PDBsum entry 1vzu

Transferase

PDB id: 1vzu

Contents

Protein chains

287 a.a. *

Ligands

BGC-GAL ×2

UDP ×2

GOL ×2

Metals

_MN ×2

Waters ×863

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Roles of active site tryptophans in substrate binding and catalysis by alpha-1,3 galactosyltransferase.

Authors

Y.Zhang, A.Deshpande, Z.Xie, R.Natesh, K.R.Acharya, K.Brew.

Ref.

Glycobiology, 2004, 14, 1295-1302.

PubMed id

15229192

Abstract

Aromatic amino acids are frequent components of the carbohydrate binding sites of lectins and enzymes. Previous structural studies have shown that in alpha-1,3 galactosyltransferase, the binding site for disaccharide acceptor substrates is encircled by four tryptophans, residues 249, 250, 314, and 356. To investigate their roles in enzyme specificity and catalysis, we expressed and characterized variants of the catalytic domain of alpha-1,3 galactosyltransferase with substitutions for each tryptophan. Substitution of glycine for tryptophan 249, whose indole ring interacts with the nonpolar B face of glucose or GlcNAc, greatly increases the K(m) for the acceptor substrate. In contrast, the substitution of tyrosine for tryptophan 314, which interacts with the beta-galactosyl moiety of the acceptor and UDP-galactose, decreases k(cat) for the galactosyltransferase reaction but does not affect the low UDP-galactose hydrolase activity. Thus, this highly conserved residue stabilizes the transition state for the galactose transfer to disaccharide but not to water. High-resolution crystallographic structures of the Trp(249)Gly mutant and the Trp(314)Tyr mutant indicate that the mutations do not affect the overall structure of the enzyme or its interactions with ligands. Substitutions for tryptophan 250 have only small effects on catalytic activity, but mutation of tryptophan 356 to threonine reduces catalytic activity for both transferase and hydrolase activities and reduces affinity for the acceptor substrate. This residue is adjacent to the flexible C-terminus that becomes ordered on binding UDP to assemble the acceptor binding site and influence catalysis. The results highlight the diverse roles of these tryptophans in enzyme action and the importance of k(cat) changes in modulating glycosyltransferase specificity.

Secondary reference #1

Title

Roles of individual enzyme-Substrate interactions by alpha-1,3-Galactosyltransferase in catalysis and specificity.

Authors

Y.Zhang, G.J.Swaminathan, A.Deshpande, E.Boix, R.Natesh, Z.Xie, K.R.Acharya, K.Brew.

Ref.

Biochemistry, 2003, 42, 13512-13521. [DOI no: 10.1021/bi035430r]

PubMed id

14621997

Secondary reference #2

Title

Bovine alpha1,3-Galactosyltransferase catalytic domain structure and its relationship with abo histo-Blood group and glycosphingolipid glycosyltransferases.

Authors

L.N.Gastinel, C.Bignon, A.K.Misra, O.Hindsgaul, J.H.Shaper, D.H.Joziasse.

Ref.

EMBO J, 2001, 20, 638-649. [DOI no: 10.1093/emboj/20.4.638]

PubMed id

11179209

Figure 2.
Figure 2 Close-up stereoview of the 3GalT UDP-Gal-binding site. (A) Hg-UDP-Gal is shown in ball-and-stick form and color coded depending on the nature of the atoms; the Mn2+ ion is shown as a pink sphere. Amino acid side chains interacting with Hg-UDP-Gal are shown in ball-and-stick form in yellow. The acidic residues from the motifs D225VD227 and the D316E317 are shown in ball-and-stick form in red. The four amino acid side chains of 3GalT residues at positions equivalent to the residues distinguishing human A-GT from B-GT are shown in ball-and-stick form in blue. (B) Stereoview of the electron density map (2F[o] - F[c], 1 ) of the Hg-UDP-Gal-binding site.

Figure 6.
Figure 6 Schematic representation of the 3GalT-retaining reaction mechanism. Steps (A) and (B) are derived from the substrate-bound 3GalT structure. The acceptor substrate schematized in steps (C) and (D) is a lactosamine-type glycan (Gal 1,4GlcNAc-R).

The above figures are reproduced from the cited reference which is an Open Access publication published by Macmillan Publishers Ltd

Secondary reference #3

Title

Structure of udp complex of udp-Galactose:beta-Galactoside-Alpha -1,3-Galactosyltransferase at 1.53-A resolution reveals a conformational change in the catalytically important c terminus.

Authors

E.Boix, G.J.Swaminathan, Y.Zhang, R.Natesh, K.Brew, K.R.Acharya.

Ref.

J Biol Chem, 2001, 276, 48608-48614. [DOI no: 10.1074/jbc.M108828200]

PubMed id

11592969

Figure 1.
Fig. 1. a, structure of 3GT with bound UDP and Mn2+ ion. The bound ligand and ion identify the location of the active site. The Mn2+ ion is shown as a magenta sphere, UDP is brown, and helices are pink, while the strands are green. This image was created using the program MOLSCRIPT (38). b, the amino acid sequence of the catalytic domain of 3GT with all secondary structure elements highlighted. UDP binding residues are marked in yellow, while the Mn2+ binding residues are shown by closed magenta spheres. This image was created using the program ALSCRIPT (39). c, stereoview comparison of the C^ atoms of form-II 3GT (present structure, in red) with the previously determined form I 3GT structure (Ref. 18; in black). The C-terminal residues 358-368 in form II show a large difference in conformation and form a lid for the active site tunnel. This image was created using the program BOBSCRIPT (40).

Figure 2.
Fig. 2. a, schematic figure showing the main hydrogen bond interactions between UDP and 3GT residues at the catalytic site of the enzyme. The Mn2+ ion and water molecules are also shown. This image was created using the program MOLSCRIPT (38) and rendered using Raster3D (41). b, the location of UDP molecule in the active site tunnel. This image was created using the program DINO (A. Philippsen; available on the World Wide Web at www.dino3d.org).

The above figures are reproduced from the cited reference with permission from the ASBMB