Mechanism of monoterpene cyclization: Stereochemical aspects of the transformation of noncyclizable substrate analogs by recombinant (-)-limonene synthase, (+)-bornyl diphosphate synthase, and (-)-pinene synthase

The tightly coupled nature of the reaction sequence catalyzed by monoterpen e synthases has prevented direct observation of the topologically required isomerization step leading from geranyl diphosphate to the presumptive, enz yme-bound, tertiary allylic intermediate linalyl diphosphate, which ultimat ely cyclizes to the various monoterpene skeletons. Previous experimental ap proaches using the noncyclizable substrate analogs 6,7-dihydrogeranyl dipho sphate and racemic methanogeranyl diphosphate, in attempts to dissect the c ryptic isomerization step from the normally coupled reaction sequence, were thwarted by the limited product available from native monoterpene synthase s and by the inability to resolve chiral monoterpene products at the micros cale. These approaches were revisited using three recombinant monoterpene s ynthases and chiral phase capillary gas chromatographic methods to separate antipodal products of the substrate analogs. The recombinant monoterpene o lefin synthases, (-)-limonene synthase from spearmint and (-)-pinene syntha se from grand fir, yielded essentially only achiral, olefin products (corre sponding to the respective analogs and homologs of myrcene, trans-ocimene a nd cis-ocimene) from 6,7-dihydrogeranyl diphosphate and (2S,3R)-methanogera nyl diphosphate; no significant amounts of terpenols or homoterpenols were formed, nor was direct evidence obtained for the formation of the anticipat ed analog and homolog of the tertiary intermediate linalyl diphosphate (i.e ., 6,7-dihydrolinalyl diphosphate and homolinalyl diphosphate, respectively ). In the case of recombinant (+)-bornyl diphosphate synthase from common s age, the achiral olefins were generated, as before, from 6,7-dihydrogeranyl diphosphate and (2R,3S)-methanogeranyl diphosphate, but 6,7-dihydrolinaloo l and homolinalool also comprised significant components of the respective product mixtures, indicating greater access of water to the active site of this enzyme compared to the olefin synthases; again, no direct evidence for the production of 6,7-dihydrolinalyl diphosphate or homolinalyl diphosphat e was obtained. Resolution of the terpenol products of (+)-bornyl diphospha te synthase, by chiral phase separation, revealed the predominant formation of (3R)-dihydrolinalool from dihydrogeranyl diphosphate and of (4S)-homoli nalool from (2R,3S)methanogeranyl diphosphate. The opposite stereochemistri es of these products indicates water trapping from opposite faces of the co rresponding tertiary carbocationic intermediates of the respective reaction s, a phenomenon that appears to result from the binding conformations of th ese substrate analogs. Although these experiments failed to provide direct evidence for the tertiary intermediate of the tightly coupled isomerization -cyclization sequence, they did reveal a mechanistic difference between the olefin synthases and bornyl diphosphate synthase involving access of water as a participant in the reaction. (C) 2001 Academic Press.