Globally, there are 10 million cases of tuberculosis (TB) cases each year. Caused by the bacterium Mycobacterium tuberculosis, TB is highly contagious and requires many months to treat. The demand for improved drugs for treating TB has increased due to the rise in TB rates caused by drug-resistance strains. As a basis for developing new drugs to treat TB, we are characterizing enzymes important for M. tuberculosis survival in the human host. One such protein is the poorly characterized S-adenosyl methionine-dependent methyltransferase encoded by the rv1405c gene (Rv1405). Transposon mutagenesis suggests that Rv1405 is essential for survival of M. tuberculosis in mouse spleen cells and transcriptomics studies in a mouse model indicate a 326-fold increase in rv1405c expression following infection. The specific objectives of this project are to clone, express, purify, assay, and determine the X-ray crystal structure of recombinant Rv1405 to afford further characterization of the enzyme function in mycobacteria. Differential Scanning Fluorimetry was employed to screen various inhibitor libraries to identify compounds that bind Rv1405. To assess the inhibitory activity of the compounds identified in the DSF screening, enzymatic studies were performed. Although knowledge of the Rv1405 methyl acceptor is lacking, initial enzymatic studies using a panel of different chemotypes as methyl acceptors in an Rv1405 catalyzed reaction showed that Tris functions as a methyl acceptor in the enzymatic assay and can be used as a surrogate substrate in inhibitor-screening experiments until the biological substrate is identified. Structural studies have been initiated through Rv1405 crystallization experiments. Initial crystals diffracting to 8 Å resolution have been obtained and are currently being optimize to improve diffraction data quality. Obtaining the X-ray crystal structure will provide crucial information to infer the identity of the methyl acceptor, the mode of methyl acceptor binding, and advance drug development of new TB drugs that target Rv1405.

Crystallization, enzymatic, and inhibitory studies of M. tuberculosis encoded methyltransferase importance in pathogenesis

2019 Fall National Meeting

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