Crystal Structure of a Histone Deacetylase Homologue from <i>Pseudomonas aeruginosa</i>

Despite the recently growing interest in the acetylation of lysine residues by prokaryotic enzymes, the underlying biological function is still not well understood. Deacetylation is accomplished by proteins that belong to the histone deacetylase (HDAC) superfamily. In this report, we present the first crystal structure of PA3774, a histone deacetylase homologue from the human pathogen <i>Pseudomonas aeruginosa</i> that shares a high degree of homology with class IIb HDACs. We determined the crystal structure of the ligand-free enzyme and protein–ligand complexes with a trifluoromethylketone inhibitor and the reaction product acetate. Moreover, we produced loss of function mutants and determined the structure of the inhibitor-free PA3774_H143A mutant, the inhibitor-free PA3774_Y313F mutant, and the PA3774_Y313F mutant in complex with the highly selective hydroxamate inhibitor PFSAHA. The overall structure reveals that the exceptionally long L1 loop mediates the formation of a tetramer composed of two “head-to-head” dimers. The distinctive dimer interface significantly confines the entrance area of the active site, suggesting a crucial role for substrate recognition and selectivity

Additional file 1: Figure S1. of Substrate specificity and function of acetylpolyamine amidohydrolases from Pseudomonas aeruginosa

Michaelis-Menten kinetics. Figure S2. Impact of SAHA and SATFMK on the growth of P. aeruginosa strain PA01 and PA14 in the presence of glucose. (DOCX 371 kb

Toward Photopharmacological Antimicrobial Chemotherapy Using Photoswitchable Amidohydrolase Inhibitors

Photopharmacological agents exhibit light-dependent biological activity and may have potential in the development of new antimicrobial agents/modalities. Amidohydrolase enzymes homologous to the well-known human histone deacetylases (HDACs) are present in bacteria, including resistant organisms responsible for a significant number of hospital-acquired infections and deaths. We report photopharmacological inhibitors of these enzymes, using two classes of photoswitches embedded in the inhibitor pharmacophore: azobenzenes and arylazopyrazoles. Although both classes of inhibitor show excellent inhibitory activity (nM IC₅₀ values) of the target enzymes and promising differential activity of the switchable <i>E</i>- and <i>Z</i>-isomeric forms, the arylazopyrazoles exhibit better intrinsic photoswitch performance (more complete switching, longer thermal lifetime of the <i>Z</i>-isomer). We also report protein–ligand crystal structures of the <i>E</i>-isomers of both an azobenzene and an arylazopyrazole inhibitor, bound to bacterial histone deacetylase-like amidohydrolases (HDAHs). These structures not only uncover interactions important for inhibitor binding but also reveal conformational differences between the two photoswitch inhibitor classes. As such, our data may pave the way for the design of improved photopharmacological agents targeting the HDAC superfamily

Snapshots of the simulation system after removal of the center-of-mass constraint (set to 0 ns).

<p>The protein is shown in cartoon representation with explicit depiction of positively charged residues (arginine: blue, lysine: red). Lipid molecules have been removed except for the head groups that are depicted as grey spheres. Potassium ions are shown in green, chloride ions in blue. The c-terminus is located on the bottom side.</p

<i>s</i>PB1-F2 generates Ca²⁺ and anion fluxes into liposomes.

<p>(A) Fluorescence of liposomes with Ca²⁺ sensitive dye Fluo3 was recorded before and after adding (at arrow) ionophore Valinomycin (triangle), sPB1-F2_pr8 alone (filled squares) or together with Valinomycin (open squares). Peptide and ionophore were added during the time gap of ca. 1 min indicated in the graph. The presence of the peptide results in an increase in fluorescence indicating an influx of Ca²⁺ into the liposomes. The ionophore enhances Ca²⁺ influx because it prevents building up of a charge, which hinders net Ca²⁺ influx. (B) Fluorescence of liposomes filled with Ca²⁺ sensitive dye Fluo-3 before and after addition (at arrow) of 1 µM peptide to incubation medium. The truncated peptide sPB1-F2_pr8^50–87 results in a fast rise in Fluo3 fluorescence. (C) Fluorescence of liposomes filled with anion sensitive dye lucigenin was measured before and after adding of anion specific ionophore TBT (filled squares, added at arrow 1), sPB1-F2_pr8 (open triangle, arrow 2). The control was left untreated (filled circles); the stepwise drop of the control signal is due to an unspecific drift of the signal. Both ionophore and sPB1-F2_pr8 generate a strong quenching of the lucigenin fluorescence well beyond the control indicating an influx of anions. Peptide and ionophore were added during the time gap of ca. 1 min indicated in the graph.</p

Dependence of various measures for protein stability over the simulation time after removal of the center-of-mass (c.o.m.) constraint (set to 0 ns).

<p>From top to bottom: Root mean square deviation (RMSD) of the protein backbone, <i>z</i> coordinate (membrane normal) of the c.o.m. of the protein (corrected by removing the total membrane drift), the protein's radius of gyration (<i>R_g</i>), and the helical fraction recognized for the fold.</p

Alignment of predicted amino acid sequences of PB1-F2 proteins.

<p>The proteins from A/Puerto Rico/8/34 (H1N1) strain (PB1-F2_pr8), the Spanish flu isolate (PB1-F2_sf) and the bird flu virus (H5N1) (PB1-F2_bf) have an overall identity (*) of ca 60%. The domains, which are predicted by structural prediction algorithms to have a high propensity for α-helixes are marked in gray. The truncated peptide sPB1-F2_pr8^50–87 is underlined.</p

I/V relation of the small (o₁) and large (o₂) <i>s</i>PB1-F2 generated current fluctuation.

<p>(A) Unitary currents were recorded in bilayer with 500 mM KCl on <i>trans</i> side and 500 mM NaCl on trans (open circles) or with 500 mM KCl on cis and 500 mM K-gluconate on trans (filled squares). (B) I/V relation obtained with 500 mM KCl on trans side and 50 mM KCl on cis side. (C) I/V relation obtained with 500 mM KCl on cis and 500 mM CaCl₂ on trans side. Currents were elicited upon adding <i>s</i>PB1-F2_pr8 (in A-C) and sPB1-F2_sf (in C) to trans side.</p

<i>s</i>PB1-F2 evoked membrane conductance.

<p>(A) Addition of <i>s</i>PB1-F2_PR8 protein at 1 µM to the trans side of a planar lipid bilayer results in current fluctuations. In the present case only occasionally clear channel like fluctuations (see expanded trace) are resolvable on the background of many unresolved fluctuations. (B) In a majority of experiments the conductance fluctuates in a channel like manner between a closed (c) and two defined conductance levels o₁ and o₂. Transitions between the two conductance levels (*) are expanded in the left inset. The channel like fluctuations are occasionally interrupted by burst like events (**), which reveal also at higher magnification (inset on the right) no resolvable conductance levels.</p

Defining the Mechanism of Action and Enzymatic Selectivity of Psammaplin A against Its Epigenetic Targets

Psammaplin A (<b>11c</b>) is a marine metabolite previously reported to be a potent inhibitor of two classes of epigenetic enzymes: histone deacetylases and DNA methyltransferases. The design and synthesis of a focused library based on the psammaplin A core has been carried out to probe the molecular features of this molecule responsible for its activity. By direct in vitro assay of the free thiol generated upon reduction of the dimeric psammaplin scaffold, we have unambiguously demonstrated that <b>11c</b> functions as a natural prodrug, with the reduced form being highly potent against HDAC1 in vitro (IC₅₀ 0.9 nM). Furthermore, we have shown it to have high isoform selectivity, being 360-fold selective for HDAC1 over HDAC6 and more than 1000-fold less potent against HDAC7 and HDAC8. SAR around our focused library revealed a number of features, most notably the oxime functionality to be important to this selectivity. Many of the compounds show significant cytotoxicity in A549, MCF7, and W138 cells, with the SAR of cytotoxicity correlating to HDAC inhibition. Furthermore, compound treatment causes upregulation of histone acetylation but little effect on tubulin acetylation. Finally, we have found no evidence for <b>11c</b> functioning as a DNMT inhibitor