Finding the Achilles heel in Pseudomonas aeruginosa
- Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria, Australia.
- Technology Development Group, National Infection Service, Public Health England, Salisbury, UK.
Pseudomonas aeruginosa is an emerging threat due to the increasing rate of multi-drug resistance. This highlights the need to both discover novel antimicrobial agents and identify promising new targets. One such target is the diaminopimelate (DAP) pathway that yields essential metabolites for bacterial survival, namely meso-diaminopimelate and L-lysine. The first committed step of the DAP pathway is the condensation of pyruvate and aspartate semi-aldehyde catalysed by the essential enzyme dihydrodipicolinate synthase (DHDPS). In bacteria, DHDPS is typically encoded for by a single dapA gene, but bioinformatic analysis of the P. aeruginosa genome reveals four putative dapA genes encoding 4 products; DapA1, DapA2, DapA3 & DapA4. In silico analyses show that only DapA1 and DapA2 contain the 7 signature residues known to be critical for DHDPS catalytic function. Not surprisingly, enzyme kinetic studies show that DapA1&2 are catalytically active, whereas DapA3&4 are void of DHDPS function. Despite similar secondary structures, analytical ultracentrifugation shows that DapA1 exists in a dimer-tetramer equilibrium, whilst DapA2 forms a stable dimer. Enzyme kinetic analyses reveal that DapA1 and DapA2 share similar substrate affinities; however, the two enzymes differ in their allosteric inhibition by L-lysine with DapA1 insensitive and DapA2 sensitive to L-lysine. Together, these studies show that P. aeruginosa contains two incorrectly annotated DHDPS enzymes (DapA3 & DapA4) and two bona fide DHDPS enzymes (DapA1 & DapA2) that interestingly differ in their L-Lysine inhibition. These results offer insight into rational approaches for the development of new antibiotic agents targeting DHDPS in P. aeruginosa.