Molecular structure of an N-formyltransferase from Providencia alcalifaciens O30

Abstract

The existence of N-formylated sugars in the O-antigens of Gram-negative bacteria has been known since the middle 1980s, but only recently have the biosynthetic pathways for their production been reported. In these pathways, glucose-1-phosphate is first activated by attachment to a dTMP moiety. This step is followed by a dehydration reaction and an amination. The last step in these pathways is catalyzed by N-formyltransferases that utilize N(10) -formyltetrahydrofolate as the carbon source. Here we describe the three-dimensional structure of one of these N-formyltransferases, namely VioF from Providencia alcalifaciens O30. Specifically, this enzyme catalyzes the conversion of dTDP-4-amino-4,6-dideoxyglucose (dTDP-Qui4N) to dTDP-4,6-dideoxy-4-formamido-d-glucose (dTDP-Qui4NFo). For this analysis, the structure of VioF was solved to 1.9 Å resolution in both its apoform and in complex with tetrahydrofolate and dTDP-Qui4N. The crystals used in the investigation belonged to the space group R32 and demonstrated reticular merohedral twinning. The overall catalytic core of the VioF subunit is characterized by a six stranded mixed β-sheet flanked on one side by three α-helices and on the other side by mostly random coil. This N-terminal domain is followed by an α-helix and a β-hairpin that form the subunit:subunit interface. The active site of the enzyme is shallow and solvent-exposed. Notably, the pyranosyl moiety of dTDP-Qui4N is positioned into the active site by only one hydrogen bond provided by Lys 77. Comparison of the VioF model to that of a previously determined N-formyltransferase suggests that substrate specificity is determined by interactions between the protein and the pyrophosphoryl group of the dTDP-sugar substrate.

Keywords: 4,6-dideoxy-4-formamido-d-glucose; N-formyltransferase; O-antigen; Providencia alcalifaciens; VioF; bacterial sugar biosynthesis; formylated sugars; lipopolysaccharide; protein structure.

Scheme 1

Predicted pathway for the production of dTDP-Qui4NF_o.

Scheme 2

Structure of tetrahydrofolate and related derivatives.

Scheme 3

Reactions catalyzed by VioF and WlaRD.

Figure 1

Crystal of VioF. All of the VioF crystals grew as six-sided stellate rods. The crystals demonstrated reticular merohedral twinning.

Figure 2

Diffraction from a VioF crystal. Shown in (a) is a diffraction pattern collected from a VioF crystal. At first glance, it appears like that from a typical single crystal. Upon X-ray data processing it became clear that the crystal was twinned. Shown in (b) are the predicted overlays for the diffraction patterns with domains 1 and 2 circled in red and yellow, respectively. A RLATT representation of the Bragg reflections harvested for indexing is displayed in (c). This view illustrates the manner in which the two lattices intersect.

Figure 3

Structure of VioF. Shown in (a) is a ribbon representation of the VioF dimer with subunits 1 and 2 displayed in light blue and cyan, respectively. The ligands are drawn in stick representations. The electron density corresponding to the bound ligands in subunit 1 is shown in (b). The electron density map was calculated with coefficients of the form F_o − F_c, where F_o was the native structure factor amplitude and F_c was the calculated structure factor amplitude. The map was contoured at 3σ. The electron density for the glutamate portion of the cofactor is weak most likely due to the fact that there are few interactions between it and the protein. A close-up view of the active site is provided in (c). Distances within 3.2 Å between the protein and ligand atoms are indicated by the dashed lines. Ordered water molecules are depicted as red spheres. All figures were prepared with the software package PyMOL.

Figure 4

Comparison of the VioF apoenzyme and the ternary complex structures. A close-up view of the active sites is shown with the models of the apoenzyme and the ternary complex highlighted in white and gray, respectively.

Figure 5

Comparison of the active sites for VioF and WbtJ. The THF cofactor, as observed binding in the VioF active site, is colored in gray (a). Those residues belonging to VioF are presented in violet whereas those belonging to WbtJ are displayed in light blue. A close-up view of the region surrounding the conserved catalytic triad is presented in (b) with the same coloring scheme as described in (a). The first and second numbers in the amino acid labels correspond to VioF and WbtJ, respectively.

Figure 6

Comparison of VioF with WlaRD. A superposition of the ribbon representations for the VioF (violet) and the WlaRD (light blue) subunits is presented in (a). A close-up view of their active sites is shown in (b). Note the close correspondence in the positions of the THF cofactors and the catalytic triads. It is the pyranosyl moieties of the dTDP-sugar substrates that assume markedly different locations within the active site regions. Simple rotations about the β-phosphoryl group of the dTDP-Qui4N ligand can position the C-4' amino group of the sugar to within 4.5 Å of His 94, the presumed catalytic base. This is shown in (c) where the observed conformation of the substrate pyranosyl group is highlighted in purple bonds and the “model” is displayed in gray bonds.