9631980 Lewis One of the most important yet poorly understood biological events in nature is that of bimolecular phenoxy radical coupling. Surprisingly, phenoxy radical coupling reactions have long been imperfectly characterized, and are frequently described as lacking stereoselectivity based on their catalytic properties in vitro. Yet even a cursory analysis of such reactions in nature indicates that some mechanism for conferring stereospecificity must exist, since the products are stereospecific. Seeking to understand how this stereoselectivity is produced, the investigators examined the coupling of two achiral molecules of E-coniferyl alcohol to give (+)-pinoresinol, one of the simplest dimeric lignan products known in nature. They established that a 78kDa protein is necessary to confer both regio- & stereospecificity on the products. This stereoselective coupling only occurs when auxiliary oxidative capacity is provided through the addition of either an oxidase or a free-radical initiator, such as ammonium peroxydisulfate or FMN. This investigation will seek to define the biochemical mechanism of this unique mode of control. The relationships between the 78 kDa protein, the substrate and an auxiliary oxidase/oxidant will be studied. That is, does the operative mechanism involve initial binding of two E-coniferyl alcohol molecules to the 78 kDa protein, or is the substrate for binding actually the free-radical species obtained via one electron oxidation? A third possibility is that some charge-transfer mechanism is in place, although the absence of any detectable active site (prosthetic group) makes this unlikely. The investigators will conduct a detailed kinetic anaylsis of the coupling reaction as well as defining the substrate specificity for binding/coupling and the effect of putative inhibitors. In this way the mechanism for conferring stereoselectivity in phenoxy radical coupling will be determined and will serve as a starting point to determine the generality of this m echanism in nature. %%% One of the most important, and yet most poorly understood, biological events in nature is that of bimolecular phenoxy radical coupling, the joining of two molecules containing phenol moieties. It is highly significant, since it eventually leads to the formation of woody and cork tissues, insect cuticles, fungal fruiting bodies, aphid pigments, and a host of defense molecules, particularly in plants. Taken together, phenolic coupling reactions account for roughly 35-40% of all organic plant material. Two phenol moieties can often be joined in numerous ways, with the individual products being mirror images of each other. Before the advances by these investigators, in vitro reactions only yielded mixtures of various mirror image products. In vivo reactions, however, often yield only one product and in one mirror image form. How is that specificity attained? The investigators have isolated a protein, which they have called a "specifier", which confers such specificity on the reactions. The goal of this project is to define the biochemical mechanism of this novel protein. ***
