The shikimate pathway produces most compounds, most prominently, the aromatic amino acids. Shikimate kinase (SK) alongside other enzymes of the pathway are essential for viability of pathogens like M. tuberculosis (M.tb). The absence of a mammalian counterpart makes its enzymes attractive targets for development of new antitubercular agents. Our aim is to develop tools to rapidly identify SK inhibitors and characterize their mechanisms of inhibition. Interestingly, M. tuberculosis shikimate kinase (MtSK) is devoid of trp. Sequence alignments and structural studies were used to guide trp substitution on strategic sites in the enzyme. Three variants were: N151W (nucleotide-binding domain), E54W (shikimate-binding domain) and V116W (Lid domain). Kinetic parameters (ATP- and shikimate-dependent) of all three variants were similar to wild-type. The three variants showed characteristic and distinct trp emission spectra. ATP titration produced hyperbolic decreases in fluorescence emission were observed for all variants, with KDs ranging from 0.2 - 0.4 µM, similar to the apparent KM for ATP. In contrast, titration with shikimate produced no change in fluorescence emission by either E54W or N151W MtSK, but there was a 30% decrease in V116W emission intensity in the presence of shikimate. V116 is part of the conformationally dynamic lid domain. This observation may point toward shikimate-induced conformational changes in MtSK. We also evaluated two inhibitors (see below). This is also corroborated by fluorescence anisotropy data. Both compounds produced a hyperbolic decrease in fluorescence intensity. KDs for Compound 1 ranged from 16 to 33 µM depending on the variant evaluated. For each variant, KDs determined for compound 2 were about two fold lower than those of compound 1. ESI-LC-MS data suggest these inhibitors form no covalent adducts with the enzyme and dilution experiments also suggest a slow reversible mechanism is operating. Together, our data suggest that these variants will serve as valuable mechanistic probes of MtSK catalysis and inhibition

Imparting intrinsic flourescence as an approach towards rapid inhibitor screening and mechanistic evaluation of tuberculosis shikimate kinase

254th National Meeting (2017)

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