Investigating IGP synthase as a potential target for mycobacterium tuberculosis
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 the world's leading cause of death by a single infectious agent, surpassing coronavirus. This epidemic is associated with the bacteria, Mycobacterium Tuberculosis and its antibiotic-resistant nature. As seen in other bacteria, Mycobacterium tuberculosis uses the enzyme indole-3-glycerol phosphate synthase (IGP Synthase) in the tryptophan biosynthetic pathway. IGP Synthase catalyzes the fourth step of this process converting 1-(o-carboxyphenylamino)-1-deoxyribulose 5’-phosphate (CdRP) into its product, indole-3-glycerol phosphate (IGP). A unique feature found in this enzyme is the β-barrel fold where the active site lies inside the channel. As this rare structure is found in only ten percent of known enzymes, it is still a growing area of research. Understanding the active site and controlling the mechanism of this enzyme with a specialized inhibitor could be a potential target to cease bacterial growth and prevent the spread of tuberculosis. We expressed and purified the protein using nickel resin affinity chromatography. Using single-point mutations found in the active site, we ran kinetic experiments to understand the residues involved in each step of catalysis. Michaelis-Menten kinetic parameters such as kcat(1/s) and Km(M) were calculated and compared to the wild type. The proteins were electrophoresed using SDS-Page, demonstrating a pure wild-type IGP Synthase that will be used in future kinetic experiments. Future studies of mutant enzymes will give more insight into the catalytic mechanism leading to specialized inhibitor design.
Investigating IGP synthase as a potential target for mycobacterium tuberculosis
Tuberculosis is the world's leading cause of death by a single infectious agent, surpassing coronavirus. This epidemic is associated with the bacteria, Mycobacterium Tuberculosis and its antibiotic-resistant nature. As seen in other bacteria, Mycobacterium tuberculosis uses the enzyme indole-3-glycerol phosphate synthase (IGP Synthase) in the tryptophan biosynthetic pathway. IGP Synthase catalyzes the fourth step of this process converting 1-(o-carboxyphenylamino)-1-deoxyribulose 5’-phosphate (CdRP) into its product, indole-3-glycerol phosphate (IGP). A unique feature found in this enzyme is the β-barrel fold where the active site lies inside the channel. As this rare structure is found in only ten percent of known enzymes, it is still a growing area of research. Understanding the active site and controlling the mechanism of this enzyme with a specialized inhibitor could be a potential target to cease bacterial growth and prevent the spread of tuberculosis. We expressed and purified the protein using nickel resin affinity chromatography. Using single-point mutations found in the active site, we ran kinetic experiments to understand the residues involved in each step of catalysis. Michaelis-Menten kinetic parameters such as kcat(1/s) and Km(M) were calculated and compared to the wild type. The proteins were electrophoresed using SDS-Page, demonstrating a pure wild-type IGP Synthase that will be used in future kinetic experiments. Future studies of mutant enzymes will give more insight into the catalytic mechanism leading to specialized inhibitor design.
Comments
Poster presentation at the 2025 Student Research Symposium.