Mycobacterial nicotinate mononucleotide adenylyltransferase: Structure, mechanism, and implications for drug discovery

Rodionova, Irina A.; Zuccola, Harmon J.; Sorci, Leonardo; Aleshin, Alexander E.; Kazanov, Marat D.; Ma, Chen -Ting; Sergienko, Eduard; Rubin, Eric J.; Locher, Christopher P.; Osterman, Andrei L.

doi:10.1074/jbc.m114.628016

Mycobacterial nicotinate mononucleotide adenylyltransferase: Structure, mechanism, and implications for drug discovery

Journal Article · Tue Jan 27 23:00:00 EST 2015 · Journal of Biological Chemistry

DOI:https://doi.org/10.1074/jbc.m114.628016· OSTI ID:1347693

Rodionova, Irina A. ^[1]; Zuccola, Harmon J. ^[2]; Sorci, Leonardo ^[3]; Aleshin, Alexander E. ^[4]; Kazanov, Marat D. ^[5]; Ma, Chen -Ting ^[4]; Sergienko, Eduard ^[4]; Rubin, Eric J. ^[6]; Locher, Christopher P. ^[2]; Osterman, Andrei L. ^[4]

Sanford-Burnham Medical Research Institute, La Jolla, CA (United States); DOE Office of Scientific and Technical Information (OSTI)
Vertex Pharmaceuticals Inc., Boston, MA (United States)
Polytechnic Univ. of Marche, Ancona (Italy)
Sanford-Burnham Medical Research Institute, La Jolla, CA (United States)
Russian Academy of Sciences, Moscow (Russia)
Harvard School of Public Health, Boston, MA (United States)

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 (K_i ~ 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. Lastly, these findings point to a possible new strategy for designing inhibitors that lock the enzyme in the inactive over-closed conformation.

View Accepted Manuscript (DOE)

Research Organization:: Univ. of California, Berkeley, CA (United States)

Sponsoring Organization:: USDOE

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1347693

Journal Information:: Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Journal Issue: 12 Vol. 290; ISSN 0021-9258

Publisher:: American Society for Biochemistry and Molecular BiologyCopyright Statement

Country of Publication:: United States

Language:: English

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