Submitted to: Journal of the American Chemical Society
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/26/2002
Publication Date: 6/26/2002
Citation: WISE, M.L., URBANSKY, M., HELMS, G.L., COATES, R.M., CROTEAU, R. SYN STEREOCHEMISTRY OF CYCLIC ETHER FORMATION IN 1,8-CINEOLE BIOSYNTHESIS CATALYZED BY RECOMBINANT SYNTHASE FROM SALVIA OFFICINALIS. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY. 2002. 124(29):8546-7.
Interpretive Summary: Monoterpenes are ten carbon metabolites found in many herbs and spices and are readily recognized as the principle flavor and aroma components of, for example, spearmint, peppermint, lemon or culinary sage. One of the compounds, 1,8-cineole (commonly referred to as eucalyptus oil), is a know allelopathic agent and served as a lead structure in the development of the commercial herbicide cinmethylin. Although a well founded paradigm exists to describe the chemical mechanisms involved in converting geranyl diphosphate, the universal precursor to all monoterpenes, into the various cyclic and bicyclic monoterpenes found in nature, gaps still remain in our understanding of key steps involved in the biosynthesis of certain members of this diverse group of natural products. In recent years, several of the enzymes involved in this metabolic pathway have been cloned and heterologously expressed thus allowing an even more detailed examination of the role of the enzyme in catalyzing these complex reactions. This abundant supply of protein will, in the near future, provide the necessary material to successfully obtain 3-dimensional structures for these enzymes by X-ray crystallographic methods. In this report, we describe the use of one of these cloned enzymes, 1,8-cineole synthase, to decipher a key chemical step in the biosynthesis of 1,8-cineole. These results provide vital information concerning the components of the enzyme's active site and will provide insight necessary to interpretation X-ray crystallographic data as they become available.
Technical Abstract: Monoterpene synthases are enzymes responsible for the biosynthetic conversion of geranyl diphosphate to the multitude of cyclic and acyclic secondary metabolites collectively referred to as monoterpenes. In this report, we describe the employment of NMR spectroscopy, in concert with regiospecifically deuterated geranyl diphosphate, to elucidate the stereochemical course of the cryptic heterocyclic ring closure in the biosynthesis of 1,8-cineole. Thus, [1,8-deuterated]generanyl diphosphate and [1,9-dueterated]generanyl diphosphate were reacted separately with recombinant cineole synthase (rCS) from Salvia offinalis. NMR experiments were conducted on the isolated reaction product to observe NOE's from the proximal ring methylenes to the gem dimethyl groups in this bicyclic compound. Interpretation of these experiments allowed determination that the attack by water on the intermediate terpinyl cation occurs from the ring side of this highly reactive intermediate. These results suggest that the enzyme precisely holds, the water molecule in the active site, preventing premature reaction with carbocationic intermediates during the course of the enzymatic reaction coordinate and specifically directs nucleophilic attack on the terpinyl cation. Moreover, reaction of the rCS with unlabeled geranyl diphosphate in deuterated buffer followed by deuterium NMR of the reaction product demonstrated that addition across the double bond of the terpineol intermediate occurs synfacially during ring closure.