title: Mycobacterial nicotinate mononucleotide adenylyltransferase: structure, mechanism, and implications for drug discovery.
abstract: 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 Å. A remarkable new feature of the MtNadD structure, compared with other members of this enzyme family, is a 310 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 (Ki ∼ 25 μM) and antimycobacterial activity (MIC80) ∼ 40-80 μM). 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.
authors: From the Sanford-Burnham Medical Research Institute, La Jolla, California 92037.
authors: Irina A
authors: IA
authors: Rodionova
authors: Vertex Pharmaceuticals Incorporated, Boston, Massachusetts 02210.
authors: Harmon J
authors: HJ
authors: Zuccola
authors: Department of Clinical Sciences, Section of Biochemistry, Polytechnic University of Marche, Ancona 60131, Italy.
authors: Leonardo
authors: L
authors: Sorci
authors: From the Sanford-Burnham Medical Research Institute, La Jolla, California 92037.
authors: Alexander E
authors: AE
authors: Aleshin
authors: A. A. Kharkevich Institute for Information Transmission Problems, Russian Academy of Sciences, 127051 Moscow, Russia, and.
authors: Marat D
authors: MD
authors: Kazanov
authors: From the Sanford-Burnham Medical Research Institute, La Jolla, California 92037.
authors: Chen-Ting
authors: CT
authors: Ma
authors: From the Sanford-Burnham Medical Research Institute, La Jolla, California 92037.
authors: Eduard
authors: E
authors: Sergienko
authors: Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts 02115.
authors: Eric J
authors: EJ
authors: Rubin
authors: Vertex Pharmaceuticals Incorporated, Boston, Massachusetts 02210.
authors: Christopher P
authors: CP
authors: Locher
authors: From the Sanford-Burnham Medical Research Institute, La Jolla, California 92037, osterman@sanfordburnham.org.
authors: Andrei L
authors: AL
authors: Osterman
mesh_terms: D000595:Amino Acid Sequence; D000995:Antitubercular Agents / Q000737:chemistry / Q000494:pharmacology*; D018360:Crystallography, X-Ray; D055808:Drug Discovery; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology*; D009612:Nicotinamide-Nucleotide Adenylyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
chemical_list: D000995:Antitubercular Agents; D004791:Enzyme Inhibitors; D009612:Nicotinamide-Nucleotide Adenylyltransferase; C038480:nicotinic acid mononucleotide adenylyltransferase
keywords: Enzyme Inhibitor; Enzyme Structure; NAD Biosynthesis; Protein Engineering; Protein Targeting
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