Evaluating the Impact of the Tyr158 pK_a on the Mechanism and Inhibition of InhA, the Enoyl-ACP Reductase from Mycobacterium tuberculosis

Abstract

InhA, the Mycobacterium tuberculosis enoyl-ACP reductase, is a target for the tuberculosis (TB) drug isoniazid (INH). InhA inhibitors that do not require KatG activation avoid the most common mechanism of INH resistance, and there are continuing efforts to fully elucidate the enzyme mechanism to drive inhibitor discovery. InhA is a member of the short-chain dehydrogenase/reductase superfamily characterized by a conserved active site Tyr, Y158 in InhA. To explore the role of Y158 in the InhA mechanism, this residue has been replaced by fluoroTyr residues that increase the acidity of Y158 up to ∼3200-fold. Replacement of Y158 with 3-fluoroTyr (3-FY) and 3,5-difluoroTyr (3,5-F 2 Y) has no effect on k cat app /K M app nor on the binding of inhibitors to the open form of the enzyme (K i app ), whereas both k cat app /K M app and K i app are altered by seven-fold for the 2,3,5-trifluoroTyr variant (2,3,5-F 3 Y158 InhA). 19 F NMR spectroscopy suggests that 2,3,5-F 3 Y158 is ionized at neutral pH indicating that neither the acidity nor ionization state of residue 158 has a major impact on catalysis or on the binding of substrate-like inhibitors. In contrast, K i * app is decreased 6-and 35-fold for the binding of the slow-onset inhibitor PT504 to 3,5-F 2 Y158 and 2,3,5-F 3 Y158 InhA, respectively, indicating that Y158 stabilizes the closed form of the enzyme adopted by EI*. The residence time of PT504 is reduced ∼four-fold for 2,3,5-F 3 Y158 InhA compared to wild-type, and thus, the hydrogen bonding interaction of the inhibitor with Y158 is an important factor in the design of InhA inhibitors with increased residence times on the enzyme.

Origin of the multi-phasic quenching dynamics in the BLUF domains across the species

“…The 19 F chemical shift is hyper-responsive to different chemical environments 41 and because of the small van der Waals radius, 19 F incorporation is generally biocompatible and structural nonperturbing 42 , 43 . As a result, an extrinsic 19 F label of fluorinated Trp allows the quantification of the Trp conformation distributions free of the protein background NMR signals 44 – 50 . We first carried out NMR experiments on 5-fluorotryptophan (5FW) substituted wild type (WT) BLUF domain in AppA, Sy PixD and Oa PAC, since 5FW is the most frequently used as a 19 F NMR probe among all fluorinated Trp amino acids 41 .…”

Activity of Bacteriophage D29 Loaded on Nanoliposomes against Macrophages Infected with Mycobacterium tuberculosis

“…This binding inhibits the production of mycolic acid, a crucial cell wall component in MTB, resulting in inhibition of cell wall synthesis and causing cell death. INH resistance (INH-R) in several strains is attributed to mutations in genes essential for cell wall synthesis [59]. The mutations are mainly found in the katG gene, the inhA gene and its promoter or in the oxiR-ahpC region.…”