Dihydrodipicolinate synthase (DHDPS) is an allosteric enzyme that catalyses the rate-limiting and first committed step in the lysine biosynthesis pathway of bacteria. This pathway produces key building blocks for the synthesis of the housekeeping protein, virulence factors and peptidoglycan cell wall. Given its essentiality to pathogenic bacteria and absence in humans DHDPS is considered a promising drug target. Structural studies show that DHDPS contains a ‘druggable’ allosteric cleft that binds the natural inhibitor and downstream product, lysine. This mediates a classic feedback inhibition process leading to reduction in the synthesis of meso-diaminopimelate and lysine. Published studies show that this occurs in plants and Gram-negative bacteria, whilst DHDPS from Gram-positive bacteria lack lysine-mediated allosteric inhibition. However, recent unpublished studies demonstrate that there are exceptions to this dogma with DHDPS from the Gram positive pathogen, Streptococcus pneumoniae, shown to have potent lysine-mediated allosteric inhibition; whereas DHDPS from the Gram negative pathogen, Legionella pneumophila, lacks allosteric inhibition. Accordingly, the aim of this study was to identify the key sequence determinants defining lysine allostery in DHDPS enzymes from Gram negative and Gram positive bacteria. Bioinformatics results show that the presence of either a His or Glu at position 56 (E. coli numbering) defines the molecular signature for DHDPS enzymes allosterically inhibited by lysine; whereas the presence of Lys at position 56 defines non-allosterically regulated sequences. To validate this assertion, we have identified several uncharacterised DHDPS sequences from Gram negative and Gram positive bacteria that contain either His/Glu at position 56 (e.g. Burkholderia pseudomallei and Enterobacter faecalis) or a Lys/Arg at this position (e.g. Coxiella burnetii and Campylobacter sp. 17-). These sequences have been cloned, expressed and the recombinant enzymes purified to homogeneity. Enzyme kinetic and biophysical thermodynamic studies are underway to ascertain the lysine allosteric binding properties of these DHDPS enzymes. The results of this study offer excellent potential to afford translational outcomes including the discovery of potent allosteric inhibitors of DHDPS as new antibiotics.