Turbek C S, Li D X, Choi G H, Schardl C L, Smith D A
Department of Plant Pathology, University of Kentucky, Lexington 40546-0091.
Phytochemistry. 1990;29(9):2841-6. doi: 10.1016/0031-9422(90)87088-c.
Fusarium solani f. sp. phaseoli is capable of detoxifying the major isoflavonoid phytoalexins produced by its host plant Phaseolus vulgaris. One of the enzymic activities involved is kievitone hydratase (KHase), a secreted glycoprotein which catalyses the conversion of kievitone to the less fungitoxic derivative, kievitone hydrate. Even under conditions of substrate induction, the enzyme is expressed at levels that are too low for satisfactory purification. Therefore, several other isoflavonoids were tested as inducers in culture. Among the phytoalexins produced by the host plant, phaseollinisoflavan was the best inducer, elevating the level of secreted enzyme eight-fold. Treatment with biochanin A, a product of chickpea, resulted in a 16-fold increase of secreted activity. The maximum rate of induction was observed 9-24 hr after addition of biochanin A, during which time several metabolites of the inducer were also present. KHase was purified from filtrates of biochanin A-induced cultures. Denaturing gel electrophoresis indicated that two species of Mr 47,000 and 49,000 copurified with the activity. N-Terminal sequence analysis indicated that the two species possessed related, or identical, polypeptide moieties. Comparison with the size of the non-denatured enzyme, previously determined to be ca 100,000, indicates that its native state is a dimer.
菜豆镰孢菌能使其寄主植物菜豆产生的主要异黄酮类植保素解毒。其中涉及的一种酶活性是基辅酮水合酶（KHase），它是一种分泌型糖蛋白，催化基辅酮转化为毒性较小的衍生物基辅酮水合物。即使在底物诱导的条件下，该酶的表达水平也很低，难以进行满意的纯化。因此，测试了几种其他异黄酮类化合物作为培养中的诱导剂。在寄主植物产生的植保素中，菜豆异黄烷是最好的诱导剂，能使分泌型酶的水平提高8倍。用鹰嘴豆的产物染料木黄酮处理，导致分泌活性增加16倍。在添加染料木黄酮后9 - 24小时观察到最大诱导率，在此期间诱导剂的几种代谢产物也存在。从染料木黄酮诱导培养物的滤液中纯化出KHase。变性凝胶电泳表明，两种分子量分别为47,000和49,000的蛋白与该活性共纯化。N端序列分析表明，这两种蛋白具有相关或相同的多肽部分。与先前测定的约100,000的非变性酶大小相比，表明其天然状态是二聚体。
