UNDERSTANDING ALLOSTERIC INHIBITION OF DIHYDRODIPICOLINATE SYNTHASE FROM CAMPYLOBACTER JEJUNI
Considering the appearance of superbugs and antibiotic-resistant bacteria there is an urgent need to design new antibiotics. This research has focused on inhibition of dihydrodipicolinate synthase (DHDPS), which is an enzyme that mediates the first unique reaction of L-lysine biosynthesis in the diaminopimelate pathway. Lysine is the natural feedback inhibitor of DHDPS and a crucial component for bacterial cell wall peptidoglycan. DHDPS is the product of an essential gene that is absent in humans. This makes DHDPS a promising target for developing new antibiotics. This research investigates molecular details of the reaction catalyzed by DHDPS from Campylobacter jejuni and its mode of inhibition with natural and synthetic inhibitors to understand more about the allosteric inhibition of this enzyme. For this purpose, a series of allosteric inhibitors of DHDPS were designed and synthesized, and their inhibition of the DHDPS-catalyzed reaction was studied using steady-state kinetics. A number of DHDPS variants including H56W, N84A, N84D, and H59K were generated by site-directed mutagenesis and studied to understand the importance of amino acids at the allosteric site. H56W was used for simple and quick screening of inhibitors on the basis of intrinsic tryptophan fluorescence change. The developed fluorescent assay delivered valuable information on the binding of inhibitors to the allosteric site of DHDPS. N84A and N84D mutants were generated to assess the importance of aspargine84 in the enzymatic function of DHDPS. It was revealed that these mutations disrupt the quaternary structure of DHDPS significantly. In addition, association of N84A/N84D subunits into higher-order oligomers improved the kinetic functionality of these mutants. Lastly, H59K, an inhibition-resistant mutant, was generated. It was found that L-histidine has improved inhibitory properties for H59K compared to lysine and bislysine, however, its IC50 is in the high milimolar range, which is due to lack of a proper hydrogen bond between L-histidine and L-lysine. These results helped to gain new insights into allosteric properties of DHDPS and can be used to improve targeted design of antimicrobial drugs.
DHDPS, Campylobacter jejuni, Fluorescence, Mutation, Inhibitor, Allosteric, Enzyme, kinetic, Assay
Doctor of Philosophy (Ph.D.)