Figure 1.
Schematic of the pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) catalyzed reactions within the folate biosynthetic pathway.
The HPPK module first uses ATP to convert 6-hydroxymethyl-7,8–dihydropterin (DHP) to 6-hydroxymethyl-7,8–dihydropterin-pyrophosphate (DHPPP) with the release of AMP, and the DHPS module then combines DHPPP with p-aminobenzoic acid (pABA) to generate dihydropteroate (DHPteroate) with the release of pyrophosphate. The pterin-ring atoms are labeled on the DHP substrate.
Figure 2.
The primary structure of the HPPK-DHPS bifunctional enzyme from Francisella tularensis and its homology to other HPPK and DHPS enzymes.
The organisms shown are Francisella tularensis (Ft), Saccharomyces cerevisiae (Sc), Yersinia pestis (Yp), Escherichia coli (Ec) and Bacillus anthracis (Ba), and numbering is with respect to the Ft enzyme. Secondary structure elements and key structural regions are labeled according to Fig. 3A. Strictly conserved regions are blocked in red, and conserved regions are boxed. Important loop regions are highlighted and labeled according to their domain association. (A) Multiple sequence alignment of the HPPK module. Residues that contribute to substrate binding are shown as blue triangles. The conserved motif that binds Mg2+ is shown as gray circles within blue triangles. (B) Alignment of the DHPS module. The inter-domain linker regions of F. tularensis and S. cerevisiae are highlighted in green and the corresponding β-hairpin of monofunctional DHPS is highlighted in orange. Residues that interact with substrates are indicated as purple triangles. Residues known to contribute to sulfonamide drug resistance are indicated by red circles. The missing Dα8 helix at the C-terminus is highlighted in purple. Sequence alignments were performed using ClustalW [39] and analyzed using ESPript2.2 [54].
Figure 3.
Analytical ultracentrifugation of the HPPK-DHPS bifunctional enzyme from Francisella tularensis.
(A) The sedimentation velocity profiles (fringe displacement) were fitted to a continuous sedimentation coefficient distribution model c(s). The experiment was conducted at a loading protein concentration of 0.69 mg/ml in at 20°C and at a rotor speed of 60,000 rpm. (B) Absorbance scans at 280 nm at equilibrium are plotted versus the distance from the axis of rotation. The protein was centrifuged at 4°C for at least 24 h at each rotor speed of 15 k (red), 22 k (blue) and 27 k (black) rpm. The solid lines represent the global nonlinear least squares best-fit of all the data sets to a monomer-dimer self-association model with a very weak KD (2.7 mM). The loading protein concentration was 20 µM and the r.m.s. deviation for this fit was 0.0037 absorbance units.
Table 1.
Data Collection and Refinement Statistics.
Figure 4.
The overall structure of the HPPK-DHPS bifunctional enzyme from Francisella tularensis.
(A) A stereo view of the overall fold and domain organization showing the secondary structure elements within each module. Each element is labeled with the prefixes ‘H’ and ‘D’ to reflect their locations in the HPPK (blue) and DHPS (purple) domains, respectively. The N- and C-termini and the linker region (green) are labeled. Note that helix Dα8 in the canonical DHPS TIM-barrel is missing. (B) A surface representation of the view shown in (A) that highlights the position of the domain linker and the cleft within the DHPS module corresponding to the missing Dα8 TIM-barrel α-helix.
Figure 5.
The FtHPPK and FtDHPS modules bound to substrates.
(A) Stereo view of the FtHPPK module showing the detailed interactions with AMPcPP and DHP. Both substrates are covered with transparent molecular surfaces and gray dashed lines indicate putative hydrogen-bond interactions. Residues that contribute to substrate binding are labeled and shown in blue sticks. The inter-domain linker is colored green and a dashed line indicates the position of the carboxy-terminal FtDHPS module. (B) Electron densities for the nucleotide analog AMPcPP (purple) and DHP (yellow) bound to the HPPK module. Two Mg2+ ions (gray spheres labeled Mg1 and Mg2), and an active site water (red sphere labeled W1) are also shown. The arrow indicates how the 6-hydroxymethyl group of DHP is appropriately oriented towards the pyrophosphate moiety of AMPcPP for in-line phosphoryl transfer. (C) Stereo view of the interactions between DHP and the FtDHPS module. DHP is bound within the TIM-barrel (light pink, β-barrel), and the residues that mediate the interaction are labeled and shown in pink sticks. Three structural water molecules are shown as red spheres and are labeled W2, W3 and W4. The location of the FtHPPK module is indicated by a dashed line that extends from the inter-domain linker (green). (D) Electron density for the molecule of DHP (yellow) which bound in the pterin pocket of the FtDHPS module. In (B) and (D), the Fo-Fc simulated-annealing omit electron densities are contoured at 3.5 σ.
Figure 6.
Interaction of the FtDHPS module with Compound 1.
(A) Schematic comparison between the scaffolds of Compound 1 and DHP-PP. Compound 1 comprises a pterin-like core and is missing half of the B-ring as highlighted in orange. (B) Stereo view of Compound 1 (orange) bound within the pterin pocket of the TIM-barrel. Residues that make van der Waals and hydrogen-bond contacts are labeled and shown as pink sticks. The Fo-Fc simulated-annealing omit electron density for Compound 1 is shown as a blue mesh contoured at 3.5 σ.
Figure 7.
The FtHPPK module bound to Compound 1.
(A) Stereo view showing the interactions of Compound 1 (Cmpd 1) within the DHP binding pocket. The orientation is the same as that shown in Fig. 5A. Putative hydrogen-bonds are indicated as gray dashes. Two water molecules (W1 and W2) are shown as red spheres bridging between Compound 1 and loop H2. Note that the side chain of Asp101 is 50∶50 in two orientations, both of which engage Compound 1. Compound 1 is enclosed by the Fo-Fc simulated-annealing omit electron density contoured at 2.5 σ (grey mesh) and 5.0 σ (royal blue). The latter indicates the most probable location of the electron-rich nitro-moiety which dictated the fit. (B) Comparison between the binding orientations of DHP (yellow) and Compound 1. As measured with respect to their ‘nitrogen faces’, the two compounds are rotated by ∼40°. The magnesium ions in the substrate complex at the first and second positions are labeled Mg1 and Mg2, respectively, and interaction with Compound 1 causes Mg2 to bind in a new location as indicated by the arrow.