Ligand Binding Interactions of M. Tuberculosis Indole-3-Glycerol Phosphate Synthase
Presentation Type
Poster
Faculty Advisor
Nina Goodey
Access Type
Event
Start Date
26-4-2024 11:15 AM
End Date
26-4-2024 12:15 PM
Description
Mycobacterium tuberculosis, a pathogenic bacteria causing tuberculosis, faces challenges with drug resistance. Indole-3-glycerol phosphate synthase (IGPS) is a potential new drug target within the tryptophan biosynthesis pathway. Steady state kinetic parameters kcat and KM were obtained for wildtype (WT) and mutant Ser220Thr (S220T) IGPS enzymes to examine the importance of the role of Ser220 in substrate binding. Expression and purification of WT and mutant was conducted to obtain protein for the study. Cell cultures of sterile LB broth with kanamycin (50 µg/mL) were inoculated with (WT) and S220T Mycobacterium tuberculosis IGPS plasmids that had been stored as glycerol stock. These plasmids served as expression vectors to produce the desired IGPS proteins in E. coli cells. Once proper targeted growth was ensured, large-scale cell cultures were grown until the cultures presented an ideal optical density of 0.5-0.6 and measured at absorbance at 600 nm. Centrifugation was then necessary to collect a pellet that contained cells with the desired proteins. Once protein expression was complete, Ni2+ resin chromatography was used for removing contaminants that could interfere with the desired protein function. Kinetic experiments were done using a 96-well plate reader to determine kcat and KM parameters for the desired proteins. If the KM of the mutant is altered compared to the wildtype, the results would support our hypothesis that S220 may play an important role in substrate binding. This information can then be leveraged in the design of potential inhibitors that resemble the substrate for this enzyme.
Ligand Binding Interactions of M. Tuberculosis Indole-3-Glycerol Phosphate Synthase
Mycobacterium tuberculosis, a pathogenic bacteria causing tuberculosis, faces challenges with drug resistance. Indole-3-glycerol phosphate synthase (IGPS) is a potential new drug target within the tryptophan biosynthesis pathway. Steady state kinetic parameters kcat and KM were obtained for wildtype (WT) and mutant Ser220Thr (S220T) IGPS enzymes to examine the importance of the role of Ser220 in substrate binding. Expression and purification of WT and mutant was conducted to obtain protein for the study. Cell cultures of sterile LB broth with kanamycin (50 µg/mL) were inoculated with (WT) and S220T Mycobacterium tuberculosis IGPS plasmids that had been stored as glycerol stock. These plasmids served as expression vectors to produce the desired IGPS proteins in E. coli cells. Once proper targeted growth was ensured, large-scale cell cultures were grown until the cultures presented an ideal optical density of 0.5-0.6 and measured at absorbance at 600 nm. Centrifugation was then necessary to collect a pellet that contained cells with the desired proteins. Once protein expression was complete, Ni2+ resin chromatography was used for removing contaminants that could interfere with the desired protein function. Kinetic experiments were done using a 96-well plate reader to determine kcat and KM parameters for the desired proteins. If the KM of the mutant is altered compared to the wildtype, the results would support our hypothesis that S220 may play an important role in substrate binding. This information can then be leveraged in the design of potential inhibitors that resemble the substrate for this enzyme.