Atropselective synthesis of gossypol by dirigent proteins from cotton

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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.