K-107. Nudix Hydrolases from Mycobacterium tuberculosis as Potential Novel Antibiotic Targets

J. G. Thomson, J. N. Ramos, S. F. O'Handley;
Rochester Inst. of Technology, Rochester, NY.

The Nudix hydrolases cleave substrates containing a nucleoside diphosphate linked to some moiety x, and are identified by the common signature sequence GX5EX7REUXEEXGU (U=I, L, or V). M. tuberculosis contains 11 potential Nudix hydrolases, and we have been systematically characterizing these enzymes as potential novel antibiotic targets. Two such enzymes are Rv2985 diadenosine polyphosphatases (ApnAase) and Rv1160 CTPase, which we have cloned, expressed, purified, and characterized. Rv2985 cleaves Ap4A, Ap5A and Ap6A, and is the major ApnAase in M. tuberculosis. Diadenosine polyphosphatases in other pathogenic bacteria have been shown to be involved in the ability of these pathogens to invade their host, and the next step will be to determine if Rv2985 complements these other ApnAase invasion enzymes. CTP is the feedback inhibitor of pyrimidine biosynthesis, and a precursor to lipid biosynthesis including cell membrane formation, thus Rv1160 may help regulate these pathways through degradation of CTP. We are currently characterizing an E. coli knockout mutant, which is less susceptible to streptomycin, a phenotype consistent with a change in the cell membrane. Preliminary results suggest that Rv1160 complements this E. coli homolog. The next step will be to analyze the cell membranes to see if this change in streptomycin susceptibility correlates with a change in cell membrane biosynthesis. Blocking the ability of a pathogen such as M. tuberculosis to be able to invade its human host, or blocking its ability to regulate cell membrane biosynthesis may be excellent targets for the development of new antibiotics.