Mechanism of the pyrophosphate migration in the enzymic cyclization of geranyl and linalyl pyrophosphates to (+)- and (-)-bornyl pyrophosphates

Abstract

Soluble enzymes from sage (Salvia officinalis) and tansy (Tanacetum vulgare), which catalyze the cyclization of geranyl pyrophosphate and the presumptive intermediate linalyl pyrophosphate to the (+) and (-) enantiomers, respectively, of 2-bornyl pyrophosphate, were employed to evaluate mechanistic alternatives for the pyrophosphate migration in monoterpene cyclization reactions. Separate incubation of [1-3H2,alpha-32P]- and [1-3H2,beta- 32P]geranyl and (+/-)-linalyl pyrophosphates with partially purified preparations of each enantiomer-generating cyclase gave [3H, 32P]bornyl pyrophosphates, which were selectively hydrolyzed to the corresponding bornyl phosphates. Measurement of 3H:32P ratios of these monophosphate esters established that two ends of the pyrophosphate moiety retained their identifies in the cyclization of both precursors to both products and also indicated that there was no appreciable exchange with exogenous inorganic pyrophosphate in the reaction. Subsequent incubations of each cyclase with [8,9-14C,1-18O]geranyl pyrophosphate and with (1E)-(+/-)-[1-3H,3-18O]linalyl pyrophosphate gave the appropriate (+)- or (-)-bornyl pyrophosphates, which were hydrolyzed in situ to the corresponding borneols. Analysis of the derived benzoates by mass spectrometry demonstrated each of the product borneols to possess an 18O enrichment essentially identical with that of the respective acyclic precursor. The absence of P alpha-P beta interchange and the complete lack of positional 18O isotope exchange of the pyrophosphate moiety are compatible with tight ion pairing of intermediates in the coupled isomerization-cyclization of geranyl pyrophosphate and establish a remarkably tight restriction on the motion of the transiently generated pyrophosphate anion with respect to its cationic terpenyl reaction partner.

Role of Arginine-304 in the Diphosphate-Triggered Active Site Closure Mechanism of Trichodiene Synthase,

“…The phosphoester oxygen of (+)-bornyl diphosphate corresponds to the original phosphoester oxygen of the substrate, geranyl diphosphate (35,36). Therefore, by the analogy evident in the alignment of the two structures in Figure 6, we propose that the O6 atom of PP i in the trichodiene synthaseMg 2+ 3 -PP i complex corresponds to the original phosphoester oxygen of the substrate FPP.…”

Monoterpene Synthases from Common Sage (Salvia officinalis )

“…Because of the unique utilization of the substrate diphosphate moiety as the terminating nucleophile by this enzyme (62,63), it was hypothesized that a serine residue may be involved in binding and transfer of the diphosphate function in the course of the reaction. Sequence comparison of SBS with the other two monoterpene synthases of sage reveals five unique serine residues at positions 67, 255, 302, 454, and 469 (the serine at position 454 is conserved in monoterpene synthases from other species).…”

Inhibition of monoterpene cyclases by inert analogues of geranyl diphosphate and linalyl diphosphate

“…Asymmetric epoxidation [33] of geraniol afforded (2S,3S)-2,3-epoxygeraniol (20a, 94:4 er), and the 2R,3R-antipode (ent-20a). The enantiomeric purities were established by conversion to the diasteromeric (S)-camphanate ester ((S)-camphanic chloride, DMAP, pyr, 2h) [34] and 1 H NMR analysis in DMSO-d 6 . Similar preparation of the (S)-camphanate diastereomers from (±)-epoxygeraniol established that no kinetic resolution occurred under the conditions used.…”

“…These mechanistic features appear to be common to nearly all monoterpene cyclase transformations, with subsequent steps involving either termination of the reaction by deprotonation or nucleophile capture, or further electrophilic cyclizations via the remaining double bond, hydride shifts, or rearrangements before termination [1][2][3]. For example, direct deprotonation from the adjacent methyl of the (4S)-α-terpinyl cation (3b) yields (−)-limonene (4) [4,5], whereas C7-C1 bridging of the (4R)-α-terpinyl intermediate (3a) with capture of the resulting cation (5) by the paired diphosphate anion generates (+)-bornyl diphosphate (6) [6,7].…”