Probing mechanisms of resistance to the tuberculosis drug isoniazid: Conformational changes caused by inhibition of InhA, the enoyl reductase from Mycobacterium tuberculosis

Abstract

The frontline tuberculosis drug isoniazid (INH) inhibits InhA, the NADH-dependent fatty acid biosynthesis (FAS-II) enoyl reductase from Mycobacterium tuberculosis (MTB), via formation of a covalent adduct with NAD + (the INH-NAD adduct). Resistance to INH can be correlated with many mutations in MTB, some of which are localized in the InhA cofactor binding site. While the InhA mutations cause a substantial decrease in the affinity of InhA for NADH, surprisingly the same mutations result in only a small impact on binding of the INH-NAD adduct. Based on the knowledge that InhA interacts in vivo with other components of the FAS-II pathway, we have initiated experiments to determine whether enzyme inhibition results in structural changes that could affect protein-protein interactions involving InhA and how these ligand-induced conformational changes are modulated in the InhA mutants. Significantly, while NADH binding to wild-type InhA is hyperbolic, the InhA mutants bind the cofactor with positive cooperativity, suggesting that the mutations permit access to a second conformational state of the protein. While cross-linking studies indicate that enzyme inhibition causes dissociation of the InhA tetramer into dimers, analytical ultracentrifugation and size exclusion chromatography reveal that ligand binding causes a conformational change in the protein that prevents cross-linking across one of the dimer-dimer interfaces in the InhA tetramer. Interestingly, a similar ligand-induced conformational change is also observed for the InhA mutants, indicating that the mutations modulate communication between the subunits without affecting the two conformational states of the protein that are present.

On the chemical mechanism of succinic semialdehyde dehydrogenase (GabD1) from Mycobacterium tuberculosis

“…This is the case for two other mycobacterial enzymes, enoyl-ACP reductase (InhA) and 3-oxoacyl-ACP reductase (MabA) [39,40]. Conformational changes were suggested as potential cause for these inverse isotope effects, and conformational changes have been described to exist in both, InhA and MabA active sites [41,42]. A similar explanation, which is fully consistent with the chemistry proposed for GabD1 and the inverse primary deuterium KIE observed is offered.…”

A Novel Interaction Linking the FAS-II and Phthiocerol Dimycocerosate (PDIM) Biosynthetic Pathways

“…Mass Spectrometry of B4M-PpsB-Whereas intact B4M-AcpM protein was analyzed directly by LC/MS, PpsB was first digested with trypsin/Glu-C before analysis using the method described previously (12). The digested protein samples were loaded onto a Zorbax 300SB-C18 column (3.5 m, 150 ϫ 0.3 mm; Agilent) and analyzed using a LTQ linear ion trap (Thermo Scientific).…”

“…Approximately 100,000 clones were harvested and isolated for use in library screening efforts. The pBT-kasA bait vector, in which KasA is expressed as a C-terminal fusion with the bacterial phage cl protein, was constructed as described previously (12).…”

“…Library Screening-The pTRG-MTB library was screened with the pBT-kasA bait vector using a standard co-transformation protocol and using nonselective, selective, or dual selective media as described previously (12). Interaction candidates were checked for nonspecific interaction by transformation with control bait proteins.…”

Abordagem racional no planejamento de novos tuberculostáticos: inibidores da InhA, enoil-ACP redutase do M. tuberculosis

“…Adicionalmente, mutações resultantes da substituição dos resíduos Ser94, já mencionados, Ile194, Ile21, Ile95 e Val78 também conferem resistência à INH (Banerjee et al, 1998;Morlock et al, 2003;Kruh et al, 2007;Dias et al, 2007).…”