Structural and biochemical characterization of PhzF-like isomerases in Pseudomonas aeruginosa PA14

P. aeruginosa is an opportunistic pathogen that causes severe infections in cystic fibrosis and immunocompromised patients. Due to its resistance against many classes of antibiotics, adequate treatment of those patients is very difficult. This circumstance urges researchers to find target structures in P. aeruginosa and develop new antibacterial compounds. One class of putative drug targets are isomerases with a similar protein fold as PhzF. PhzF and PhzF-like isomerases are often involved in important metabolic pathways such as phenazine biosynthesis, lysine biosynthesis, proline or energy metabolism. In this study, the PhzF-like isomerase genes and proteins PA14_46630, PA14_62410, PA14_28280 and PA14_18020, without associated function, were investigated with X-ray crystallography, biochemical and biological methods. Additionally, the diaminopimelate epimerase PA14_69690 and hydroxyproline epimerase PA14_47840 were investigated with similar methods. The structural analysis for PA14_46630, PA14_62410, PA14_28280 and PA14_18020 revealed for all four proteins an active site with residues very similar to PhzF, enabling them to accept and convert the PhzF substrate (2S,3S)-2,3-dihydro-3-hydroxyanthranilic acid. However, no complex structure with the substrate was obtained. Deleting the gene PA14_46630 in P. aeruginosa, a change in colony morphology was observed on agar plates, but the conducted metabolic and transcriptomic analysis could not explain this observation. Furthermore, an impact on P. aeruginosa virulence or growth was not found for any of these genes in growth studies and Galleria mellonella assays. Even if no specific function could be annotated to these genes and proteins, the results acquired in this work provide starting points for future follow-up studies. For the diaminopimelate epimerase PA14_69690, a crystal structure in the open conformation was obtained and kinetic and thermal stability experiments were performed. Complex crystal structures of the hydroxyproline epimerase PA14_47840 with proline analogues in the active site provide a deeper insight into the ligand binding mode. Additionally, an assay investigating the substrate spectrum of the hydroxyproline epimerase revealed that it is not strictly limited to hydroxyprolines. Altogether, the information provided here about diaminopimelate epimerase PA14_69690 and hydroxyproline epimerase PA14_47840 help to complete the knowledge about these proteins. Determined crystal structures may serve as templates for drug design and help to combat antibiotic resistance of the nosocomial pathogen P. aeruginosa.