Abstract 1616 The Role of N189 in M. tuberculosis IGP Synthase, a Potential Tuberculosis Drug Target

Tuberculosis is one of the world's leading infectious diseases. In 2021, 1.6 million people died from tuberculosis around the world. Over the years, new drug resistant strains of tuberculosis have emerged, making the long and difficult treatments that some patients have to go through virtually ineffective. The bacteria that causes tuberculosis is M. tuberculosis. One enzyme inside this bacteria is IGP Synthase (MtIGPS). MtIGPS interacts with its substrate (CdRP) and produces IGP, a metabolic intermediate in the production of tryptophan, an essential amino acid for tuberculosis. We study MtIGPS to learn and understand how it operates and to predict potential inhibitors that would interfere with the interaction between the MtIGPS and CdRP. I have studied the mutated MtIGPS enzymes N189D and N189L, where the 189th amino acid was changed from a asparagine (N) to aspartic acid (D) and leucine (L), respectively. E. coli cells were transformed with the plasmids that coded for the unmutated and the mutated MtIGPS enzymes and grown to express the enzymes. Later, the desired MtIGPS was extracted from the E. coli cells and purified by column chromatography using a nickel resin that binds a His-tag on the MtIGPS. The substrate CdRP was provided by Dr. David Konas and his lab at Montclair State. Steady-state kinetics for the wildtype and mutated MtIGPS enzymes were measured using a microplate reader at 25 °C. The N189D mutation changed the residue's neutral amide charge to a negatively charged carboxyl group. This mutation resulted in a 910 times slower catalytic activity compared to the natural enzyme. The N189L mutation did not change the charge but changed the amide functional group in the sidechain to an isobutyl group. The N189L mutant was found to have a 2,000 times slower catalytic activity than the wild type enzyme. This decrease in activity may be caused by the loss of hydrogen bonds between the substrate or intermediates and MtIGPS mutants compared to the wild type. To further study MtIGPS, I will continue to examine the mutants N189D, N189L and other active site mutations and evaluate steady state kinetics, pH profiles, and determine the rate limiting step of the original and mutated enzymes. We express our sincere gratitude to the National Institute of General Medical Sciences of the National Institutes of Health (#R15GM126467), The American Society for Biochemistry and Molecular Biology, the National Science Foundation through the Garden State-LSAMP (NSF Award 1909824), and the American Heart Association for funding our research.