Uncovering the mode of electron transport within the 3-ketosteroid-9-hydroxylase, KshAB, enzyme complex from Mycobacterium tuberculosis will reveal previously unknown possibilities towards curing tuberculosis infections. The KshB protein was mutated at one possible site of electron transfer – an arginine at location 286 – to rupture its cooperation with the KshA complex to prevent the bacteria from gaining energy from the breakdown of steroids as a fuel source. The arginine was mutated to an aspartic acid and alanine to get varying chemical properties to replace the original amino acid. Experimentation included over-expressing the wildtype and mutant proteins in E. coli, purifying them through Nickel-NTA column chromatography, and analyzing them for size and purity through SDS-PAGE. The kinetics of KshA and mutant KshB proteins’ electron transfer were quantified through UV-vis kinetic analysis and utilizing the Michaelis-Menten equation to extrapolate the kinetic parameters, KM, Vmax, and kcat. Data collected from these experiments were used to compare the wtKshA/KshB-R286D mutant to the wildtype KshAB data to determine whether or not the mutation disrupted the electron transfer between the two subunits. It was concluded based off of the kinetic data that the rate at which the mutant KshAB enzyme hydroxylates a steroid structure is significantly lower than that of the wildtype KshAB complex. This leads to the observation that the site of mutation is crucial to the electron transfer pathway between the enzyme’s subunits. The overall function is not completely inhibited, but it is decreased significantly enough to assume a successful outcome. Obstructing the interaction between the A and B subunits effectively can open pathways towards a new antibiotic treatment for TB by targeting the bacteria’s mechanism for energy production, benefitting the pharmaceutical industry, medical field, and those infected or at risk of TB infection.
Department of Chemical Sciences
Dr. Sarah R. Soltau, Thesis Advisor
Dr. Saritha Nellutla, Committee Member
Dr. Samer Lone, Committee Member
Copyright and Permissions
Original document was submitted as an Honors Program requirement. Copyright is held by the author.
Esteireiro, Olivia. (2020). Disruption of the electron transfer system within the protein 3-ketosteroid-9α-hydroxylase (KshAB) of Mycobacterium tuberculosis. In BSU Honors Program Theses and Projects. Item 324. Available at: https://vc.bridgew.edu/honors_proj/324
Copyright © 2020 Olivia Esteireiro