Reynolds 9418581 Streptomyces collinus produces cylohexanecarboxylic acid (CHC) from shikimic acid and adds this unit to the side chain of the antifungal agent ansatrienin. Streptomyces hygroscopicus is thought to produce a 3(R),4(R)-dihydroxy-1(R)-cyclohexanecarboxylic acid (DHCHC) from shikimic acid and to use this compound as the biosynthetic starter unit for the formation of the novel immunosuppressant, FK520. The long term aims of this research are to both investigate and compare these two pathways. Preliminary in vivo results indicate that the initial steps of both pathways follow the same sequence of enzymes catalyzed reactions but that the stereochemical course of the reduction steps is different. The unusual secondary metabolic pathways from shikimic acid to CHC and DHCHC therefore present an interesting example of both metabolic similarity and stereochemical diversity. In this research project enoyl CoA reductases involved in catalyzing the reduction steps in both the CHC and CHCHC pathway will be analyzed from a chemical, biochemical, stereochemical and genetic perspective. Homology between these enzymes will indicate if they have evolved by divergent evolution from a common ancestor. This finding would be unusual based on the observation of the stereochemical diversity (there is no precedent of homologous enoyl CoA reductases catalyzing reactions with different stereochemical courses). Alternatively these reductases might demonstrate no homology indicating an independent evolution of two similar unusual pathways in Streptomyces. %%% Soil bacteria often produce a vast array of secondary metabolites, complex organic molecules that have pharmaceutical relevance but that serve no apparent function in bacteria. How and why bacteria came to produce these metabolites has been the subject of much research and discussion. Initial evidence has indicated that many of the biochemical pathways which produce these are related on an evolutionary basis. This resear ch project concerns two different soil bacteria. Streptomyces collinus converts shikimic acid to cyclohexanecarboxylic acid in order to produce the secondary metabolic ansatrienin (an antifungal agent). Streptomyces hygroscopicus converts shikimic acid to dihydroxycyclohexanecarboxylic acid in order to produce the secondary metabolite immunomycin (an immunosuppressant). These two conversions of shikimic acid are similar and would be expected to be related on an evolutionary basis. However initial evidence has suggested that in the two bacteria the proteins that catalyze these conversion do so with different stereochemical outcomes (one is "left handed" an the other "right handed"). This project will determine if these proteins do have a different handedness and if they are related. The discovery that the proteins are related would be unusual because of this handedness difference. The discovery that the proteins are unrelated would also be unusual because it would indicate that the similar conversions of shikimic acid have arisen by convergent rather than divergent evolution. ***
