DESCRIPTION: (Principla Investigator's): Monoterpene synthases provide the focus for study of prenyl diphosphate cyclization, the reaction of principal importance in C-C bond formation in the biosynthesis of numerous terpenoid natural products of pharmacological significance. A stereochemical model for the coupled isomerization-cyclization of the universal isoprenoid precursor, geranyl diphosphate, was developed through studies on the origin of the seven major monoterpene skeletal types. Selected synthases ((+)- and (-)-limonene synthases, (+)- and (-)-pinene synthases, and (+)- and (-)-bornyl diphosphate synthases from the same species (common sage) and from a phylogenetically distant source (grand fir)) that differ significantly in mechanistic and stereochemical features will be employed to examine active site structure-function relationships that underlie the formation of olefin isomers, oxygenated derivatives, and their enantiomers. Molecular cloning of (-)-4S-limonene synthase, catalyzing the simplest of all terpenoid cyclizations, has provided access to cDNAs encoding the other target synthases, and functional expression of the truncated enzymes, in which the troublesome plastidial transit peptides have been deleted, allows the examination of active site structure and function. The first two specific aims of the proposal are to utilize the similarity-based cloning strategy to acquire the remaining target cDNAs, and to express the appropriate truncations for high yield production of fully active 'pseudo-mature' enzymes for X-ray crystallographic studies and related investigations. In the third aim, the active sites of the recombinant synthases (with limonene synthase as the prototype) will be located using cysteine- and histidine-directed reagents and substrate protection/deprotection strategies, a mechanism-based alkylator, and photolabile substrate analogs to target hydrophobic binding pockets. Information from active site location, plus that gained by primary sequence comparisons, will be used in specific aim four to target selected residues and hydrophobic domains for mutagenesis. The mutants will be evaluated for kinetic behavior and product outcome to deduce which steps of the reaction cascade have been altered. In the final aim, substrate analogs will be used to examine the cryptic isomerization step of the reaction and to explore the catalytic repertoire of the cloned synthases. These studies will provide new information on the relationship of structure to reaction mechanism for these novel catalysts, and allow a clearer understanding of this important aspect of prenyl diphosphate metabolism.