Fungal β-tubulin, expressed as a fusion protein, binds benzimidazole and phenylcarbamate fungicides
Fungal β-tubulin, expressed as a fusion protein, binds benzimidazole and phenylcarbamate fungicides
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DOI：
10.1128/aac.42.9.2171
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发表时间：
1998-09-01
影响因子：
4.9
通讯作者：
Hall, L
中科院分区：
文献类型：
--
作者：
Hollomon, DW;Butters, JA;Hall, L
苯并咪唑类化合物是兽医和植物病害防治中重要的抗微管蛋白药物。耐药性是一个实际问题，与β-微管蛋白中单一氨基酸的变化有关，而且往往与对苯氨基甲酸酯更敏感有关。这种负面的交叉阻力为持久的反耐药性策略创造了机会。由于无法从丝状真菌中提纯微管蛋白，试图了解苯并咪唑耐药性的分子基础一直受到阻碍。我们通过将β-微管蛋白表达为与麦芽糖结合蛋白的融合来克服其中的一些问题。这种融合蛋白是可溶的，我们第一次使用凝胶过滤实验证实了苯并咪唑确实与β-微管蛋白结合。这种结合被突变Glu(198)-->；Gly(198)减少，这也会产生抗药性。苯氨基甲酸酯的结合则完全相反，反映了它们的生物活性和负的交叉抗性。这表明杀菌剂结合位点在融合蛋白中正确折叠。
Benzimidazoles are important antitubulin agents used in veterinary medicine and plant disease control. Resistance is a practical problem correlated with single amino acid changes in beta-tubulin and is often linked to greater sensitivity to phenylcarbamates. This negative cross-resistance creates opportunities for durable antiresistance strategies. Attempts to understand the molecular basis of benzimidazole resistance have been hampered by the inability to purify tubulin from filamentous fungi. We have overcome some of these problems by expressing beta-tubulin as a fusion with a maltose binding protein. This fusion protein is soluble, and we confirm for the first time using a gel filtration assay that benzimidazoles indeed bind to beta-tubulin. This binding is reduced by the mutation Glu(198)-->Gly(198), which also confers resistance. Binding of phenylcarbamates is the complete opposite, reflecting their biological activity and the negative cross-resistance. This suggests that the fungicide binding sites fold correctly in the fusion protein.