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 aspartic acid and alanine to get varying chemical properties to replace the original amino acid. Experimentation previously 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. Data collected from these experiments are being used to compare the mutants to the wildtype KshAB data, to determine whether or not the mutations disrupted the electron transfer between the two subunits. Further experimentation will include size exclusion chromatography to determine the presence or lack of interaction between subunits, and computational modeling to theorize possible drugs or ligands that will disrupt the enzyme’s function. Future goals include performing experiments within other bacteria similar to M. tuberculosis that use this enzyme complex. These experiments would use the mutated KshB enzyme to compare how this affects the growth rate on a larger organismal scale. Obstructing the interaction between the A and B subunits successfully 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 infection of TB.

Disruption of the electron transfer system within the protein 3-ketosteroid-9-hydroxylase (KshAB) of Mycobacterium tuberculosis

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.