17659
PubChem 23930
445675
6031
1omxA00 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Unbound Unbound Unbound
1omxB00 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Unbound Unbound Unbound
1omzA00 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Bound:_MN Analogue:UD2 Unbound Unbound Unbound
1omzB00 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Bound:_MN Analogue:UD2 Unbound Unbound Unbound
1on6A00 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Bound:_MN Bound:UD1 Unbound Unbound Unbound
1on6B00 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Bound:_MN Bound:UD1 Unbound Unbound Unbound
1on8A00 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Bound:_MN Unbound Analogue:BDP-GAL Bound:UDP Unbound
1on8B00 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Bound:_MN Unbound Analogue:BDP-GAL Bound:UDP Unbound

Reference for Active-site residues
resource references E.C.
Literature [4]

Active-site residues
PDB Catalytic residues Cofactor-binding residues Modified residues Main-chain involved in catalysis Comment
1omxA00 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain ASN 243;ASP 246;ARG 293 ASP 153(Manganese binding) TRP 284 invisible 274-286
1omxB00 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain ASN 243;ASP 246;ARG 293 ASP 153(Manganese binding) TRP 284 invisible 274-287
1omzA00 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain ASN 243;ASP 246;ARG 293 ASP 153(Manganese binding) TRP 284 invisible 275-286
1omzB00 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain ASN 243;ASP 246;ARG 293 ASP 153(Manganese binding) TRP 284 invisible 275-285
1on6A00 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain ASN 243;ASP 246;ARG 293 ASP 153(Manganese binding) TRP 284 invisible 275-285
1on6B00 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain ASN 243;ASP 246;ARG 293 ASP 153(Manganese binding) TRP 284 invisible 275-285
1on8A00 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain ASN 243;ASP 246;ARG 293 ASP 153(Manganese binding) TRP 284
1on8B00 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain ASN 243;ASP 246;ARG 293 ASP 153(Manganese binding) TRP 284 invisible 278-280

References for Catalytic Mechanism
References Sections No. of steps in catalysis
[4]
FIG.7
[7]
p.241
[8]
Figure 10

References
[1]
Resource
Comments
Medline ID
PubMed ID 10318803
Journal J Biol Chem
Year 1999
Volume 274
Pages 13933-7
Authors Kitagawa H, Shimakawa H, Sugahara K
Title The 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.
Related PDB
Related UniProtKB
[2]
Resource
Comments
Medline ID
PubMed ID 11042448
Journal Curr Opin Struct Biol
Year 2000
Volume 10
Pages 518-27
Authors Sugahara K, Kitagawa H
Title Recent advances in the study of the biosynthesis and functions of sulfated glycosaminoglycans.
Related PDB
Related UniProtKB
[3]
Resource
Comments
Medline ID
PubMed ID 12659829
Journal Biochem Biophys Res Commun
Year 2003
Volume 303
Pages 393-8
Authors Negishi M, Dong J, Darden TA, Pedersen LG, Pedersen LC
Title Glucosaminylglycan biosynthesis: what we can learn from the X-ray crystal structures of glycosyltransferases GlcAT1 and EXTL2.
Related PDB
Related UniProtKB
[4]
Resource
Comments X-RAY CRYSTALLOGRAPHY (2.1 ANGSTROMS) OF 38-330, METAL-BINDING, DISULFIDE BOND.
Medline ID
PubMed ID 12562774
Journal J Biol Chem
Year 2003
Volume 278
Pages 14420-8
Authors Pedersen LC, Dong J, Taniguchi F, Kitagawa H, Krahn JM, Pedersen LG, Sugahara K, Negishi M
Title Crystal structure of an alpha 1,4-N-acetylhexosaminyltransferase (EXTL2), a member of the exostosin gene family involved in heparan sulfate biosynthesis.
Related PDB 1omx 1omz 1on6 1on8
Related UniProtKB Q9ES89
[5]
Resource
Comments
Medline ID
PubMed ID 15489968
Journal Chem Commun (Camb)
Year 2004
Volume (20)
Pages 2243-8
Authors Lairson LL, Withers SG
Title Mechanistic analogies amongst carbohydrate modifying enzymes.
Related PDB
Related UniProtKB
[6]
Resource
Comments
Medline ID
PubMed ID 15831490
Journal J Biol Chem
Year 2005
Volume 280
Pages 23441-5
Authors Sobhany M, Dong J, Negishi M
Title Two-step mechanism that determines the donor binding specificity of human UDP-N-acetylhexosaminyltransferase.
Related PDB
Related UniProtKB
[7]
Resource
Comments
Medline ID
PubMed ID
Journal Top Curr Chem
Year 2007
Volume 272
Pages 217-57
Authors Schuman B, Alfaro JA, Evans SV
Title Glycosyltransferase structure and function.
Related PDB
Related UniProtKB
[8]
Resource
Comments
Medline ID
PubMed ID 18518825
Journal Annu Rev Biochem
Year 2008
Volume 77
Pages 521-55
Authors Lairson LL, Henrissat B, Davies GJ, Withers SG
Title Glycosyltransferases: structures, functions, and mechanisms.
Related PDB
Related UniProtKB

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.

Created Updated
2010-08-19 2011-10-05