3-Ketosteroid-9α-hydroxylase (KshAB) is a two component enzyme that hydroxylates the 9-position of steroid intermediates found in many bacterial species including <i>Mycobacterium tuberculosis</i>.  The mechanism of hydroxylation in the oxygenase component, KshA, has been examined previously, while the mechanism of electron transfer through the reductase component is less well understood.  In this presentation, computational and experimental approaches to locate the protein-protein binding site of the reductase, KshB, and the oxygenase, KshA, will be discussed as well as their implications for the mechanism of protein electron transfer through a series of iron-sulfur clusters and a non-heme iron center.  A homology model of KshB was generated from the sequence and the structure of phthalate dioxygenase reductase. This homology model was used in protein-protein docking studies to generate potential binding sites for KshB at its interface with KshA.  Using results of the docking studies we were able to predict binding interactions of the two domains in the protein complex.  Studies are underway to obtain protein crystals of the complex and KshB for further structural analysis.  In addition, site-directed mutagenesis studies were used to interrogate these potential sites of protein-protein interactions for their effects on the hydroxylase activity of KshA.  This presentation will also discuss ongoing size-exclusion chromatography experiments and the additional insights they provide to the oligomeric size and interactions in the KshAB complex.  These ongoing studies will provide insights toward the electron transfer pathway in KshAB and the shape and location of the KshA - KshB binding pocket which may ultimately lead to the design of small molecule targets to block this electron transfer pathway.<br/>

Characterization of protein-protein interactions in 3-ketosteroid-9α-hydroxylase necessary for protein electron transfer

256th National Meeting (2018)

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