Crystal structure of TDP-fucosamine acetyltransferase (WecD) from Escherichia coli, an enzyme required for enterobacterial common antigen synthesis

Abstract

Enterobacterial common antigen (ECA) is a polysaccharide found on the outer membrane of virtually all gram-negative enteric bacteria and consists of three sugars, N-acetyl-d-glucosamine, N-acetyl-d-mannosaminuronic acid, and 4-acetamido-4,6-dideoxy-d-galactose, organized into trisaccharide repeating units having the sequence -->3)-alpha-d-Fuc4NAc-(1-->4)-beta-d-ManNAcA-(1-->4)-alpha-d-GlcNAc-(1-->. While the precise function of ECA is unknown, it has been linked to the resistance of Shiga-toxin-producing Escherichia coli (STEC) O157:H7 to organic acids and the resistance of Salmonella enterica to bile salts. The final step in the synthesis of 4-acetamido-4,6-dideoxy-d-galactose, the acetyl-coenzyme A (CoA)-dependent acetylation of the 4-amino group, is carried out by TDP-fucosamine acetyltransferase (WecD). We have determined the crystal structure of WecD in apo form at a 1.95-Angstrom resolution and bound to acetyl-CoA at a 1.66-Angstrom resolution. WecD is a dimeric enzyme, with each monomer adopting the GNAT N-acetyltransferase fold, common to a number of enzymes involved in acetylation of histones, aminoglycoside antibiotics, serotonin, and sugars. The crystal structure of WecD, however, represents the first structure of a GNAT family member that acts on nucleotide sugars. Based on this cocrystal structure, we have used flexible docking to generate a WecD-bound model of the acetyl-CoA-TDP-fucosamine tetrahedral intermediate, representing the structure during acetyl transfer. Our structural data show that WecD does not possess a residue that directly functions as a catalytic base, although Tyr208 is well positioned to function as a general acid by protonating the thiolate anion of coenzyme A.

FIG. 1.

Enzymatic reaction catalyzed by TDP-fucosamine acetyltransferase (WecD). The portion of the CoA cofactor utilized in computational molecular modeling is boxed.

FIG.2.

Three-dimensional structure of E. coli WecD. (a) Stereo view of the WecD monomer. The domains are differently colored; N-terminal domain is shown in cyan and the C-terminal GNAT domain in light pink. (b) The N-terminal domain with secondary structure elements labeled. (c) The C-terminal domain with secondary structure elements labeled. These and subsequent figures were prepared using PyMol (www.pymol.org). (d) Sequence alignment of selected WecD sequences, highlighting the N-terminal extended sequence present in WecD from E. coli CFT073 and Salmonella enterica (blue). The potential general acid, Tyr 208, is highlighted as a green star, and other AcCoA-binding residues are highlighted by cyan squares. Sequence alignment was carried out using ClustalW (7) and formatted using ESPript (23).

FIG.2.

Three-dimensional structure of E. coli WecD. (a) Stereo view of the WecD monomer. The domains are differently colored; N-terminal domain is shown in cyan and the C-terminal GNAT domain in light pink. (b) The N-terminal domain with secondary structure elements labeled. (c) The C-terminal domain with secondary structure elements labeled. These and subsequent figures were prepared using PyMol (www.pymol.org). (d) Sequence alignment of selected WecD sequences, highlighting the N-terminal extended sequence present in WecD from E. coli CFT073 and Salmonella enterica (blue). The potential general acid, Tyr 208, is highlighted as a green star, and other AcCoA-binding residues are highlighted by cyan squares. Sequence alignment was carried out using ClustalW (7) and formatted using ESPript (23).

FIG. 3.

Structure of the WecD dimer. (a) Ribbon representation of the WecD dimer with chain A colored green and chain B colored magenta. (b) Surface representation of the WecD dimer, with acetyl-CoA shown in stick representation. The arrow defines the location of the narrow side of the tunnel in monomer B, while the wide funnel-like side of the tunnel is towards the viewer in monomer A.

FIG. 4.

WecD acetyl-CoA binding site. (a) Stereo view of the Fo-Fc (omit) electron density around AcCoA from subunit A contoured at the 2.5 σ level. Residues interacting with AcCoA are indicated. (b) Interactions between AcCoA and WecD. Residues within 4 Å of CoA are shown. Hydrogen bonds are marked by dashed lines.

FIG. 5.

Structural model of the WecD tetrahedral intermediate resulting from nucleophilic attack of the 4-amino group of TDP-fucosamine at the acetyl-CoA thioester carbon atom (stereo view). The backbone of the WecD protein is rendered as a tube. Select WecD residues lining the putative binding site for TDP-Fuc4N, together with the putative general acid Tyr208, are displayed. Carbon atoms are colored in cyan for the TDP-Fuc4N part, green for the AcCoA part, and yellow for the displayed WecD residues. The other atom types are colored as follows: N, blue; O, red; S, yellow; P, magenta. Intermolecular hydrogen bonds are indicated by green dashed lines. Hydrogen atoms are omitted for clarity.