Layre, EmilieLee, Ho JunYoung, David C.Martinot, Amanda JezekButer, JeffreyMinnaard, Adriaan J.Annand, John W.Fortune, Sarah M.Snider, Barry B.Matsunaga, IsamuRubin, Eric J.Alber, TomMoody, D. Branch2019-10-152014Layre, E., H. J. Lee, D. C. Young, A. Jezek Martinot, J. Buter, A. J. Minnaard, J. W. Annand, et al. 2014. “Molecular Profiling of Mycobacterium Tuberculosis Identifies Tuberculosinyl Nucleoside Products of the Virulence-Associated Enzyme Rv3378c.” Proceedings of the National Academy of Sciences 111 (8): 2978–83. https://doi.org/10.1073/pnas.1315883111.0027-84240744-28311091-6490http://nrs.harvard.edu/urn-3:HUL.InstRepos:41552031To identify lipids with roles in tuberculosis disease, we systematically compared the lipid content of virulent Mycobacterium tuberculosis with the attenuated vaccine strain Mycobacterium bovis bacillus Calmette-Guerin. Comparative lipidomics analysis identified more than 1,000 molecular differences, including a previously unknown, Mycobacterium tuberculosis-specific lipid that is composed of a diterpene unit linked to adenosine. We established the complete structure of the natural product as 1-tuberculosiny-ladenosine (1-TbAd) using mass spectrometry and NMR spectroscopy. A screen for 1-TbAd mutants, complementation studies, and gene transfer identified Rv3378c as necessary for 1-TbAd biosynthesis. Whereas Rv3378c was previously thought to function as a phosphatase, these studies establish its role as a tuberculosinyl transferase and suggest a revised biosynthetic pathway for the sequential action of Rv3377c-Rv3378c. In agreement with this model, recombinant Rv3378c protein produced 1-TbAd, and its crystal structure revealed a cis-prenyl transferase fold with hydrophobic residues for isoprenoid binding and a second binding pocket suitable for the nucleoside substrate. The dual-substrate pocket distinguishes Rv3378c from classical cis-prenyl transferases, providing a unique model for the prenylation of diverse metabolites. Terpene nucleosides are rare in nature, and 1-TbAd is known only in Mycobacterium tuberculosis. Thus, this intersection of nucleoside and terpene pathways likely arose late in the evolution of the Mycobacterium tuberculosis complex; 1-TbAd serves as an abundant chemical marker of Mycobacterium tuberculosis, and the extracellular export of this amphipathic molecule likely accounts for the known virulence-promoting effects of the Rv3378c enzyme.en-USJournal Article2019-10-1510.1073/pnas.1315883111