Atropselective synthesis of gossypol by dirigent proteins from cotton

苯氧基自由基偶联反应在体外缺乏严格的区域和立体选择性控制。因此，如果存在多个偶联部位，则观察到不同的区域化学，如果形成手性中心，则产生外消旋产物。另一方面，天然产物的生物合成需要严格的立体化学控制。作为木脂素生物合成的第一步，作为木脂素生物合成的第一步，发现了介导两个针叶醇自由基对(+)或(-)-松脂醇的区域和对映体选择性偶联的衍生蛋白，从而提出了衍生蛋白可能在植物次生代谢的立体化学控制中发挥普遍作用的假说。然而，这一假设仍然没有得到证实，因为到目前为止还没有发现其他底物或偶联产物。我们的目标是通过鉴定有助于棉酚生物合成的新型引导性蛋白来填补这一知识空白。棉酚是由半棉酚氧化二聚而成的。偶联产物的轴向手性是由于围绕中心联萘键的旋转受阻而获得的。在不同的棉花品种中观察到过量的(+)-或(-)-棉酚对映体，这表明存在有利于形成一种或另一种阿托品的对映体互补调节蛋白。这些转导蛋白将被克隆，在异源系统中表达，纯化，鉴定和优化(+)-棉酚和(-)-棉酚的形成。X射线结构分析将在分子水平上深入了解底物专一性和对映体选择性的机制。棉花DIR的不同区域将以互换的方式进行交换，以研究它们对偶联反应的立体化学控制的贡献，并朝着设计具有修饰选择性和更广泛底物专一性的介导性蛋白质迈出第一步。

Phenoxy radical coupling reactions lack strict regio- and stereoselective control in vitro. Therefore, different regiochemistries are observed if several coupling sites are present, and racemic products arise if chiral centers are formed. Natural product biosynthesis, on the other hand, requires strict stereochemical control. The discovery of dirigent proteins mediating the regio- und enantioselective coupling of two coniferylalcohol radicals to either (+)- or (-)-pinosresinol as the first step during lignan biosynthesis led to the hypothesis that dirigent proteins might play a general role in the stereochemical control of plant secondary metabolism. However, this hypothesis is still unsubstantiated, since no other substrates or coupling products could be identified so far. We aim to fill this gap in knowledge by identification of novel dirigent proteins that contribute to the biosynthesis of gossypol in cotton plants. Gossypol is formed by oxidative dimerization of hemigossypol. The coupling product gains axial chirality because of hindered rotation around the central binaphthyl bond. An enantiomeric excess of either (+)- or (-)-gossypol is observed in different cotton species, suggesting the presence of enantiocomplementary dirigent proteins favoring the formation of one or the other atropisomer. These dirigent proteins will be cloned, expressed in heterologous systems, purified, characterized and optimized with respect to the formation of (+)- and (-)-gossypol, respectively. X-ray structure analysis will be performed to gain insight into the mechanisms underlying substrate specificity and enantioselectivity at the molecular level. Different regions of cotton DIRs will be exchanged in a reciprocal manner to investigate their contribution to stereochemical control of the coupling reaction, and to make a first step towards the engineering of dirigent proteins with modified selectivity and broader substrate specificity.