This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Trichodiene synthase (TS) is a Mg(II)-dependent sesquiterpene cyclase. Sesquiterpene cyclases catalyze the conversion of a universal acyclic substrate farnesyl diphosphate to one of numerous cyclic and chiral sesquiterpene hydrocarbons or alcohols with exquisite geometric and stereochemical precision. These sesquiterpenes are the precursors of several molecules that are of great importance to us in our everyday life as antibiotics, culinary additives, perfumes etc. TS evolved to exclusively form only trichodiene. However, conservative site-specific mutations in the active site of the enzyme not only affect the activity of the enzyme but also derail the enzyme into making additional products that resemble the intermediates more than the product or the substrate. Therefore, the study of these products and how a certain mutation makes certain aberrant products sheds light on the mechanism of the enzyme. We are studying the structural basis for aberrant product formation by the study of the mutant TS enzymes and their complexes with various ligands. Based on the structures of various mutants, we found that the aberrant product formation is mainly a function of the active site that does not form a perfectly tailored template for the exclusive formation of trichodiene. The volumes of ligand-bound active sites are generally much larger in the mutants than in the wild-type enzyme. We have studied an aspartate-rich motif that is conserved across sesquiterpene cyclases from all species. We are currently studying the importance of a second metal-binding motif, often called the NSE (or DTE) motif. The ultimate goal of these studies on this and other sesquiterpene cyclases is to be able to predict the products of a given sesquiterpene cyclases and to design novel sesquiterpenes that will form novel sesquiterpenes with various biological and commercial applications.
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