Mycobacterial Nicotinate Mononucleotide Adenylyltransferase: STRUCTURE, MECHANISM, AND IMPLICATIONS FOR DRUG DISCOVERY

Background: NAD biosynthesis was implicated as an antibacterial target pathway. Results: Structure-functional analysis of Mycobacterium tuberculosis NadD revealed an over-closed conformation and a sequential kinetic mechanism. Conclusion:M. tuberculosis NadD is a potentially druggable target suitable for the development of selective inhibitors. Significance: This study describes a novel regulatory mechanism and points to a specific strategy for targeting mycobacterial NadD.Nicotinate mononucleotide adenylyltransferase NadD is an essential enzyme in the biosynthesis of the NAD cofactor, which has been implicated as a target for developing new antimycobacterial therapies. Here we report the crystal structure of Mycobacterium tuberculosis NadD (MtNadD) at a resolution of 2.4 angstrom. A remarkable new feature of the MtNadD structure, compared with other members of this enzyme family, is a 3(10) helix that locks the active site in an over-closed conformation. As a result, MtNadD is rendered inactive as it is topologically incompatible with substrate binding and catalysis. Directed mutagenesis was also used to further dissect the structural elements that contribute to the interactions of the two MtNadD substrates, i.e. ATP and nicotinic acid mononucleotide (NaMN). For inhibitory profiling of partially active mutants and wild type MtNadD, we used a small molecule inhibitor of MtNadD with moderate affinity (K-i approximate to 25 m) and antimycobacterial activity (MIC80) approximate to 40-80 m). This analysis revealed interferences with some of the residues in the NaMN binding subsite consistent with the competitive inhibition observed for the NaMN substrate (but not ATP). A detailed steady-state kinetic analysis of MtNadD suggests that ATP must first bind to allow efficient NaMN binding and catalysis. This sequential mechanism is consistent with the requirement of transition to catalytically competent (open) conformation hypothesized from structural modeling. A possible physiological significance of this mechanism is to enable the down-regulation of NAD synthesis under ATP-limiting dormancy conditions. These findings point to a possible new strategy for designing inhibitors that lock the enzyme in the inactive over-closed conformation.