Crystal Structure of the 6-Hydroxymethyl-7,8-Dihydropterin Pyrophosphokinase•Dihydropteroate Synthase Bifunctional Enzyme from Francisella tularensis

The 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) enzymes catalyze sequential metabolic reactions in the folate biosynthetic pathway of bacteria and lower eukaryotes. Both enzymes represent validated targets for the development of novel anti-microbial therapies. We report herein that the genes which encode FtHPPK and FtDHPS from the biowarfare agent Francisella tularensis are fused into a single polypeptide. The potential of simultaneously targeting both modules with pterin binding inhibitors prompted us to characterize the molecular details of the multifunctional complex. Our high resolution crystallographic analyses reveal the structural organization between FtHPPK and FtDHPS which are tethered together by a short linker. Additional structural analyses of substrate complexes reveal that the active sites of each module are virtually indistinguishable from those of the monofunctional enzymes. The fused bifunctional enzyme therefore represents an excellent vehicle for finding inhibitors that engage the pterin binding pockets of both modules that have entirely different architectures. To demonstrate that this approach has the potential of producing novel two-hit inhibitors of the folate pathway, we identify and structurally characterize a fragment-like molecule that simultaneously engages both active sites. Our study provides a molecular framework to study the enzyme mechanisms of HPPK and DHPS, and to design novel and much needed therapeutic compounds to treat infectious diseases

Interaction of the ^FtDHPS module with Compound 1.

<p>(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 <i>F</i>o-<i>F</i>c simulated-annealing omit electron density for Compound 1 is shown as a blue mesh contoured at 3.5 σ.</p

The overall structure of the HPPK-DHPS bifunctional enzyme from <i>Francisella tularensis</i>.

<p>(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.</p

Data Collection and Refinement Statistics.

<p>*Data were collected from a single crystal. Values in parentheses are for the highest-resolution shell.</p>a<p>R_free was calculated using 5% of the reflections.</p

Analytical ultracentrifugation of the HPPK-DHPS bifunctional enzyme from <i>Francisella tularensis</i>.

<p>(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 <i>versus</i> 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 <i>solid lines</i> represent the global nonlinear least squares best-fit of all the data sets to a monomer-dimer self-association model with a very weak K_D (2.7 mM). The loading protein concentration was 20 µM and the r.m.s. deviation for this fit was 0.0037 absorbance units.</p

The primary structure of the HPPK-DHPS bifunctional enzyme from <i>Francisella tularensis</i> and its homology to other HPPK and DHPS enzymes.

<p>The organisms shown are <i>Francisella tularensis</i> (Ft), <i>Saccharomyces cerevisiae</i> (Sc), <i>Yersinia pestis</i> (Yp), <i>Escherichia coli</i> (Ec) and <i>Bacillus anthracis</i> (Ba), and numbering is with respect to the Ft enzyme. Secondary structure elements and key structural regions are labeled according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014165#pone-0014165-g003" target="_blank">Fig. 3A</a>. 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 <i>F. tularensis</i> and <i>S. cerevisiae</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014165#pone.0014165-Thompson1" target="_blank">[39]</a> and analyzed using ESPript2.2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014165#pone.0014165-Gouet1" target="_blank">[54]</a>.</p