Hollomon D W, Butters J A, Barker H, Hall L
IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol BS41 9AF, United Kingdom.
Antimicrob Agents Chemother. 1998 Sep;42(9):2171-3. doi: 10.1128/AAC.42.9.2171.
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 Glu198-->Gly198, 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.
苯并咪唑是用于兽医学和植物病害防治的重要抗微管蛋白剂。耐药性是一个与β-微管蛋白中的单个氨基酸变化相关的实际问题，并且通常与对苯基氨基甲酸盐的更高敏感性相关。这种负交叉耐药性为持久的抗耐药策略创造了机会。由于无法从丝状真菌中纯化微管蛋白，了解苯并咪唑耐药性分子基础的尝试受到了阻碍。我们通过将β-微管蛋白与麦芽糖结合蛋白融合表达克服了其中一些问题。这种融合蛋白是可溶的，并且我们首次使用凝胶过滤分析证实苯并咪唑确实与β-微管蛋白结合。这种结合因Glu198→Gly198突变而减少，该突变也赋予耐药性。苯基氨基甲酸盐的结合情况则完全相反，这反映了它们的生物活性和负交叉耐药性。这表明杀真菌剂结合位点在融合蛋白中正确折叠。
