PDBsum entry 1v83

Transferase

PDB id: 1v83

Contents

Protein chains

245 a.a. *

Ligands

TLA

UDP ×2

Metals

_MN ×2

Waters ×414

* Residue conservation analysis

PDB id:

1v83

Name:

Transferase

Title:

Crystal structure of human glcat-p in complex with udp and mn2+

Structure:

Galactosylgalactosylxylosylprotein 3-beta- glucuronosyltransferase 1. Chain: a, b. Fragment: catalytic domain. Synonym: beta-1,3-glucuronyltransferase 1, glucuronosyltransferase-p, glcat-p, udp-glcua:glycoprotein beta- 1,3-glucuronyltransferase. Engineered: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Dimer (from PQS)

Resolution:

1.90Å R-factor: 0.201 R-free: 0.229

Authors:

S.Kakuda,T.Shiba,M.Ishiguro,H.Tagawa,S.Oka,Y.Kajihara,T.Kawasaki, S.Wakatsuki,R.Kato

Key ref:

S.Kakuda et al. (2004). Structural basis for acceptor substrate recognition of a human glucuronyltransferase, GlcAT-P, an enzyme critical in the biosynthesis of the carbohydrate epitope HNK-1. J Biol Chem, 279, 22693-22703. PubMed id: 14993226 DOI: 10.1074/jbc.M400622200

Date:

27-Dec-03 Release date: 25-May-04

Protein chains

Q9P2W7  (B3GA1_HUMAN) -  Galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase 1 from Homo sapiens

Seq: 334 a.a.

Struc: 245 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.2.4.1.135 - galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase.
• Pathway: Heparan and Chondroitin Biosynthesis; (early stages)
• Reaction: 3-O-(beta-D-galactosyl-(1->3)-beta-D-galactosyl-(1->4)-beta-D-xylosyl)-L- seryl-[protein] + UDP-alpha-D-glucuronate = 3-O-(beta-D-GlcA-(1->3)-beta- D-Gal-(1->3)-beta-D-Gal-(1->4)-beta-D-Xyl)-L-seryl-[protein] + UDP + H⁺

3-O-(beta-D-galactosyl-(1->3)-beta-D-galactosyl-(1->4)-beta-D-xylosyl)-L- seryl-[protein] + UDP-alpha-D-glucuronate = 3-O-(beta-D-GlcA-(1->3)-beta- D-Gal-(1->3)-beta-D-Gal-(1->4)-beta-D-Xyl)-L-seryl-[protein] + UDP + H(+) (Bound ligand (Het Group name = UDP) corresponds exactly)

• Cofactor: Mn(2+)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Added reference

DOI no: 10.1074/jbc.M400622200

J Biol Chem 279:22693-22703 (2004)

PubMed id: 14993226

Structural basis for acceptor substrate recognition of a human glucuronyltransferase, GlcAT-P, an enzyme critical in the biosynthesis of the carbohydrate epitope HNK-1.

S.Kakuda, T.Shiba, M.Ishiguro, H.Tagawa, S.Oka, Y.Kajihara, T.Kawasaki, S.Wakatsuki, R.Kato.

ABSTRACT

The HNK-1 carbohydrate epitope is found on many neural cell adhesion molecules. Its structure is characterized by a terminal sulfated glucuronyl acid. The glucuronyltransferases, GlcAT-P and GlcAT-S, are involved in the biosynthesis of the HNK-1 epitope, GlcAT-P as the major enzyme. We overexpressed and purified the recombinant human GlcAT-P from Escherichia coli. Analysis of its enzymatic activity showed that it catalyzed the transfer reaction for N-acetyllactosamine (Galbeta1-4GlcNAc) but not lacto-N-biose (Galbeta1-3GlcNAc) as an acceptor substrate. Subsequently, we determined the first x-ray crystal structures of human GlcAT-P, in the absence and presence of a donor substrate product UDP, catalytic Mn(2+), and an acceptor substrate analogue N-acetyllactosamine (Galbeta1-4GlcNAc) or an asparagine-linked biantennary nonasaccharide. The asymmetric unit contains two independent molecules. Each molecule is an alpha/beta protein with two regions that constitute the donor and acceptor substrate binding sites. The UDP moiety of donor nucleotide sugar is recognized by conserved amino acid residues including a DXD motif (Asp(195)-Asp(196)-Asp(197)). Other conserved amino acid residues interact with the terminal galactose moiety of the acceptor substrate. In addition, Val(320) and Asn(321), which are located on the C-terminal long loop from a neighboring molecule, and Phe(245) contribute to the interaction with GlcNAc moiety. These three residues play a key role in establishing the acceptor substrate specificity.

Selected figure(s)

Figure 3.
FIG. 3. The electron density maps of the substrates and cofactor. A, the omit F[O]-F[C] electron density map of the UDP molecule and Mn2+ ion, contoured at 1.6 (gray) and 6.0 (blue), respectively, superimposed with a ball-and-stick model colored according to atom types (nitrogen, blue; carbon, black; oxygen, red; phosphorous, purple; manganese, orange). B, the omit F[O]-F[C] electron density map of the N-acetyllactosamine, contoured at 1.6 (gray), superimposed with a ball-and-stick model. C, the interactions between Mn2+, UDP, and Asp197 side chain of GlcAT-P. The Mn2+ interactions are shown in blue dashed lines.

Figure 4.
FIG. 4. Comparison of GlcAT-P quaternary complex (UDP, Mn2+, and N-acetyllactosamine) with GlcAT-I quaternary complex (UDP, Mn2+, and Gal 1-3Gal). A, dimer structure of GlcAT-P complex. Each monomer is colored blue and yellow, respectively. Substrate molecules are shown in ball-and-stick models. B, dimer structure of GlcAT-I complex is shown in the same orientation as in A. C, dimer surface of GlcAT-P complex is colored according to the electrostatic surface potential (blue, positive; red, negative; scale from -10 to +10 kT/e). D, surface representation of GlcAT-I complex in dimer is shown in the same orientation as in C.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2004, 279, 22693-22703) copyright 2004.

Figures were selected by an automated process.