Abstract:We report the first X-ray structure of the unique "head-to-middle" monoterpene synthase,l avandulyl diphosphate synthase (LPPS). LPPS catalyzes the condensation of two molecules of dimethylallyl diphosphate (DMAPP) to form lavandulyl diphosphate,aprecursor to the fragrance lavandulol. The structure is similar to that of the bacterial cis-prenyl synthase,u ndecaprenyl diphosphate synthase (UPPS), and contains an allylic site (S1) in which DMAPP ionizesa nd asecond site (S2) which houses the DMAPP nucleophile.Both S-thiolo-dimethylallyl diphosphate and S-thiolo-isopentenyl diphosphate bind intact to S2, but are cleaved to (thio)diphosphate,inS1. His78 (Asn in UPPS) is essential for catalysis and is proposed to facilitate diphosphate release in S1, while the P1 phosphate in S2 abstracts ap roton from the lavandulyl carbocation to form the LPP product. The results are of interest since they provide the first structure and structurebased mechanism of this unusual prenyl synthase.There are > 65 000 terpene or isoprene-like compounds known.[1] In general, there are three main classes of enzymes [2] involved in their biosynthesis: trans-prenyltransferases such as farnesyl diphosphate (FPP;S cheme 1) synthase,i nvolved in for example,protein prenylation and quinone biosynthesis;cis-prenyltransferases such as undecaprenyl diphosphate synthase (UPPS), involved in bacterial cell wall biosynthesis; and ad iverse range of terpene cyclases,s ome of which are modular proteins. [3] With the cis and trans prenyltransferases, an allylic diphosphate condenses with the homoallylic species isopentenyl diphosphate (IPP,S cheme 1) to form longerchain isoprenoids such as FPP,many of which are then acted on by other synthases.F or example,s qualene synthase produces presqualene diphosphate (PSPP,S cheme 1) and then squalene,aprecursor to sterols.T here are also many more unusual prenyl synthases.F or example,iridoid synthase converts 8-oxo-geranial to nepetalactol (Scheme 1), ak ey component in formation of the indole-terpenoid drugs vincristine and vinblastine. [4,5] There are also "irregular" terpene synthases such as lavandulyl diphosphate synthase (LPPS) in which two DMAPP molecules-both allylic diphosphates-condense via as o-called "head-to-middle" condensation [6] to form the (C 10 )m onterpene,l avandulyl diphosphate (LPP,Scheme 1), the precursor of the fragrances (R)-lavandulol and (R)-lavandulyl acetate.T he structure of this enzyme is unknown. Based on aB LAST (basic local alignment search tool) [7] search of all reported genomes,LPPS has closest homology (ca. 47 %identity over 234 residues) to (plant) dehydrodolichyl diphosphate synthases,b ut none of these structures have been reported. When compared with just bacterial genomes,t here is also close homology to undecaprenyl diphosphate synthases (ca. 42 %i dentity) for acatalytic "core" of 230 LPPS residues,and the structures of several UPPS proteins are known. [8,9] However,i th as also been shown that LPP can be made by ac himeric terpenoid synthase with the conventi...