Inhibition of Mycobacterium tuberculosis InhA by 3‐nitropropanoic acid

3‐Nitropropanoic acid (3NP), a bioactive fungal natural product, was previously demonstrated to inhibit growth of Mycobacterium tuberculosis. Here we demonstrate that 3NP inhibits the 2‐trans‐enoyl‐acyl carrier protein reductase (InhA) from Mycobacterium tuberculosis with an IC50 value of 71 μM, and present the crystal structure of the ternary InhA‐NAD+‐3NP complex. The complex contains the InhA substrate‐binding loop in an ordered, open conformation with Tyr158, a catalytically important residue whose orientation defines different InhA substrate/inhibitor complex conformations, in the “out” position. 3NP occupies a hydrophobic binding site adjacent to the NAD+ cofactor and close to that utilized by the diphenyl ether triclosan, but binds predominantly via electrostatic and water‐mediated hydrogen‐bonding interactions with the protein backbone and NAD+ cofactor. The identified mode of 3NP binding provides opportunities to improve inhibitory activity toward InhA.