Cholera is an enteric disease caused by the Gram negative pathogen Vibrio cholerae (Vc). Promising anti-cholera targets include the enzymes functioning in the diaminopimelate (DAP) pathway. The pathway is responsible for de novo synthesis of lysine and its immediate metabolic precursor meso-DAP, which are important building blocks for protein and cell wall synthesis. The first committed step of the DAP pathway is catalysed by dihydrodipicolinate synthase (DHDPS). DHDPS is an allosteric enzyme that binds lysine, the natural inhibitor and end product of the pathway. Structural analysis of DHDPS co-crystallised with lysine reveals that two lysine molecules bind in close proximity (~4Å). This offers the scope for the development of dilysine based compounds as a novel class of DHDPS inhibitors.
Analytical ultracentrifugation studies of Vc-DHDPS show that the enzyme exists as a dimer in solution, but tetramerises in the presence of lysine. SAXS analyses subsequently demonstrate that the lysine-bound Vc-DHDPS tetramer surprisingly mimics the canonical plant DHDPS structure. We, therefore propose a new mechanism of allosteric regulation of Vc-DHDPS coined the Ligand-Induced associatioN by lysine (K) (LINK) model. Given that DHDPS enzymes from plants are more tightly regulated by lysine (IC50: 10-100 µM) compared to bacterial orthologues (IC50: 0.25-1.0 mM), the exciting discovery that Vc-DHDPS adopts a canonical plant architecture in the presence of lysine offers potential to develop high affinity allosteric inhibitors. Indeed, we show using enzyme kinetics studies that Vc-DHDPS exhibits a ping-pong kinetic mechanism (KM Pyruvate = 0.15 +/- 0.01 mM, KM ASA = 0.09 +/- 0.01 mM, kcat = 46 s-1) and displays plant DHDPS-like sensitivity towards lysine inhibition with IC50 = 69.5 +/- 0.064 µM. Crystal trials in the presence and absence of lysine are underway to validate the proposed LINK model. Results of this study offer excellent potential for the design of potent allosteric inhibitors of Vc-DHDPS as novel anti-cholera agent