Investigating the role of Ser220 mutations in m. tuberculosis IGPS
Presentation Type
Abstract
Faculty Advisor
Nina Goodey
Access Type
Event
Start Date
25-4-2025 12:00 PM
End Date
25-4-2025 1:00 PM
Description
Tuberculosis is an infectious disease that affects millions of people worldwide. In recent years, antibiotic resistance has become a growing concern in treating tuberculosis. Mycobacterium tuberculosis is the bacterial pathogen responsible for tuberculosis. Indole-3-glycerol phosphate synthase (IGPS) catalyzes the fourth step of the tryptophan biosynthesis pathway, an essential amino acid for the survival of M. tuberculosis. IGPS has been suggested as a novel drug target in treating tuberculosis. It catalyzes the conversion of the substrate, CdRP to the product, IGP. Understanding the structural and functional roles of active site amino acid residues is essential for developing a potential inhibitor of IGPS. Ser220 has been hypothesized as an important amino acid residue for the binding of IGPS to CdRP. In order to determine the significance of Ser220 in substrate binding, steady state kinetic parameters were investigated after expression and purification of mutants Ser220Thr and Ser220Ala. Michaelis-Menten kinetic experiments of Wildtype, Ser220Thr, and Ser220Ala IGPS were conducted in order to determine KM and kcat values. This provides an understanding of the effect of these mutations on substrate binding and catalytic activity of MtIGPS. Ser220Thr exhibited weaker substrate binding, indicated by a 22-fold increase in KM and a 10-fold decrease in catalytic activity compared to the wild-type enzyme. Preliminary kinetic studies of Ser220Ala demonstrate a significant increase of KM, however further kinetic studies are needed to draw a clear conclusion on how this mutant interacts with CdRP. These results demonstrate the significance of Ser220Thr in the conversion of CdRP to IGP by MtIGPS.
Investigating the role of Ser220 mutations in m. tuberculosis IGPS
Tuberculosis is an infectious disease that affects millions of people worldwide. In recent years, antibiotic resistance has become a growing concern in treating tuberculosis. Mycobacterium tuberculosis is the bacterial pathogen responsible for tuberculosis. Indole-3-glycerol phosphate synthase (IGPS) catalyzes the fourth step of the tryptophan biosynthesis pathway, an essential amino acid for the survival of M. tuberculosis. IGPS has been suggested as a novel drug target in treating tuberculosis. It catalyzes the conversion of the substrate, CdRP to the product, IGP. Understanding the structural and functional roles of active site amino acid residues is essential for developing a potential inhibitor of IGPS. Ser220 has been hypothesized as an important amino acid residue for the binding of IGPS to CdRP. In order to determine the significance of Ser220 in substrate binding, steady state kinetic parameters were investigated after expression and purification of mutants Ser220Thr and Ser220Ala. Michaelis-Menten kinetic experiments of Wildtype, Ser220Thr, and Ser220Ala IGPS were conducted in order to determine KM and kcat values. This provides an understanding of the effect of these mutations on substrate binding and catalytic activity of MtIGPS. Ser220Thr exhibited weaker substrate binding, indicated by a 22-fold increase in KM and a 10-fold decrease in catalytic activity compared to the wild-type enzyme. Preliminary kinetic studies of Ser220Ala demonstrate a significant increase of KM, however further kinetic studies are needed to draw a clear conclusion on how this mutant interacts with CdRP. These results demonstrate the significance of Ser220Thr in the conversion of CdRP to IGP by MtIGPS.
Comments
Poster presentation at the 2025 Student Research Symposium.