Many bacteria have adapted to common medicines and overcome antibiotics via the development of resistance through the emergence of defensive mechanisms. One of these drug-resistant bacteria, Mycobacterium tuberculosis, along with several other bacterial species, possesses one of many defensive mechanisms where the protein-protein complex 3-Ketosteroid-9α-hydroxylase (KshAB) performs a hydroxylation reaction on the 9-position of steroid intermediates. Steroid intermediates, such as cholesterol, become food sources for the bacteria that allow it to remain undamaged inside white blood cells. This defensive mechanism is one of the many degradation pathways that bacteria, including M. tuberculosis, rely on to maintain its infection process despite human immune-cell responses. The protein-protein complex consists of a trimeric oxygenase component (KshA) and a monomeric reductase component (KshB). KshB is proposed to transfer electrons to KshA where the hydroxylation reaction occurs; however, the structure and interactions of KshA and KshB in the KshAB binding site are not still well understood. Therefore, this study investigates the overall shape and structure of the KshAB complex. To reach these goals, plasmid DNA was transformed into BL21-DE3 cells and expressed with IPTG via cell culture. Purification of the proteins was performed through Ni-NTA chromatography. Subsequently the structure and molecular weight of the KshAB complex was repeatedly analyzed by size-exclusion chromatography and native gel electrophoresis. These experiments showed two bands: one band is approximately 250 kDa, which corresponds to the size of a KshA trimer each bound to three KshB monomers, and another with a much higher molecular weight. The higher molecular weight band suggests that there may be protein aggregation occurring. Current work is underway to remove this aggregation through changing buffer conditions including pH, ion concentration, and the addition of non-ionic detergents. Successfully characterizing this enzyme complex will allow for future development of enzyme inhibitors and potentially new antibiotics for tuberculosis.

Investigation to locate the binding site and mode of electron transfer between two proteins, KshA and KshB

2019 Spring National Meeting

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