Structural and Biochemical Characterization of the Diaminopimelate Epimerase DapF From Pseudomonas aeruginosa PA14.

The emergence of antibiotic resistance in bacteria is a major threat to global public health. To combat increasingly drug-resistant bacteria like Pseudomonas aeruginosa, new targets for pharmaceuticals need to be explored. One such potential target for P. aeruginosa infections is the diaminopimelate epimerase DapF, a protein that plays an essential role in bacterial lysine and peptidoglycan synthesis. In this study, the diaminopimelate epimerase of Pseudomonas aeruginosa PA14 (PA14_69690) is characterized by a crystal structure at 1.69 Å resolution, revealing a homodimer in the open conformation in which one of the monomers was found inactivated by disulfide formation between the two catalytic cysteines of the active site. Further, the influence of substrate and product on the stability of the enzyme has been investigated, finding that both significantly increase the melting temperature. We determined enzyme kinetic parameters with a coupled enzyme assay. With KM = 211 μM (95% confidence interval: 169-267 μM) and kcat of 76 s-1 (95% confidence interval: 69-85 s-1), P. aeruginosa PA14 DapF has activity levels comparable to homologues from other bacterial species. Finally, we show that a previously observed interaction between DapF and the Nudix hydrolase RppH in E. coli is conserved in P. aeruginosa. Soluble recombinant P. aeruginosa RppH could only be obtained when the hydrolase was co-expressed with DapF, hinting at a moonlighting chaperone activity of P. aeruginosa DapF. The catalytic activity of DapF was not affected by complex formation with RppH.