Using M. marinum T6PP as a Model for M. tuberculosis Inhibitor Design

Abstract

Trehalose-6-phosphate phosphatase (T6PP) is a member of the haloalkanoic acid dehalogenase superfamily (HADSF) acting upon trehalose 6-phosphate, an intermediate in the pathway by which glucose 6-phosphate is converted to trehalose (1). The gene encoding T6PP, otsB, is conserved among bacterial pathogens, such as Mycobacterium tuberculosis, but absent in mammalian species. Bacterial pathogens of the Mycolata taxon utilize trehalose to synthesize cord factor, a mycolic acid-derived membrane glycolipid enabling toxicity, pathogenicity, and phagocyte survival (2). The otsB-knockout of M. tuberculosis precludes pathogen growth in vitro and virulence in a mouse model (2); thus, T6PP inhibitors are desired as therapeutics. Due to the 76% sequence identity between M. marinum and M. tuberculosis T6PP, our study investigates the molecular structure of T6PP from the model organism M. marinum in order to understand the binding determinants of the M. tuberculosis ortholog. Toward this goal, we have determined the unliganded structure of M. marinum to 1.97 □ resolution with a magnesium ion cofactor. The structure consists of 3 domains: the N-terminal, Rossmann-like core and cap domains. Like many other unliganded T6PP structures, the active site, located at cap-core interface, is exposed to solvent and is not in a conformation competent to bind substrate. However, comparison of the individual domains with M. tuberculosis T6PP results in an RMSD score of 0.64, 0.60, and 0.85 for the N-terminal, core and cap domains, respectively. We have measured the steady-state kinetic constants of M. marinum T6PP as kcat of 13 s−1 and a Km of 150 μM, which are comparable to those from the M. tuberculosis enzyme (kcat of 10 s−1 and Km of 500 μM) (3). Together these data confirm that the M. marinum enzyme is a model for structural and kinetic studies. To date, collaboration with The Scripps Research Institute facilitated the discovery of two T6PP inhibitors: clinically employed compounds Closantel and Cephalosporin C. We have determined a Ki for M. marinum T6PP of 39 μM in the presence of Cephalosporin C. Based on clinical data reporting the usage of the drug for tuberculosis therapy (4), we postulate that Cephalosporin C inhibits T6PP by preventing cord factor synthesis and thereby bacterial pathogenicity. In the future, we seek to determine the liganded structure of M. marinum T6PP with potential inhibitors bound. Support or Funding Information This work was funded by the Arnold and Mabel Beckman Foundation and Boston University Undergraduate Research Opportunities Program. Newly determined structure of M. marinum T6PP overlaid onto known structure of M. tuberculosis T6PP. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.