PDBsum entry 3ee5

Transferase

PDB id: 3ee5

Contents

Protein chain

273 a.a. *

Ligands

GAL-NAG ×3

UDH ×3

2NA ×3

SO4 ×14

GOL ×7

DIO

MES

Metals

_MN ×3

Waters ×475

* Residue conservation analysis

PDB id:

3ee5

Name:

Transferase

Title:

Crystal structure of human m340h-beta1,4-galactosyltransferase-i (m340h-b4gal-t1) in complex with glcnac-beta1,3-gal-beta- naphthalenemethanol

Structure:

Beta-1,4-galactosyltransferase 1. Chain: a, b, c. Fragment: catalytic domain of beta-1,4-galactosyltransferase. Synonym: beta-1,4-galtase 1, beta4gal-t1, b4gal-t1, udp- galactose:beta-n-acetylglucosamine beta-1,4-galactosyltransferase 1, udp-gal:beta-glcnac beta-1,4-galactosyltransferase 1. Engineered: yes. Mutation: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: b4galt1, ggtb2. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.20Å R-factor: 0.206 R-free: 0.240

Authors:

B.Ramakrishnan,P.K.Qasba

Key ref:

J.R.Brown et al. (2009). Deoxygenated disaccharide analogs as specific inhibitors of beta1-4-galactosyltransferase 1 and selectin-mediated tumor metastasis. J Biol Chem, 284, 4952-4959. PubMed id: 19106107 DOI: 10.1074/jbc.M805782200

Date:

04-Sep-08 Release date: 06-Jan-09

Protein chains

P15291  (B4GT1_HUMAN) -  Beta-1,4-galactosyltransferase 1 from Homo sapiens

Seq: 398 a.a.

Struc: 273 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

Enzyme reactions

• Enzyme class 1: E.C.2.4.1.- - ?????
• Enzyme class 2: E.C.2.4.1.22 - lactose synthase.
• Reaction: D-glucose + UDP-alpha-D-galactose = lactose + UDP + H⁺

D-glucose (Bound ligand (Het Group name = GAL) matches with 91.67% similarity) + UDP-alpha-D-galactose = lactose + UDP + H(+) (Bound ligand (Het Group name = UDH) matches with 78.12% similarity)

• Enzyme class 3: E.C.2.4.1.275 - neolactotriaosylceramide beta-1,4-galactosyltransferase.
• Reaction: a beta-D-GlcNAc-(1->3)-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1)- Cer(d18:1(4E)) + UDP-alpha-D-galactose = a neolactoside nLc4Cer(d18:1(4E)) + UDP + H⁺

beta-D-GlcNAc-(1->3)-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1)- Cer(d18:1(4E)) + UDP-alpha-D-galactose = neolactoside nLc4Cer(d18:1(4E)) + UDP + H(+) (Bound ligand (Het Group name = UDH) matches with 78.12% similarity)

• Enzyme class 4: E.C.2.4.1.38 - beta-N-acetylglucosaminylglycopeptide beta-1,4-galactosyltransferase.
• Reaction: an N-acetyl-beta-D-glucosaminyl derivative + UDP-alpha-D-galactose = a beta-D-galactosyl-(1->4)-N-acetyl-beta-D-glucosaminyl derivative + UDP + H⁺

N-acetyl-beta-D-glucosaminyl derivative + UDP-alpha-D-galactose = beta-D-galactosyl-(1->4)-N-acetyl-beta-D-glucosaminyl derivative + UDP + H(+) (Bound ligand (Het Group name = UDH) matches with 78.12% similarity)

• Enzyme class 5: E.C.2.4.1.90 - N-acetyllactosamine synthase.
• Reaction: N-acetyl-D-glucosamine + UDP-alpha-D-galactose = beta-D-galactosyl- (1->4)-N-acetyl-D-glucosamine + UDP + H⁺

N-acetyl-D-glucosamine (Bound ligand (Het Group name = NAG) matches with 93.33% similarity) + UDP-alpha-D-galactose = beta-D-galactosyl- (1->4)-N-acetyl-D-glucosamine + UDP + H(+) (Bound ligand (Het Group name = UDH) matches with 78.12% similarity)

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

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

reference

DOI no: 10.1074/jbc.M805782200

J Biol Chem 284:4952-4959 (2009)

PubMed id: 19106107

Deoxygenated disaccharide analogs as specific inhibitors of beta1-4-galactosyltransferase 1 and selectin-mediated tumor metastasis.

J.R.Brown, F.Yang, A.Sinha, B.Ramakrishnan, Y.Tor, P.K.Qasba, J.D.Esko.

ABSTRACT

The disaccharide peracetylated GlcNAcbeta1-3Galbeta-O-naphthalenemethanol (disaccharide 1) diminishes the formation of the glycan sialyl Lewis X (Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3) GlcNAc; sLe(X)) in tumor cells. Previous studies showed that the mechanism of action of disaccharide 1 involves three steps: (i) deacetylation by carboxyesterases, (ii) action as a biosynthetic intermediate for downstream enzymes involved in sLe(X) assembly, and (iii) generation of several glycans related to sLe(X). In this report, we show that GlcNAcbeta1-3Galbeta-O-naphthalenemethanol binds to the acceptor site of human beta1-4-galactosyltransferase much like the acceptor trisaccharide, GlcNAcbeta1-2Manbeta1-6Man, which is present on N-linked glycans. The 4'-deoxy analog, in which the acceptor hydroxyl group was replaced by -H, did not act as a substrate but instead acted as a competitive inhibitor of the enzyme. The acetylated form of this compound inhibited sLe(X) formation in U937 monocytic leukemia cells, suggesting that it had inhibitory activity in vivo as well. A series of synthetic acetylated analogs of 1 containing -H, -F, -N(3), -NH(2), or -OCH(3) instead of the hydroxyl groups at C-3'- and C-4'-positions of the terminal N-acetylglucosamine residue also blocked sLe(X) formation in cells. The reduction of sLe(X) by the 4'-deoxy analog also diminished experimental tumor metastasis by Lewis lung carcinoma in vivo. These data suggest that nonsubstrate disaccharides have therapeutic potential through their ability to bind to glycosyltransferases in vivo and to alter glycan-dependent pathologic processes.

Selected figure(s)

Figure 1.
Chemical structure of per-O-acetylated GlcNAcβ1–3Galβ-O-naphthalenemethanol ( 1 ) and C-3′ and C-4′ hydroxyl-modified analogs ( 2 – 9 ).

Figure 2.
Co-crystallization of deacetylated 1 with the open form of the human enzyme, hM340H-Gal-T1. a, binding of deacetylated 1 to the catalytic domain of β4Gal-T1, in the presence of Mn^2+ (purple sphere) and UDP-hexanolamine (UDP-H). The β4Gal-T1 molecule is found in the closed conformation with its Trp-310 side chain (red) placed inside the catalytic pocket, interacting with the phosphate oxygen atom of UDP-hexanolamine molecule, whereas the long flexible loop (blue) covers the UDP-hexanolamine and exposes the acceptor binding site to facilitate binding to the enzyme. b, the molecular interactions of deacetylated 1 (blue) with the β4Gal-T1 (green). The hydrogen bonds are shown in black dotted lines. The GlcNAc moiety of deacetylated 1 is bound in the acceptor sugar binding site. The Gal residue forms hydrophobic interactions with the aromatic side chain of the Tyr-282 residue, whereas the naphthalenemethanol extends out of the sugar binding site, weakly interacting with the β4Gal-T1 molecule. There is a structural water molecule (W) indicated with black dotted lines that, in addition to forming a hydrogen bond with the side-chain amino group of Arg-355, bridges the GlcNAc and Gal via hydrogen bonds. c, molecular surface (van der Waals) diagram showing the binding of deacetylated trisaccharide GlcNAcβ1–2Manα1–6Manβ-O-R (where R represents -CH[2]–CH[2]–CH[2]–CH=CH[2] (15)) to β4Gal-T1 (light blue). d, molecular surface (van der Waals) diagram showing the binding of deacetylated disaccharide 1 to β4Gal-T1 (light blue). e, superposition of the bound deacetylated disaccharide 1 (blue) with the bound trisaccharide, GlcNAcβ1–2Manα1–6Manβ-O-R (yellow) in the respective acceptor substrate complexes with β4Gal-T1 (blue).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2009, 284, 4952-4959) copyright 2009.

Figures were selected by an automated process.