Institute of Life Science, School of Medicine, Swansea University, Swansea SA2 8PP, United Kingdom.
Appl Environ Microbiol. 2011 Feb;77(4):1460-5. doi: 10.1128/AEM.01332-10. Epub 2010 Dec 17.
Prothioconazole is one of the most important commercially available demethylase inhibitors (DMIs) used to treat Mycosphaerella graminicola infection of wheat, but specific information regarding its mode of action is not available in the scientific literature. Treatment of wild-type M. graminicola (strain IPO323) with 5 μg of epoxiconazole, tebuconazole, triadimenol, or prothioconazole ml(-1) resulted in inhibition of M. graminicola CYP51 (MgCYP51), as evidenced by the accumulation of 14α-methylated sterol substrates (lanosterol and eburicol) and the depletion of ergosterol in azole-treated cells. Successful expression of MgCYP51 in Escherichia coli enabled us to conduct spectrophotometric assays using purified 62-kDa MgCYP51 protein. Antifungal-binding studies revealed that epoxiconazole, tebuconazole, and triadimenol all bound tightly to MgCYP51, producing strong type II difference spectra (peak at 423 to 429 nm and trough at 406 to 409 nm) indicative of the formation of classical low-spin sixth-ligand complexes. Interaction of prothioconazole with MgCYP51 exhibited a novel spectrum with a peak and trough observed at 410 nm and 428 nm, respectively, indicating a different mechanism of inhibition. Prothioconazole bound to MgCYP51 with 840-fold less affinity than epoxiconazole and, unlike epoxiconazole, tebuconazole, and triadimenol, which are noncompetitive inhibitors, prothioconazole was found to be a competitive inhibitor of substrate binding. This represents the first study to validate the effect of prothioconazole on the sterol composition of M. graminicola and the first on the successful heterologous expression of active MgCYP51 protein. The binding affinity studies documented here provide novel insights into the interaction of MgCYP51 with DMIs, especially for the new triazolinethione derivative prothioconazole.
丙硫菌唑是一种重要的商品化脱甲基酶抑制剂（DMIs），用于治疗小麦上的禾谷丝核菌感染，但关于其作用模式的具体信息在科学文献中尚未报道。用 5 μg 的环丙唑醇、戊唑醇、三唑醇或丙硫菌唑/ml 处理野生型禾谷丝核菌（菌株 IPO323），导致禾谷丝核菌 CYP51（MgCYP51）被抑制，这表现在 14α-甲基化甾醇底物（羊毛甾醇和 eburicol）的积累和唑处理细胞中麦角固醇的耗尽。MgCYP51 在大肠杆菌中的成功表达使我们能够使用纯化的 62 kDa MgCYP51 蛋白进行分光光度法测定。抗真菌结合研究表明，环丙唑醇、戊唑醇和三唑醇都与 MgCYP51 紧密结合，产生强烈的 II 型差光谱（峰在 423 到 429nm，谷在 406 到 409nm），表明形成了经典的低自旋第六配体络合物。丙硫菌唑与 MgCYP51 的相互作用表现出一种新的光谱，其峰和谷分别在 410nm 和 428nm 处观察到，表明存在不同的抑制机制。丙硫菌唑与 MgCYP51 的结合亲和力比环丙唑醇低 840 倍，与非竞争性抑制剂环丙唑醇、戊唑醇和三唑醇不同，丙硫菌唑是底物结合的竞争性抑制剂。这是首次研究丙硫菌唑对禾谷丝核菌甾醇组成的影响，也是首次成功异源表达活性 MgCYP51 蛋白。这里的结合亲和力研究为 MgCYP51 与 DMIs 的相互作用提供了新的见解，特别是对新的三唑啉硫酮衍生物丙硫菌唑。
