STEREOCHEMICAL STUDIES OF ISOPRENOID BIOSYNTHESIS

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Ongoing studies of cell-free terpenoid cyclizations will be continued and extended. A general stereochemical model of monoterpene and sesquiterpene biosynthesis will be tested by examining the action of a group of sesquiterpene synthetases isolated from a variety of microbial and plant sources. It is expected that the information gained from such studies will provide fundamental understanding of the catalysis of biological carbon- carbon bond formation and the nature of enzymatic catalysis and control in general. 1) We have demonstrated the intermediacy of nerolidyl pyrophosphate (1) in the enzymatic cyclization of farnesyl pyrophosphate (2) to trichodiene (3) and established the detailed stereochemical course of this cyclization. In order to further dissect the complex isomerization - cyclization sequence mediated by trichodiene synthetase, a series of strategically chosen substrate analogs will be incubated with homogeneous trichodiene synthetase, in collaboration with Dr. Thomas Hohn of the USDA. The goal in these experiments will be to disrupt normal bond-making processes, thereby leading to premature release of abortive cyclization products thus revealing the otherwise cryptic bond-forming and rearrangement events which take place during normal cyclization. In parallel with these studies we will also carry out incubations of many of the same substrate analogs with farnesyl pyrophosphate - nerolidyl pyrophosphate isomerase, an enzyme isolated from Gibberella fujikuroi which catalyzes a simple allylic rearrangement corresponding to the initial isomerization step of many terpenoid cyclizations. 2) Two recently isolated new microbial sesquiterpene cyclases, bergamotene (4) synthetase from Pseudeurotium ovalis and aristolochene (5) synthetase from Aspergillus terreus will be studied. The latter enzyme has also been isolated by Dr. Hohn from Penicillium roquefortii and purified to homogeneity. Proposed stereochemical models for each cyclization will be tested using specifically labelled samples of farnesyl and nerolidyl pyrophosphate as well as selected substrate analogs. The study of bergamotene synthetase provides an opportunity to examine cyclizations involving intermediates antipodal to those involved in trichodiene biosynthesis and closely related mechanistically to the formation of the monoterpene beta-pinene, while the aristolochene synthetase reaction provides a platform for the study of the conversion of farnesyl pyrophosphate to all-trans 10-membered ring intermediates. 3) Professor Rodney Croteau of Washington State University has recently succeeded in purifying humulene (6) synthetase isolated from sage (Salvia officinalis). Collaborative studies of the stereochemistry of the formation of this 11-membered ring sesquiterpene, using specifically labelled samples of farnesyl pyrophosphate will be continued. Finally, (- )-gamma-cadinene synthetase will be isolated from A. terreus and the mechanism of cyclization of farnesyl and nerolidyl pyrophosphate to a cis, trans-germacradienyl intermediate will be examined.