Mutational analysis of the triclosan-binding region of enoyl-ACP (acyl-carrier protein) reductase from Plasmodium falciparum
Mutational analysis of the triclosan-binding region of enoyl-ACP (acyl-carrier protein) reductase from Plasmodium falciparum
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DOI：
10.1042/bj20040302
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发表时间：
2004-08-01
影响因子：
4.1
通讯作者：
Surolia, A
中科院分区：
文献类型：
--
作者：
Kapoor, M;Gopalakrishnapai, J;Surolia, A
三氯生是一种已知的抗菌剂，通过抑制烯酰-ACP（酰基载体蛋白）还原酶（ENR）发挥作用，ENR是II型脂肪酸合成（FAS）系统的关键酶。相反，引起人类疟疾的寄生虫恶性疟原虫携带II型FAS。其人类宿主利用I型FAS。由于这一显著差异，ENR已成为开发新抗疟药物的一个重要目标。模型研究和恶性疟原虫ENR的晶体结构突出了酶、三氯生和NAD（+）之间三元复合物形成的特征[Suguna. A. Surolia和N. Surolia（2001）Biochem. Biophys.通信资源283，224-228; Perozzo，Kuo，Sidhu，Valiyaveettil，Bittman，Jacobs，Fidock和Sacchettini（2002）J.Biol.Chem.277，13106-13114;和Swarnamukhi，Kapoor，N. Surolia，A. Surolia和Suguna（2003）PDB 1UH 5]。为了解决三氯生与恶性疟原虫ENR的强特异性和化学计量结合所涉及的残基的重要性问题，我们突变了以下残基：Ala-217、Asn-218、Met-281和Phe-368。与野生型酶相比，所有突变体对三氯生的亲和力都有不同程度的降低。最显著的突变是A217 V，与野生型PfENR相比，其导致三氯生结合亲和力降低7000倍以上。A217 G显示结合亲和力仅降低10倍。因此，这些研究指出恶性疟原虫酶的三氯生结合区域与其细菌对应物的三氯生结合区域存在显著差异。
Triclosan, a known antibacterial, acts by inhibiting enoyl-ACP (acyl-carrier protein) reductase (ENR), a key enzyme of the type II fatty acid synthesis (FAS) system. Plasmodium falciparum, the human malaria-causing parasite, harbours the type II FAS; in contrast. its human host utilizes type I FAS. Due to this striking difference, ENR has emerged as an important target for the development of new antimalarials. Modelling studies, and the crystal structure of P. falciparum ENR, have highlighted the features of ternary complex formation between the enzyme, triclosan and NAD(+) [Suguna. A. Surolia and N. Surolia (2001) Biochem. Biophys. Res. Commun. 283, 224-228; Perozzo, Kuo, Sidhu, Valiyaveettil, Bittman, Jacobs, Fidock, and Sacchettini (2002) J. Biol. Chem. 277, 13106-13114; and Swarnamukhi, Kapoor, N. Surolia, A. Surolia and Suguna (2003) PDB1UH5]. To address the issue of the importance of the residues involved in strong specific and stoichiometric binding of triclosan to P. falciparum ENR, we mutated the following residues: Ala-217, Asn-218, Met-281, and Phe-368. The affinity of all the mutants was reduced for triclosan as compared with the wild-type enzyme to different extents. The most significant mutation was A217V, which led to a greater than 7000-fold decrease in the binding affinity for triclosan as compared with wild-type PfENR. A217G showed only 10-fold reduction in the binding affinity. Thus, these studies point out significant differences in the triclosan-binding region of the P. falciparum enzyme from those of its bacterial counterparts.