Disruption of key NADH-binding pocket residues of the Mycobacterium tuberculosis InhA affects DD-CoA binding ability

Abstract

Tuberculosis (TB) is a global health problem that affects over 10 million people. There is an urgent need to develop novel antimicrobial therapies to combat TB. To achieve this, a thorough understanding of key validated drug targets is required. The enoyl reductase InhA, responsible for synthesis of essential mycolic acids in the mycobacterial cell wall, is the target for the frontline anti-TB drug isoniazid. To better understand the activity of this protein a series of mutants, targeted to the NADH co-factor binding pocket were created. Residues P193 and W222 comprise a series of hydrophobic residues surrounding the cofactor binding site and mutation of both residues negatively affect InhA function. Construction of an M155A mutant of InhA results in increased affinity for NADH and DD-CoA turnover but with a reduction in Vmax for DD-CoA, impairing overall activity. This suggests that NADH-binding geometry of InhA likely permits long-range interactions between residues in the NADH-binding pocket to facilitate substrate turnover in the DD-CoA binding region of the protein. Understanding the precise details of substrate binding and turnover in InhA and how this may affect protein-protein interactions may facilitate the development of improved inhibitors enabling the development of novel anti-TB drugs.

Examining the role of protein structural dynamics in drug resistance inMycobacterium tuberculosis

“…InhA was prepared via over-expression in E. coli and purified using methods described previously. 22 Enzyme assay data were consistent with previous studies of InhA and have been published elsewhere. 22 To ensure that the prepared enzymes were free of NADH prior to subsequent experiments, InhA samples were checked after purification and subsequent concentration by means of UV-vis absorption.…”

“… 22 Enzyme assay data were consistent with previous studies of InhA and have been published elsewhere. 22 To ensure that the prepared enzymes were free of NADH prior to subsequent experiments, InhA samples were checked after purification and subsequent concentration by means of UV-vis absorption. NADH presence would be indicated by an absorption peak at 340 nm, a feature that was absent in the UV-vis spectra recorded for our samples (see Fig.…”

“…To reaffirm both structural integrity and activity, 1–2 μl of the enzyme was removed, re-diluted to ∼2 μM, and assayed. 22 The highly concentrated InhA was also checked for consistency with low concentration samples via UV-vis spectroscopy. Confirming activity at 0.8 mM was challenging due to the large absorbances of the assay constituents, but when InhA was added to 150 μM NADH and 50 μM DD–CoA, an almost immediate drop to zero of the NADH absorbance was observed, indicating that the InhA was active at 0.8 mM concentration.…”

“…To study this we explored the capability of 2D-IR to reveal changes as a result of ligand binding to an unlabeled protein by examining the amide I spectroscopy of the whole molecule. Combining this with mutational studies of the S94A mutation of InhA that is clinically-implicated in resistance, the INH-susceptible W222A and the biologically-inactive P193A control, which still retains a similar secondary structure to the wild-type, 22 allowed deeper insight into the mechanism of action of INH with InhA ( Fig. 1(c) ).…”

Mycobacterial Cell Wall: A Source of Successful Targets for Old and New Drugs

“…This mutant led to the hypothesis that InhA was the primary target of both INH and ETH [57]. Introduction of the Ser94Ala mutation into wild-type M. tuberculosis H37Rv was shown to be sufficient to confer INH and ETH resistance [60] and to decrease the binding of the INH-NAD adduct to InhA [60,78]. These observations strongly supported the conclusion that INH and ETH target InhA.…”