geranyl-pyrophosphate and linalyl-pyrophosphate

Topics: Acyclic Monoterpenes; Catalytic Domain; Citrus sinensis; Crystallography, X-Ray; Diphosphates; Diterpenes; Enzyme Assays; Enzyme Inhibitors; Intramolecular Lyases; Polyisoprenyl Phosphates

The tightly coupled nature of the reaction sequence catalyzed by monoterpene synthases has prevented direct observation of the topologically required isomerization step leading from geranyl diphosphate to the enzyme-bound, tertiary allylic intermediate linalyl diphosphate, which then cyclizes to the various monoterpene skeletons. X-ray crystal structures of these enzymes complexed with suitable analogues of the substrate and intermediate could provide a clearer view of this universal, but cryptic, step of monoterpenoid cyclase catalysis. Toward this end, the functionally inert analogues 2-fluorogeranyl diphosphate, (+/-)-2-fluorolinalyl diphosphate, and (3R)- and (3S)-homolinalyl diphosphates (2,6-dimethyl-2-vinyl-5-heptenyl diphosphates) were prepared, and compared to the previously described substrate analogue 3-azageranyl diphosphate (3-aza-2,3-dihydrogeranyl diphosphate) as inhibitors and potential crystallization aids with two representative monoterpenoid cyclases, (-)-limonene synthase and (+)-bornyl diphosphate synthase. Although these enantioselective synthases readily distinguished between (3R)- and (3S)-homolinalyl diphosphates, both of which were more effective inhibitors than was 3-azageranyl diphosphate, the fluorinated analogues proved to be the most potent competitive inhibitors and have recently yielded informative liganded structures with limonene synthase.

Topics: Acyclic Monoterpenes; Diphosphates; Diterpenes; Enzyme Activation; Enzyme Inhibitors; Enzyme Stability; Intramolecular Lyases; Monoterpenes; Polyisoprenyl Phosphates

The conversion of geranyl pyrophosphate to (+)-bornyl pyrophosphate and (+)-camphene is considered to proceed by the initial isomerization of the substrate to (-)-(3R)-linalyl pyrophosphate and the subsequent cyclization of this bound intermediate. In the case of (-)-bornyl pyrophosphate and (-)-camphene, isomerization of the substrate to the (+)-(3S)-linalyl intermediate precedes cyclization. The geranyl and linalyl precursors were shown to be mutually competitive substrates (inhibitors) of the relevant cyclization enzymes isolated from Salvia officinalis (sage) and Tanacetum vulgare (tansy) by the mixed substrate analysis method, demonstrating that isomerization and cyclization take place at the same active site. Incubation of partially purified enzyme preparations with (3R)-[1Z-3H]linalyl pyrophosphate plus [1-14C]geranyl pyrophosphate gave rise to double-labeled (+)-bornyl pyrophosphate and (+)-camphene, whereas incubation of enzyme preparations catalyzing the antipodal cyclizations with (3S)-[1Z-3H]-linalyl pyrophosphate plus [1-14C]geranyl pyrophosphate yielded double-labeled (-)-bornyl pyrophosphate and (-)-camphene. Each product was then transformed to the corresponding (+)- or (-)-camphor without change in the 3H:14C isotope ratio, and the location of the tritium label was deduced in each case by stereoselective, base-catalyzed exchange of the exo-alpha-hydrogen of the derived ketone. The finding that the 1Z-3H of the linalyl precursor was positioned at the endo-alpha-hydrogen of the corresponding camphor in all cases, coupled to the previously demonstrated retention of configuration at C1 of the geranyl substrate in these transformations, confirmed the syn-isomerization of geranyl pyrophosphate to linalyl pyrophosphate and the cyclization of the latter via the anti,endo- conformer. These relative stereochemical elements, in combination with the observed enantiospecificities of the enzymes for the linalyl intermediates, allows definition of the overall absolute stereochemistry of the coupled isomerization and cyclization of geranyl pyrophosphate to the antipodal camphane (bornane) and isocamphane monoterpenoids.

Topics: Acyclic Monoterpenes; Camphanes; Carbon Radioisotopes; Chemical Phenomena; Chemistry; Hydrolysis; Isomerism; Monoterpenes; Plants; Polyisoprenyl Phosphates; Radioisotope Dilution Technique; Terpenes; Tritium

The conversion of geranyl pyrophosphate to (-)-endo-fenchol is considered to proceed by the initial isomerization of the substrate to (-)-(3R)-linalyl pyrophosphate and the subsequent cyclization of this bound intermediate. To test this stereochemical scheme, phosphatase-free preparations of (-)-endo-fenchol cyclase from fennel (Foeniculum vulgare M.) fruit were repeatedly incubated with a sample of (3RS)-[1-3H2]linalyl pyrophosphate until approximately 50% of this precursor was converted to the bicyclic monoterpenol end product. The residual linalyl pyrophosphate was isolated and enzymatically hydrolyzed to the free alcohol, linalool, which was resolved by chiral phase capillary gas-liquid chromatography of the derived threo and erythro mixture of 1,2-epoxides. The predominance of the (3S)-enantiomer in the residual substrate indicated that the (3R)-enantiomer was preferred for the cyclization to (-)-(1S)-endo-fenchol. This conclusion was subsequently confirmed by the preparation and direct testing of (3R)-1Z-[1-3H] linalyl pyrophosphate, which afforded a Km value lower than that observed for geranyl pyrophosphate and a relative velocity nearly three times higher. (3S)-1Z-[1-3H]Linalyl pyrophosphate was not an effective substrate for (-)-endo-fenchol biosynthesis but did, by an anomalous cyclization, give rise to low levels of the enantiomeric (+)-(1R)-endo-fenchol as well as to other products. These results support the proposed stereochemical model and also suggest that the isomerization step is rate limiting in the coupled isomerization-cyclization of geranyl pyrophosphate to (-)-endo-fenchol.

Topics: Acyclic Monoterpenes; Camphanes; Intramolecular Lyases; Isomerases; Monoterpenes; Norbornanes; Plants; Polyisoprenyl Phosphates; Stereoisomerism; Substrate Specificity; Terpenes

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.

Topics: Acyclic Monoterpenes; Carbon Radioisotopes; Intramolecular Lyases; Isomerases; Isotope Labeling; Monoterpenes; Oxygen Isotopes; Phosphorus Radioisotopes; Plants; Polyisoprenyl Phosphates; Stereoisomerism; Substrate Specificity; Tritium