Molecular architecture of an N-formyltransferase from Salmonella enterica O60

Abstract

N-formylated sugars are found on the lipopolysaccharides of various pathogenic Gram negative bacteria including Campylobacter jejuni 81116, Francisella tularensis, Providencia alcalifaciens O30, and Providencia alcalifaciens O40. The last step in the biosynthetic pathways for these unusual sugars is catalyzed by N-formyltransferases that utilize N¹⁰-formyltetrahydrofolate as the carbon source. The substrates are dTDP-linked amino sugars with the functional groups installed at either the C-3' or C-4' positions of the pyranosyl rings. Here we describe a structural and enzymological investigation of the putative N-formyltransferase, FdtF, from Salmonella enterica O60. In keeping with its proposed role in the organism, the kinetic data reveal that the enzyme is more active with dTDP-3-amino-3,6-dideoxy-d-galactose than with dTDP-3-amino-3,6-dideoxy-d-glucose. The structural data demonstrate that the enzyme contains, in addition to the canonical N-formyltransferase fold, an ankyrin repeat moiety that houses a second dTDP-sugar binding pocket. This is only the second time an ankyrin repeat has been shown to be involved in small molecule binding. The research described herein represents the first structural analysis of a sugar N-formyltransferase that specifically functions on dTDP-3-amino-3,6-dideoxy-d-galactose in vivo and thus adds to our understanding of these intriguing enzymes.

Keywords: Ankyrin repeat; Lipopolysaccharide; N(10)-Formyltetrahydrofolate; N-Formyltransferase; O-antigen; dTDP-3-formamido-3,6-dideoxy-d-galactose.

Fig. 1

Overall structure of FdtF. A ribbon representation of the FdtF subunit is presented in stereo in (a). The N-terminal and middle domains are highlighted in violet and wheat, respectively. The ankyrin repeat is displayed in light blue. The electron densities shown were calculated with (F_o-F_c) coefficients and contoured at 3σ. The ligands were not included in the X-ray coordinate file used to calculate the omit map, and thus there is no model bias. The FdtF dimer, shown in a ribbon representation, is displayed in (b). The subunit:subunit interface lies between the N-terminal and middle domains. All figures were prepared with the software package PyMOL (DeLano, 2002).

Fig. 2

Close-up stereo views of the dTDP ligand binding pockets. Shown in (a) and (b) are the protein regions surrounding the ligands in the active site and the auxiliary binding pockets, respectively. Ordered water molecules are represented by the red spheres. Possible hydrogen bonding interactions are indicated by the dashed lines. It was not possible to place a label for Asn 334 in (b) without obscuring its side chain position or the position of β-phosphoryl group of the ligand. Thus, for the sake of clarity, the position of Asn 334 is marked by an asterisk.

Fig. 3

Electron densities corresponding to the bound ligands. The electron densities corresponding to the bound dTDP-Fuc3N (green) and N⁵-formyl-THF (blue) ligands in the active site are displayed in (a). The electron density for the dTDP-Fuc3N ligand located in the auxiliary binding pocket is presented in (b). The electron density maps were calculated with (F_o -F_c) coefficients and contoured at 3σ. The ligands were not included in the X-ray coordinate file used to calculate the omit map, and thus there is no model bias.

Fig. 4

Close-up stereo views of the ligand binding pockets in the FdtF/dTDP-Fuc3N/N⁵-formyl-THF ternary complex. Shown in (a) and (b) are the active site and the auxiliary binding pocket regions, respectively. Ordered water molecules are represented by the red spheres. Possible hydrogen bonding interactions are indicated by the dashed lines.

Fig. 4

Close-up stereo views of the ligand binding pockets in the FdtF/dTDP-Fuc3N/N⁵-formyl-THF ternary complex. Shown in (a) and (b) are the active site and the auxiliary binding pocket regions, respectively. Ordered water molecules are represented by the red spheres. Possible hydrogen bonding interactions are indicated by the dashed lines.

Fig. 5

Comparison of the binding of N⁵-formyl-THF versus THF. Shown in stereo is a superposition of the observed binding locations for the ligands in the FdtF/ dTDP-Fuc3N/N⁵-formyl-THF and FdtF/dTDP-Fuc3N/THF ternary complexes. The N⁵-formyl-THF and THF cofactors are depicted in gold and white bonds, respectively. The location of N5 in the N⁵-formyl-THF ligand is indicated by the asterisk.

Fig. 6

Comparison of the binding of dTDP-Fuc3N versus dTDP-Qui3N. A stereo view of the FdtF active sites with either bound dTDP-Fuc3N or dTDP-Qui3N is presented in (a). The blue highlighted bonds correspond to the FdtF model with bound dTDP-Qui3N. Shown in (b) is a stereo view of the FdtF auxiliary binding pocket with either bound dTDP-Fuc3N or dTDP-Qui3N. The FdtF/dTDP-Qui3N model is displayed in blue bonds.

Fig. 7

Initial velocities experiments. Shown are plots of the initial velocities of wild-type FdtF (a) and the E395A mutant variant (b) versus dTDP-Fuc3N concentrations. Plot of initial velocity of the E395A mutant variant against dTDP-Qui3N is shown in (c). Finally, plots of the initial velocities of the W305A/E395A mutant variant against either dTDP-Fuc3N or dTDP-Qui3N are presented in (d) and (e), respectively. Note that the graphs presented have different y-axis scales.