Structure-activity studies of the inhibition of FabI, the enoyl reductase from Escherichia coli, by triclosan:: Kinetic analysis of mutant
Structure-activity studies of the inhibition of FabI, the enoyl reductase from Escherichia coli, by triclosan:: Kinetic analysis of mutant
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DOI：
10.1021/bi0300229
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发表时间：
2003-04-22
期刊：
影响因子：
2.9
通讯作者：
Tonge, PJ
中科院分区：
文献类型：
--
作者：
Sivaraman, S;Zwahlen, J;Tonge, PJ
三氯生是消费品中常用的抗菌添加剂，是FabI的抑制剂，FabI是11型细菌脂肪酸生物合成的烯酰还原酶。与以前的研究[沃德，W。H、霍尔盖特湾一、Rowsell，S.，姆克林，E. G.，波普蒂特河一、克莱顿，尼科尔斯，W。W.，Colls，J.G.，明舒尔角一、Jude，D.一、Mistry，A.，Timms，D.，坎布尔河Hales，N. J.，布里顿角J.，泰勒，我。W.（1999）Biochemistry 38，12514-12525]，我们在此报道三氯生是来自大肠杆菌的FabI的缓慢、可逆、紧密结合抑制剂。三氯生优先结合野生型酶的E-NAD+形式，Ki值为23 pM。与遗传选择实验[McMurry，L. M.，Oethinger，M.，和Levy，S. B。（1998）Nature 394，531-532]，三氯生对FabI突变体G93 V、M159 T和F203 L的亲和力显著降低，优先结合E.NAD（+）形式的G93 V、M159 T和F203 L，K-1值分别为0.2 μ M、4 nM和0.9 nM。也可以检测到三氯生与F203 L的E.NADH形式的结合，并通过51 nM的K-2值进行定义。我们还对Y156 F和A197 M突变体进行了表征，以比较和对比三氯生与结核分枝杆菌同源烯酰还原酶InhA的结合。如InhA所观察到的，Y156 F FabI对三氯生的亲和力降低，抑制剂结合酶的E.NAD（+）和E.NADH形式，K-1和K-2值分别为3和30 nM。用Met替换A 197对三氯生亲和力没有影响，表明保守活性位点环序列的差异不能解释FabI和InhA对三氯生亲和力的10000倍差异。
Triclosan, a common antibacterial additive used in consumer products, is an inhibitor of FabI, the enoyl reductase enzyme from type 11 bacterial fatty acid biosynthesis. In agreement with previous studies [Ward, W. H., Holdgate, G. A., Rowsell, S., McLean, E. G., Pauptit, R. A., Clayton, E., Nichols, W. W., Colls, J. G., Minshull, C. A., Jude, D. A., Mistry, A., Timms, D., Camble, R., Hales, N. J., Britton, C. J., and Taylor, I. W. (1999) Biochemistry 38, 12514-12525], we report here that triclosan is a slow, reversible, tight binding inhibitor of the FabI from Escherichia coli. Triclosan binds preferentially to the E-NAD+ form of the wild-type enzyme with a K, value of 23 pM. In agreement with genetic selection experiments [McMurry, L. M., Oethinger, M., and Levy, S. B. (1998) Nature 394, 531-532], the affinity of triclosan for the FabI mutants G93V, M159T, and F203L is substantially reduced, binding preferentially to the E.NAD(+) forms of G93V, M159T, and F203L with K-1 values of 0.2 muM, 4 nM, and 0.9 nM, respectively. Triclosan binding to the E.NADH form of F203L can also be detected and is defined by a K-2 value of 51 nM. We have also characterized the Y156F and A197M mutants to compare and contrast the binding of triclosan to InhA, the homologous enoyl reductase from Mycobacterium tuberculosis. As observed for InhA, Y156F FabI has a decreased affinity for triclosan and the inhibitor binds to both E.NAD(+) and E.NADH forms of the enzyme with K-1 and K-2 values of 3 and 30 nM, respectively. The replacement of A 197 with Met has no impact on triclosan affinity, indicating that differences in the sequence of the conserved active site loop cannot explain the 10000-fold difference in affinities of FabI and InhA for triclosan.