Journal
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
Volume 143, Issue 42, Pages 17666-17676Publisher
AMER CHEMICAL SOC
DOI: 10.1021/jacs.1c07970
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Funding
- Texas A&M Agrilife Research
- NIAID-NIH [P01AI095208]
- Welch Foundation [A-0015]
- NIH [P41GM128577]
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The research shows that 5-NIC can serve as an effective inactivator of ICL1. Its design aims to lower the overall charge for improved cell permeability, facilitating the synthesis of pro-drug forms for M. tuberculosis cell-infection models.
The isocitrate lyase paralogs of Mycobacterium tuberculosis (ICL1 and 2) are essential for mycobacterial persistence and constitute targets for the development of antituberculosis agents. We report that (2R,3S)-2-hydroxy-3-(nitromethyl)succinic acid (5-NIC) undergoes apparent retroaldol cleavage as catalyzed by ICL1 to produce glyoxylate and 3-nitropropionic acid (3-NP), the latter of which is a covalent-inactivating agent of ICL1. Kinetic analysis of this reaction identified that 5-NIC serves as a robust and efficient mechanism-based inactivator of ICL1 (k(inact)/K-I = (1.3 +/- 0.1) x 10(3) M-1 s(-1)) with a partition ratio <1. Using enzyme kinetics, mass spectrometry, and X-ray crystallography, we identified that the reaction of the 5-NIC-derived 3-NP with the Cys(191) thiolate of ICL1 results in formation of an ICL1-thiohydroxamate adduct as predicted. One aspect of the design of 5-NIC was to lower its overall charge compared to isocitrate to assist with cell permeability. Accordingly, the absence of the third carboxylate group will simplify the synthesis of pro-drug forms of 5-NIC for characterization in cell-infection models of M. tuberculosis.
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