Pseudomonas aeruginosa is a Gram negative bacterium that is emerging as a multi-drug resistant human pathogen. New antimicrobial agents for treating P. aeruginosa infections are thus urgently required as too the need to identify novel drug targets. Once such target is the enzyme dihydrodipicolinate synthase (DHDPS), which is encoded for by the dapA gene and catalyses the rate-limiting step in the lysine biosynthesis pathway of bacteria. Surprisingly, bioinformatic analyses reveal the presence of two dapA genes in P. aeruginosa that encode for PaDHDPS1 and PaDHDPS2. In order to evaluate these enzymes as potential drug targets, we aimed to compare the structure, function and regulation of PaDHDPS1 and PaDHDPS2. Both PaDHDPS1 and PaDHDPS2 were cloned, expressed and purified to yield >15mg/L of >95% pure products with molecular masses determined by mass spectrometry of 34408 Da and 34863 Da, respectively, agreeing well with theoretical values derived from sequence. Both structures possess the canonical α/β secondary structure as demonstrated by circular dichroism spectroscopy; whilst analytical ultracentrifugation analyses indicate that PaDHDPS1 exists primarily as a tetramer in solution, whilst PaDHDPS2 is dimeric. Enzyme kinetic studies show the enzymes differ in catalytic function with PaDHDPS1 having a higher KMapp and lower maximal rate than PaDHDPS2. Furthermore, kinetic studies also reveal that only PaDHDPS2 is allosterically inhibited by lysine. These studies have elucidated key differences in the structure, function and allosteric regulation of DHDPS1 and DHDPS2 from P. aeruginosa, which provides insight into the development of novel inhibitors against this significant pathogen.