Date

11-21-2023

Document Type

Thesis

Abstract

With drug-resistant bacteria such as Mycobacterium tuberculosis being a serious public health problem, infected individuals have minimal treatment options. By investigating the electron transfer within 3-ketosteroid-9��-hydroxylase (KshAB), a protein involved in the energy production pathway of this bacteria, it will allow for potential inhibitors of this enzyme to be designed as antibiotics against tuberculosis. The purpose of this project is to stop the interaction between the KshA and KshB complexes to prevent the bacteria from gaining energy by breaking down steroids like cholesterol. To achieve this goal, an arginine at the 286th position of the KshB protein was mutated to alanine as a possible site of electron transfer. The methodology included expressing the KshA wildtype and the KshB mutant proteins in E. coli, purifying both proteins through Nickel-NTA chromatography, confirming the presence and purity for each protein using SDS-PAGE, performing Bradford Assays to calculate their concentrations, and determining the amount of iron in both proteins through Flame Atomic Absorption Spectroscopy. Kinetic assays were performed to further study the enzymatic activity and dictate whether the mutant was successful in stopping the electron transfer between the KshA complex and the KshB complex through Michaelis-Menten kinetic analysis. Using the Origin 2017 program, the following kinetic values for the KshA-KshB-R286A mutant were obtained and calculated: maximum velocity (Vmax) of 130 ± 2.80 µM/min, Michaelis constant (KM) of 48.9 ± 3.77 µM, turnover number (kcat) of 676 ± 16.0 sec-1, and the ratio of kcat/KM being 13.8 ± 4.24 sec-1*μM-1. This mutant does not compare well to the previously studied mutant of KshA-KshB-R286D as well as the wildtype KshAB complex due to discrepancies in iron incorporation necessary for electron transfer. Obstructing this interaction of electron transfer will lead to new antibiotics for those infected by tuberculosis and give professionals an option for treating those individuals.

Department

Chemical Sciences

Thesis Comittee

Dr. Sarah Soltau, Thesis Advisor
Dr. Saritha Nellutla, Committee Member
Dr. Samer Lone, Committee Member

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