Aspartate semialdehyde dehydrogenase (ASADH) is an essential enzyme that functions in the biosynthesis of isoleucine, lysine, methionine and threonine in bacteria. ASADH catalyzes the NADPH-dependent reduction of ß-aspartyl phosphate to aspartate ß-semialdehyde (ASA), also forming inorganic phosphate (Pi) as a product. Gene knockout studies in Bacillus subtilis demonstrates that the gene encoding ASADH (asd) is one of only 271 essential genes, providing scope to target the enzyme for microbial intervention. We therefore set out to assess whether asd is also essential to the Gram positive human pathogen, Staphylococcus aureus. The asd gene was knocked out by homologous recombination in the S. aureus Newman strain and shown to be essential in minimal media, with subsequent growth studies demonstrating that the Δasd strain was auxotrophic for lysine, methionine and threonine as expected. We therefore set out next to characterise the structure and function of the asd gene product. S. aureus ASADH was cloned, expressed, and purified to homogeneity as a His-tagged enzyme. We show by circular dichroism spectroscopy and analytical ultracentrifugation that recombinant S. aureus ASADH is folded and exists as a dimer in solution with a modal sedimentation coefficient of 4.0 S. The enzymatic activity of the purified enzyme was then assessed by measuring the ASADH-mediated oxidation of NADPH spectrophotometrically, which shows that the enzyme has a K_M(ASA) = 0.19 mM and a k_cat = 23 sec^-1. We are currently exploring the stability of recombinant S. aureus ASADH in solution and have initiated crystallisation trials in order to determine the high resolution structure of this promising antibiotic target.