<i>Chlamydia trachomatis</i> CT771 (<i>nudH</i>) Is an Asymmetric Ap₄A Hydrolase

Asymmetric diadenosine 5′,5‴-P1,P4-tetraphosphate (Ap4A) hydrolases are members of the Nudix superfamily that asymmetrically cleave the metabolite Ap4A into ATP and AMP while facilitating homeostasis. The obligate intracellular mammalian pathogen Chlamydia trachomatis possesses a single Nudix family protein, CT771. As pathogens that rely on a host for replication and dissemination typically have one or zero Nudix family proteins, this suggests that CT771 could be critical for chlamydial biology and pathogenesis. We identified orthologues to CT771 within environmental Chlamydiales that share active site residues suggesting a common function. Crystal structures of both apo- and ligand-bound CT771 were determined to 2.6 Å and 1.9 Å resolution, respectively. The structure of CT771 shows a αβα-sandwich motif with many conserved elements lining the putative Nudix active site. Numerous aspects of the ligand-bound CT771 structure mirror those observed in the ligand-bound structure of the Ap4A hydrolase from Caenorhabditis elegans. These structures represent only the second Ap4A hydrolase enzyme member determined from eubacteria and suggest that mammalian and bacterial Ap4A hydrolases might be more similar than previously thought. The aforementioned structural similarities, in tandem with molecular docking, guided the enzymatic characterization of CT771. Together, these studies provide the molecular details for substrate binding and specificity, supporting the analysis that CT771 is an Ap4A hydrolase (nudH)

<i>Chlamydia trachomatis</i> CT771 (<i>nudH</i>) Is an Asymmetric Ap₄A Hydrolase

Asymmetric diadenosine 5′,5‴-<i>P</i>¹,<i>P</i>⁴-tetraphosphate (Ap₄A) hydrolases are members of the Nudix superfamily that asymmetrically cleave the metabolite Ap₄A into ATP and AMP while facilitating homeostasis. The obligate intracellular mammalian pathogen <i>Chlamydia trachomatis</i> possesses a single Nudix family protein, CT771. As pathogens that rely on a host for replication and dissemination typically have one or zero Nudix family proteins, this suggests that CT771 could be critical for chlamydial biology and pathogenesis. We identified orthologues to CT771 within environmental <i>Chlamydiales</i> that share active site residues suggesting a common function. Crystal structures of both apo- and ligand-bound CT771 were determined to 2.6 Å and 1.9 Å resolution, respectively. The structure of CT771 shows a αβα-sandwich motif with many conserved elements lining the putative Nudix active site. Numerous aspects of the ligand-bound CT771 structure mirror those observed in the ligand-bound structure of the Ap₄A hydrolase from <i>Caenorhabditis elegans</i>. These structures represent only the second Ap₄A hydrolase enzyme member determined from eubacteria and suggest that mammalian and bacterial Ap₄A hydrolases might be more similar than previously thought. The aforementioned structural similarities, in tandem with molecular docking, guided the enzymatic characterization of CT771. Together, these studies provide the molecular details for substrate binding and specificity, supporting the analysis that CT771 is an Ap₄A hydrolase (<i>nudH</i>)

Two Distinct Ferritin-like Molecules in <i>Pseudomonas aeruginosa</i>: The Product of the <i>bfrA</i> Gene Is a Bacterial Ferritin (FtnA) and Not a Bacterioferritin (Bfr)

Two distinct types of ferritin-like molecules often coexist in bacteria, the heme binding bacterioferritins (Bfr) and the non-heme binding bacterial ferritins (Ftn). The early isolation of a ferritin-like molecule from Pseudomonas aeruginosa suggested the possibility of a bacterioferritin assembled from two different subunits [Moore, G. R., et al. (1994) Biochem. J. 304, 493–497]. Subsequent studies demonstrated the presence of two genes encoding ferritin-like molecules in P. aeruginosa, designated bfrA and bfrB, and suggested that two distinct bacterioferritins may coexist [Ma, J.-F., et al. (1999) J. Bacteriol. 181, 3730–3742]. In this report, we present structural evidence demonstrating that the product of the bfrA gene is a ferritin-like molecule not capable of binding heme that harbors a catalytically active ferroxidase center with structural properties similar to those characteristic of bacterial and archaeal Ftns and clearly distinct from those of the ferroxidase center typical of Bfrs. Consequently, the product of the bfrA gene in P. aeruginosa is a bacterial ferritin, which we propose should be termed Pa FtnA. These results, together with the previous characterization of the product of the bfrB gene as a genuine bacterioferritin (Pa BfrB) [Weeratunga, S. J., et al. (2010) Biochemistry 49, 1160–1175], indicate the coexistence of a bacterial ferritin (Pa FtnA) and a bacterioferritin (Pa BfrB) in P. aeruginosa. In agreement with this idea, we also obtained evidence demonstrating that release of iron from Pa BfrB and Pa FtnA is likely subject to different regulation in P. aerugionsa. Whereas the efficient release of iron stored in Pa FtnA requires only the input of electrons from a ferredoxin NADP reductase (Pa Fpr), the release of iron stored in Pa BfrB requires not only electron delivery by Pa Fpr but also the presence of a “regulator”, the apo form of a bacterioferritin-associated ferredoxin (apo Pa Bfd). Finally, structural analysis of iron uptake in crystallo suggests a possible pathway for the internalization of ferroxidase iron into the interior cavity of Pa FtnA

Structural Studies of Bacterioferritin B from <i>Pseudomonas aeruginosa</i> Suggest a Gating Mechanism for Iron Uptake via the Ferroxidase Center,

The structure of recombinant Pseudomonas aeruginosa bacterioferritin B (Pa BfrB) has been determined from crystals grown from protein devoid of core mineral iron (as-isolated) and from protein mineralized with ∼600 iron atoms (mineralized). Structures were also obtained from crystals grown from mineralized BfrB after they had been soaked in an FeSO4 solution (Fe soak) and in separate experiments after they had been soaked in an FeSO4 solution followed by a soak in a crystallization solution (double soak). Although the structures consist of a typical bacterioferritin fold comprised of a nearly spherical 24-mer assembly that binds 12 heme molecules, comparison of microenvironments observed in the distinct structures provided interesting insights. The ferroxidase center in the as-isolated, mineralized, and double-soak structures is empty. The ferroxidase ligands (except His130) are poised to bind iron with minimal conformational changes. The His130 side chain, on the other hand, must rotate toward the ferroxidase center to coordinate iron. In comparison, the structure obtained from crystals soaked in an FeSO4 solution displays a fully occupied ferroxidase center and iron bound to the internal, Fe(in), and external, Fe(out), surfaces of Pa BfrB. The conformation of His130 in this structure is rotated toward the ferroxidase center and coordinates an iron ion. The structures also revealed a pore on the surface of Pa BfrB that likely serves as a port of entry for Fe2+ to the ferroxidase center. On its opposite end, the pore is capped by the side chain of His130 when it adopts its “gate-closed” conformation that enables coordination to a ferroxidase iron. A change to its “gate-open”, noncoordinative conformation creates a path for the translocation of iron from the ferroxidase center to the interior cavity. These structural observations, together with findings obtained from iron incorporation measurements in solution, suggest that the ferroxidase pore is the dominant entry route for the uptake of iron by Pa BfrB. These findings, which are clearly distinct from those made with Escherichia coli Bfr [Crow, A. C., Lawson, T. L., Lewin, A., Moore, G. R., and Le Brun, N. E. (2009) J. Am. Chem. Soc. 131, 6808−6813], indicate that not all bacterioferritins operate in the same manner

Superposition of ADH-NADPH (green) with YADH (coral, PDB 2HCY) and HADH (blue, PDB 5ADH).

<p>Active site ligands are drawn as cylinders and Zn²⁺ ions are represented as spheres.</p

The Zn²⁺ binding site of PyAeADHII.

<p><b>a) Zn²⁺ binding in the active site of ADH-WT subunit A.</b> The phased anomalous map contoured at 5σ calculated using the Zn-peak data, is shown as blue mesh. b) Metal binding site for Co-substituted PyAeADHII with the cobalt ion drawn as a red sphere. Phased anomalous difference maps contoured at 5σ were calculated using Co-peak data (orange) and Zn²⁺-peak data (blue) and revealed that both ions were present.</p

Active site view of ADH-NADPH (green) with YADH (coral, PDB 2HCY) and HADH (blue, PDB 5ADH) (a).

<p>YADH and HADH have adenosine-5-diphosphoribose and nicotinamide-8-iodo-adenine-dinucleotide bound in the active site respectively. Active site ligands are drawn as cylinders and active site Zn²⁺ ions for YADH and HADH are represented as spheres. b) Superposition of ADH-WT (magenta) and ADH-NADPH (green). The NADPH molecule and Cys residues in the metal binding site are drawn as cylinders. The Zn²⁺ ions associated with ADH-WT and ADH-NADPH are drawn as grey and blue spheres respectively. Regions with the largest conformational differences are highlighted.</p

Replacing the Axial Ligand Tyrosine 75 or Its Hydrogen Bond Partner Histidine 83 Minimally Affects Hemin Acquisition by the Hemophore HasAp from <i>Pseudomonas aeruginosa</i>

Hemophores from <i>Pseudomonas aeruginosa</i> (HasAp), <i>Serratia marcescens</i> (HasA_sm), and <i>Yersinia pestis</i> (HasA_yp) bind hemin between two loops. One of the loops harbors conserved axial ligand Tyr75 (Y75 loop) in all three structures, whereas the second loop (H32 loop) contains axial ligand His32 in HasAp and HasA_sm, but a noncoordinating Gln32 in HasA_yp. Binding of hemin to the Y75 loop of HasAp or HasA_sm causes a large rearrangement of the H32 loop that allows His32 coordination. The Q32 loop in apo-HasA_yp is already in the closed conformation, such that binding of hemin to the conserved Y75 loop occurs with minimal structural rearrangement and without coordinative interaction with the Q32 loop. In this study, structural and spectroscopic investigations of the hemophore HasAp were conducted to probe (i) the role of the conserved Tyr75 loop in hemin binding and (ii) the proposed requirement of the His83–Tyr75 hydrogen bond to allow the coordination of hemin by Tyr75. High-resolution crystal structures of H83A holo-HasAp obtained at pH 6.5 (0.89 Å) and pH 5.4 (1.25 Å) show that Tyr75 remains coordinated to the heme iron, and that a water molecule can substitute for N_δ of His83 to interact with the O_η atom of Tyr75, likely stabilizing the Tyr75–Fe interaction. Nuclear magnetic resonance spectroscopy revealed that in apo-Y75A and apo-H83A HasAp, the Y75 loop is disordered, and that disorder propagates to nearby elements of secondary structure, suggesting that His83 N_δ–Tyr75 O_η interaction is important to the organization of the Y75 loop in apo-HasA. Kinetic analysis of hemin loading conducted via stopped-flow UV–vis and rapid-freeze-quench resonance Raman shows that both mutants load hemin with biphasic kinetic parameters that are not significantly dissimilar from those previously observed for wild-type HasAp. When the structural and kinetic data are taken together, a tentative model emerges, which suggests that HasA hemophores utilize hydrophobic, π–π stacking, and van der Waals interactions to load hemin efficiently, while axial ligation likely functions to slow hemin release, thus allowing the hemophore to meet the challenge of capturing hemin under inhospitable conditions and delivering it selectively to its cognate receptor

Binding interaction of α-tetralone in the substrate binding site of PyAeADHII/NADPH predicted by AutoDock.

<p>a) Binding mode 1, α-tetralone was positioned on top of the nicotinamide ring as a stacking interaction, and the oxygen atom formed a H-bonding interaction with the side chain of residue Asn-39 (3.5 Å). b) α-tetralone again forms stacking interaction with nicotinamide ring, but the oxygen atom was orientated towards residue Arg-88 forming a H-bonding interaction (3.0 Å). PyAeADHII is shown as a cartoon (green) and residues in the substrate binding site are shown as sticks (carbon colored in yellow, nitrogen in blue, oxygen in red). NADPH (carbons are in grey) and the substrate α-tetralone (carbon are in orange) are shown as sticks.</p

Absorption spectra of PyAeADHII Co-complex.

<p>The lack of a weak band at 520 nm and the presence of the 740 nm band indicates that cobalt was substituted at the structural site.</p