ENZYMATIC CYCLIZATION TO TERPENOID NATURAL PRODUCTS

Washington State University, Pullman WA

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Linked publications & trials

Abstract

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