Bacterial 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (DAHPS) is an antimicrobial target. A transition state (TS) mimic inhibitor of DAHPS, DAHP oxime, was developed in our lab, with Ki = 1.5 μM. Despite being a potent inhibitor, some of DAHP oxime’s properties could hinder its efficacy in blocking bacterial growth. One problem was that DAHP oxime bound competitively with respect to the metal cofactor, Mn2+. Identifying the origin of this competition was crucial for in vivo success of DAHPS inhibitors, as the abundance of metal ions in living cells could hinder DAHP oxime’s effectiveness. Mutant enzymes and fragment-based inhibitors demonstrated that the competition originated in interactions involving the O4 hydroxyl of DAHP oxime and residues Asp326, and Cys61. This suggested improved inhibitor designs to avoid metal competition. Another drawback was the highly negatively charged nature of DAHP oxime and other DAHPS inhibitors. The charge hindered cell membrane penetration, and therefore its effectiveness in cells. Truncating DAHP oxime to a fragment reduced its hydrophilicity and charge, while the introduction of fluorine in the oxime’s α-position increased potency. DAHPS-specific bacterial growth inhibition was obtained with this fragment. DAHPS inhibition by high DAHP oxime concentrations resulted in a 15% residual activity. This residual activity would be sufficient for bacterial survival, so two derivatives, DAHP hydrazone and DAHP O-(2-fluoroethyl) oxime that showed complete inhibition were characterized. DAHP hydrazone was successfully co-crystallized with DAHPS. Its 100-fold higher potency relative to DAHP oxime was attributed to an extra water bound in the active site. The inhibitor bound in all four of the enzyme’s subunits. However, the observed subunit asymmetry showed that the subunits communicate with each other and that there is a significant energetic penalty to enforcing full subunit symmetry. / Thesis / Doctor of Philosophy (PhD) / Antibiotic resistance is imposing a growing disease burden on society that the United Nations calls “…a global crisis that we cannot ignore”. Many traditional antibiotics have now lost their efficacy as bacteria increasingly develop mechanisms of resistance. The development of new treatment options is therefore crucial. The first step towards this goal is the characterization of new antimicrobial targets. DAHPS is an enzyme that is exclusively expressed by bacteria, fungi and plants. Its absence in mammalian cells is expected to reduce side effects in humans. DAHPS inhibitors have previously been developed, but none of them has shown sufficient effectiveness in bacterial cell culture. Possible reasons for this failure were, among others, reduced inhibition in the presence of metal ions, low cell membrane penetration and incomplete enzyme inhibition. This thesis examines DAHPS’s properties and provides solutions on how to successfully inhibit this enzyme, to fight antibiotic resistance with new treatment options.