Nicotinate mononucleotide adenylyltransferase as a potential drug target against Bacillus anthracis

In bacteria, biosynthesis of NAD(P) occurs through either the de novo pathway or by salvaging of precursors, which converge at the point where the reaction of nicotinate mononucleotide (NaMN) with adenosine triphosphate (ATP), is coupled to the formation of nicotinate adenine dinucleotide (NaAD) and inorganic pyrophosphate (PPi). This reaction is catalyzed by nicotinate mononucleotide adenylyltransferase (NMAT) which is essential for bacterial growth making it an attractive drug target for the development of new antibiotics. Steady-state kinetic and direct binding studies on NMAT from Bacillus anthracis suggest a random sequential Bi-Bi kinetic mechanism. Interestingly, the interactions of NaMN and ATP with NMAT were observed to exhibit negative cooperativity, i.e. Hill coefficients < 1.0. Negative cooperativity in binding is also supported from direct binding studies using isothermal titration calorimetry (ITC) and from X-ray crystallographic studies. X-ray structures of the B. anthracis NMAT apoenzyme, and the NaMN and NaAD-bound complexes were determined to resolutions of 2.50 A, 2.60 A and 1.75 A, respectively. The X-ray structure of the NMAT-NaMN complex revealed only one NaMN molecule bound in the biological dimer supporting negative cooperativity in substrate binding. The kinetic, direct-binding, and X-ray structural studies support a model whereby the binding affinity of substrates to the first monomer of NMAT is stronger than to the second and analysis of the three X-ray structures reveals significant conformational changes of NMAT along the enzymatic reaction coordinate. The negative cooperativity observed in B. anthracis NMAT substrate binding is a unique property that has so far not yet been observed in other prokaryotic NMAT enzymes. We propose that regulation of the NAD(P) biosynthetic pathway may in part occur at the reaction catalyzed by NMAT.;High throughput screening methods were utilized to screen two 50,000 compound combinatorial libraries. Three potential lead compounds were identified and mode of inhibitions studies determined that the compounds are mixed-type inhibitors of NMAT.