Parikh S L, Xiao G, Tonge P J
Department of Chemistry and Graduate Programs in Biophysics and Molecular and Cellular Biochemistry, State University of New York at Stony Brook, New York 11794-3400, USA.
Biochemistry. 2000 Jul 4;39(26):7645-50. doi: 10.1021/bi0008940.
Structural and genetic studies indicate that the antibacterial compound triclosan, an additive in many personal care products, is an inhibitor of EnvM, the enoyl reductase from Escherichia coli. Here we show that triclosan specifically inhibits InhA, the enoyl reductase from Mycobacterium tuberculosis and a target for the antitubercular drug isoniazid. Binding of triclosan to wild-type InhA is uncompetitive with respect to both NADH and trans-2-dodecenoyl-CoA, with K(i)' values of 0.22+/-0.02 and 0.21+/-0.01 microM, respectively. Replacement of Y158, the catalytic tyrosine residue, with Phe, reduces the affinity of triclosan for the enzyme and results in noncompetitive inhibition, with K(i) and K(i)' values of 36+/-5 and 47+/-5 microM, respectively. Consequently, the Y158 hydroxyl group is important for triclosan binding, suggesting that triclosan binds in similar ways to both InhA and EnvM. In addition, the M161V and A124V InhA mutants, which result in resistance of Mycobacterium smegmatis to triclosan, show significantly reduced affinity for triclosan. Inhibition of M161V is noncompetitive with K(i)' = 4.3+/-0.5 microM and K(i) = 4.4+/-0.9 microM, while inhibition of A124V is uncompetitive with K(i)' = 0. 81 +/- 0.11 microM. These data support the hypothesis that the mycobacterial enoyl reductases are targets for triclosan. The M161V and A124V enzymes are also much less sensitive to isoniazid compared to the wild-type enzyme, indicating that triclosan can stimulate the emergence of isoniazid-resistant enoyl reductases. In contrast, I47T and I21V, two InhA mutations that occur in isoniazid-resistant clinical isolates of M. tuberculosis, show unimpaired inhibition by triclosan, with uncompetitive inhibition constants (K(i)') of 0.18+/-0.01 and 0.12+/- 0.01 microM, respectively. The latter result indicates that InhA inhibitors targeted at the enoyl substrate binding site may be effective against existing isoniazid-resistant strains of M. tuberculosis.
结构和遗传学研究表明，抗菌化合物三氯生（许多个人护理产品中的一种添加剂）是大肠杆菌烯酰还原酶EnvM的抑制剂。在此我们表明，三氯生特异性抑制结核分枝杆菌的烯酰还原酶InhA，它也是抗结核药物异烟肼的作用靶点。三氯生与野生型InhA的结合对于NADH和反式-2-十二碳烯酰辅酶A而言均为非竞争性，其抑制常数（K(i)'）分别为0.22±0.02和0.21±0.01微摩尔。将催化性酪氨酸残基Y158替换为苯丙氨酸，降低了三氯生对该酶的亲和力，并导致非竞争性抑制，其K(i)和K(i)'值分别为36±5和47±5微摩尔。因此，Y158羟基对于三氯生的结合很重要，这表明三氯生以相似的方式与InhA和EnvM结合。此外，耻垢分枝杆菌中导致对三氯生耐药的M161V和A124V InhA突变体，对三氯生的亲和力显著降低。M161V的抑制为非竞争性，K(i)' = 4.3±0.5微摩尔，K(i) = 4.4±0.9微摩尔，而A124V的抑制为非竞争性，K(i)' = 0.81±0.11微摩尔。这些数据支持了结核分枝杆菌烯酰还原酶是三氯生作用靶点的假说。与野生型酶相比，M161V和A124V酶对异烟肼也不太敏感，这表明三氯生可促使异烟肼耐药的烯酰还原酶出现。相比之下，结核分枝杆菌异烟肼耐药临床分离株中出现的两种InhA突变I47T和I21V，对三氯生的抑制未受影响，非竞争性抑制常数（K(i)'）分别为0.18±0.01和0.12±0.01微摩尔。后一结果表明，靶向烯酰底物结合位点的InhA抑制剂可能对现有的结核分枝杆菌异烟肼耐药菌株有效。
