Monoterpene biosynthesis: mechanism and stereochemistry of the enzymatic cyclization of geranyl pyrophosphate to (+)-cis- and (+)-trans-sabinene hydrate

The conversion of geranyl pyrophosphate to (+)-cis- and (+)-trans-sabinene hydrate by a partially purified cyclase from sweet marjoram (Majorana hortensis) is considered to proceed by the initial ionization and isomerization of the substrate to (-)-(3R)-linalyl pyrophosphate and the subsequent cyclization of this enzyme-bound tertiary allylic intermediate to the monocyclic (+)-(4R)-alpha-terpinyl cation. A 1,2-hydride shift and a second cyclization with water capture of the resulting cation complete the reaction sequence. [6-3H, 14C]Geranyl pyrophosphate, coupled with selective chemical degradation of the resulting sabinene hydrate products, was employed to demonstrate the hydride shift, while separate testing of the linalyl pyrophosphate enantiomers confirmed the involvement of the (3R)-antipode in the cyclization and indicated the cyclization of linalyl pyrophosphate to be faster than the coupled isomerization-cyclization of the geranyl substrate. (1R)- and (1S)-[1-3H, 14C]geranyl pyrophosphates, in conjunction with stereoselective degradations of the biosynthetic products to locate the 3H, were exploited to deduce that configuration at C1 of the substrate was retained in the reaction. These findings suggest the isomerization of the geranyl substrate to be a suprafacial process and the cyclization of the (3R)-linalyl intermediate to proceed via the anti,endo-conformation consistent with the stereo-chemistry of other monoterpene cyclizations and with chemical model studies. Sulfonium ion analogs of the presumptive linalyl and alpha-terpinyl cationic intermediates of the isomerization-cyclization sequence were shown to be potent inhibitors of the enzymatic reaction (Ki = 0.3 and 2.8 microM, respectively), and inhibition was synergized by the presence of inorganic pyrophosphate, indicating that the enzyme recognized and bound more tightly to these ion-paired species than to either cationic or anionic partner alone. Additionally, the enzyme was capable of ionizing (solvolyzing) the noncyclizable substrate analogs 6,7-dihydrogeranyl pyrophosphate and 2,3-methanogeranyl pyrophosphate. These results define the overall stereochemistry of the coupled isomerization-cyclization to sabinene hydrate, demonstrate the 1,2-hydride shift, and confirm the electrophilic nature of this enzymatic reaction type.     

Biosynthesis of monoterpenes. Enantioselectivity in the enzymatic cyclization of linalyl pyrophosphate to (-)-endo-fenchol

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.     

Evidence for the ionization steps in monoterpene cyclization reactions using 2-fluorogeranyl and 2-fluorolinalyl pyrophosphates as substrates

Conversion of geranyl pyrophosphate to cyclic monoterpenes is considered to involve the preliminary isomerization of this acyclic precursor to enzyme-bound linalyl pyrophosphate and the cyclization of this tertiary intermediate. 2-Fluorogeranyl pyrophosphate and 2-fluorolinalyl pyrophosphate are effective competitive inhibitors of the cyclization of geranyl pyrophosphate by several different monoterpene cyclases, and the electron withdrawing alpha-fluorine substituent was shown to suppress the rate of cyclic product formation from both tritium-labeled analogs by at least two orders of cyclic These results indicate that both steps of the coupled isomerization-cyclization sequence are initiated by ionization of an allylic pyrophosphate, and they confirm the electrophilic nature of this enzymatic reaction type and its similarity to the prenyltransferase reaction.     

Biosynthesis of monoterpenes. Stereochemistry of the enzymatic cyclization of geranyl pyrophosphate to (-)-endo-fenchol

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. Incubation of (1R)-[2-14C,1-3H]- and (1S)-[2-14C,1-3H]geranyl pyrophosphate with a preparation of (-)-endo-fenchol cyclase (synthase) from common fennel (Foeniculum vulgare) gave labeled product of unchanged 3H:14C ratio in both cases, and each was dehydrated to a mixture of alpha- and beta-fenchene which were oxidized to the corresponding alpha- and beta-fenchocamphorones, again without change in isotope ratio. 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 findings indicated that the configuration at C1 of the substrate was retained in the enzymatic transformation to (-)-endo-fenchol which is entirely consistent with the syn-isomerization of geranyl pyrophosphate to (3R)-linalyl pyrophosphate and cyclization of the latter via the anti-endo-conformer. These absolute stereochemical elements of the reaction sequence were confirmed by the enzymatic conversion of (3R)-1Z-[1-3H]linalyl pyrophosphate to (-)-endo-fenchol and by the location of the tritium in the derived fenchocamphorones as before. The summation of the results fully defines the overall stereochemistry of the coupled isomerization and cyclization of geranyl pyrophosphate to (-)-endo-fenchol.     

Inhibition of monoterpene cyclases by sulfonium analogs of presumptive carbocationic intermediates of the cyclization reaction

The enzymatic cyclization of geranyl pyrophosphate to monoterpenes is thought to proceed through a series of carbocation-pyrophosphate anion paired intermediates. Sulfonium analogs of two putative carbocationic intermediates of the cyclization sequence were shown to be inhibitors of the conversion of the acyclic precursor to the bicyclic monoterpenes (+)-alpha-pinene and (+)-bornyl pyrophosphate by partially purified cyclase preparations from sage (Salvia officinalis). The sulfonium analog of the tertiary allylic, linalyl, intermediate (i.e. methyl-(4-methylpent-3-en-1-yl)vinyl-sulfonium perchlorate) provided respective Ki values of 2.5 microM and 3.0 microM against the cyclization to alpha-pinene and bornyl pyrophosphate at a substrate concentration of 5 microM, whereas the sulfonium analog of the monocyclic, alpha-terpinyl, intermediate (i.e. dimethyl-(4-methylcyclohex-3-en-1-yl) sulfonium iodide) exhibited respective Ki values of 3.4 microM and 3.9 microM against the same two cyclizations. The potency of inhibition in all cases increased with increasing substrate concentration, indicating that the affinity of the enzymes for the sulfonium analogs was increased by the presence of the pyrophosphate ester. Inorganic pyrophosphate at a concentration of 50 microM, which alone had little influence on the cyclizations, increased the effectiveness of inhibition of the sulfonium analogs severalfold, and the apparent Ki for inorganic pyrophosphate was reduced manyfold by the presence of either analog at 5 microM. That the combination of sulfonium analog and pyrophosphate provided synergistic inhibition of the electrophilic cyclizations indicated that the cyclases bind the paired species more tightly than either partner alone. Specificity studies suggested that inhibition by the above sulfonium ion:pyrophosphate pairs was due to both electronic and structural resemblance to intermediates of the reaction.     

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 monoterpene synthases has prevented direct observation of the topologically required isomerization step leading from geranyl diphosphate to the presumptive, enzyme-bound, tertiary allylic intermediate linalyl diphosphate, which ultimately cyclizes to the various monoterpene skeletons. Previous experimental approaches using the noncyclizable substrate analogs 6,7-dihydrogeranyl diphosphate and racemic methanogeranyl diphosphate, in attempts to dissect the cryptic isomerization step from the normally coupled reaction sequence, were thwarted by the limited product available from native monoterpene synthases and by the inability to resolve chiral monoterpene products at the microscale. These approaches were revisited using three recombinant monoterpene synthases and chiral phase capillary gas chromatographic methods to separate antipodal products of the substrate analogs. The recombinant monoterpene olefin synthases, (-)-limonene synthase from spearmint and (-)-pinene synthase from grand fir, yielded essentially only achiral, olefin products (corresponding to the respective analogs and homologs of myrcene, trans-ocimene and cis-ocimene) from 6,7-dihydrogeranyl diphosphate and (2S,3R)-methanogeranyl diphosphate; no significant amounts of terpenols or homoterpenols were formed, nor was direct evidence obtained for the formation of the anticipated 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 sage, the achiral olefins were generated, as before, from 6,7-dihydrogeranyl diphosphate and (2R,3S)-methanogeranyl diphosphate, but 6,7-dihydrolinalool 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 diphosphate was obtained. Resolution of the terpenol products of (+)-bornyl diphosphate synthase, by chiral phase separation, revealed the predominant formation of (3R)-dihydrolinalool from dihydrogeranyl diphosphate and of (4S)-homolinalool from (2R,3S)-methanogeranyl diphosphate. The opposite stereochemistries of these products indicates water trapping from opposite faces of the corresponding tertiary carbocationic intermediates of the respective reactions, a phenomenon that appears to result from the binding conformations of these 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.     

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

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.     

Biosynthesis of monoterpenes. Stereochemical implications of acyclic and monocyclic olefin formation by (+)- and (-)-pinene cyclases from sage

(+)-Pinene cyclase from sage (Salvia officinalis) catalyzes the isomerization and cyclization of geranyl pyrophosphate to (+)-alpha-pinene and (+)-camphene, and to lesser amounts of (+)-limonene, myrcene, and terpinolene, whereas (-)-pinene cyclase from this tissue catalyzes the conversion of the acyclic precursor to (-)-alpha-pinene, (-)-beta-pinene, and (-)-camphene, and to lesser quantities of (-)-limonene, myrcene, and terpinolene. The bicyclic products of these enzymes (pinene and camphene) are derived via the cyclization of the cisoid, anti-endo-conformers of the bound, tertiary allylic intermediates (3R)-linalyl pyrophosphate [+)-pinene cyclase) and (3S)-linalyl pyrophosphate [-)-pinene cyclase). When challenged with either enantiomer of linalyl pyrophosphate or with neryl pyrophosphate (cis-isomer of geranyl pyrophosphate) as substrate, both pinene cyclases synthesize disproportionately high levels of acyclic olefins (myrcene and ocimene) and monocyclic olefins (limonene and terpinolene), compared with the product mixtures generated from the natural geranyl precursor. Resolution of the limonene derived from linalyl pyrophosphate and neryl pyrophosphate demonstrated that this monocyclic olefin was formed via conformational foldings in addition to the cisoid,anti-endo-pattern. These results indicate that the alternate substrates are ionized by the cyclases prior to their achieving the optimum orientation for bicyclization. In the case of geranyl pyrophosphate, a preassociation mechanism is suggested in which optimum folding of the terpenyl chain precedes the initial ionization step.     

Characterization and mechanism of (4S)-limonene synthase, a monoterpene cyclase from the glandular trichomes of peppermint (Mentha x piperita).

(4S)-Limonene synthase, a monoterpene cyclase isolated from the secretory cells of the glandular trichomes of Mentha x piperita (peppermint), catalyzes the cyclization of geranyl pyrophosphate to (4S)-limonene, a key intermediate in the biosynthesis of p-menthane monoterpenes in Mentha species. The enzyme synthesizes principally (-)-(4S)-limonene (greater than 94% of the total products), plus several other monoterpene olefins. The general properties of (4S)-limonene synthase resemble those of other monoterpene cyclases. The enzyme shows a pH optimum near 6.7, an isoelectric point of 4.35, and requires a divalent metal ion for catalysis, either Mg2+ or Mn2+, with Mn2+ preferred. The Km value measured for geranyl pyrophosphate was 1.8 microM. The activity of (4S)-limonene synthase was inhibited by sodium phosphate, sodium pyrophosphate, and reagents directed against the amino acids cysteine, methionine, and histidine. In the presence of Mn2+, geranyl pyrophosphate protected against cysteine-directed inhibition, suggesting that at least one cysteine residue is located at or near the active site. Experiments with alternate substrates and substrate analogs confirmed many elements of the proposed reaction mechanism, including the binding of geranyl pyrophosphate in the form of a complex with the divalent metal ion, the preliminary isomerization of geranyl pyrophosphate to linalyl pyrophosphate (a bound intermediate capable of cyclization), and the participation of a series of carbocation:pyrophosphate anion pairs in the reaction sequence.     

Monoterpene cyclases: use of the noncyclizable substrate analog 6,7-dihydrogeranyl pyrophosphate to uncouple the isomerization step of the coupled isomerization-cyclization reaction

Enzymes from Salvia officinalis capable of catalyzing the isomerization and subsequent cyclization of geranyl pyrophosphate to the monoterpenes (+)-alpha-pinene and (+)-bornyl pyrophosphate were examined with the noncyclizable substrate analog 6,7-dihydrogeranyl pyrophosphate in an attempt to dissect the cryptic isomerization step from the normally coupled reaction sequence. The analog inhibited the cyclization of geranyl pyrophosphate and was itself catalytically active, affording acyclic terpene olefins and alcohols as products. The enzymatic products generated from 6,7-dihydrogeranyl pyrophosphate qualitatively resembled the solvolysis products of 6,7-dihydrolinalyl pyrophosphate, yet they constituted a far higher proportion of olefins, suggesting that enzymatic product formation occurs in an environment relatively inaccessible to water. Since the normal cyclization of geranyl pyrophosphate is considered to proceed via preliminary isomerization to the bound tertiary intermediate (3R)-linalyl pyrophosphate, the results suggest that the analog undergoes the normal pyrophosphate ionization-migration step, giving rise in this case to (3R)-6,7-dihydrolinalyl pyrophosphate which is reionized, and because the subsequent cyclizations are precluded, the resulting cation is either deprotonated or captured by water. In divalent metal ion requirement, pH optimum, and other characteristics, the enzymatic transformation of the analog resembles the normal monoterpene cyclase reaction.     

Uncompetitive inhibition of monoterpene cyclases by an analog of the substrate geranyl pyrophosphate and inhibition of monoterpene biosynthesis in vivo by an analog of geraniol

Monoterpene cyclases catalyze the divalent metal ion-dependent conversion of the acyclic precursor geranyl pyrophosphate to a variety of monocyclic and bicyclic monoterpene skeletons. Examination of the kinetics of inhibition of cyclization by the pyrophosphate ester of (E)-4-[2-diazo-3-trifluoropropionyloxy]-3-methyl-2-buten-1-o l, a photolabile structural analog of the substrate, using a partially purified preparation of geranyl pyrophosphate:(+)-pinene cyclase and geranyl pyrophosphate:(+)-bornyl pyrophosphate cyclase from common sage (Salvia officinalis) evidenced (under dark conditions) strictly uncompetitive inhibition with K'i values of 3.2 and 4.7 microM, respectively. These values are close to the corresponding Km values for the substrate with these two enzymes. This novel property of the substrate analog was also examined in the presence of two other inhibitors which bind to different domains of the cyclase active site (inorganic pyrophosphate and a sulfonium ion analog of a cyclic carbocationic intermediate of the reaction sequence (dimethyl-(4-methylcyclohex-3-en-1-yl)sulfonium iodide)) in order to address the mechanistic origins of the uncompetitive inhibition of cyclization. It was not possible, however, to rule out either an induced-fit mechanism or a sequential binding mechanism since the substrate is recognized by at least two binding domains and because direct examination of the effects of binding on cyclase conformation is currently not feasible. The substrate analog, although photoactive, did not give rise to light-dependent enzyme inactivation of greater magnitude than that obtained from ultraviolet light alone. The unusual behavior of the analog was attributed to intramolecular interaction of the electron-rich carbonyl group of the diazoester with the required divalent metal ion that is chelated by the pyrophosphate group. A photostable analog of geraniol that resembled the photoactive substrate analog in bearing a carbonyl function at C6 (6-oxo-3,7-dimethyloct-2(trans)en-1-ol) was prepared. Following foliar application to rapidly growing sage plants, this analog was seemingly activated to the corresponding pyrophosphate ester in vivo and selectively inhibited the activity of several cyclases in this tissue as evidenced by diminished production of the corresponding monoterpene end products.     

Biosynthesis of monoterpenes. Stereochemistry of the enzymatic cyclizations of geranyl pyrophosphate to (+)-alpha-pinene and (-)-beta-pinene

The conversion of geranyl pyrophosphate to (+)-alpha-pinene and to (-)-beta-pinene is considered to proceed by the initial isomerization of the substrate to (-)-(3R)- and to (+)-(3S)-linalyl pyrophosphate, respectively, and the subsequent cyclization of the anti, endo-conformer of these bound intermediates by mirror-image sequences which should result in the net retention of configuration at C1 of the geranyl precursor. Incubation of (1R)-[2-14C,1-3H]- and (1S)-[2-14C,1-3H]geranyl pyrophosphate with (+)-pinene cyclase and with (-)-pinene cyclase from common sage (Salvia officinalis) gave labeled (+)-alpha- and (-)-beta-pinene of unchanged 3H/14C ratio in all cases, and the (+)- and (-)-olefins were stereoselectively converted to (+)- and (-)-borneol, respectively, which were oxidized to the corresponding (+)- and (-)-isomers of camphor, again without change in isotope ratio. The location of the tritium was determined in each case by stereoselective, base-catalyzed exchange of the exo-alpha-hydrogens of these derived ketones. The results indicated that the configuration at C1 of the substrate was retained in the enzymatic transformations to the (+)- and (-)-pinenes, which is entirely consistent with the syn-isomerization of geranyl pyrophosphate to linalyl pyrophosphate, transoid to cisoid rotation, and anti, endo-cyclization of the latter. The absolute stereochemical elements of the antipodal reaction sequences were confirmed by the selective enzymatic conversions of (3R)- and (3S)-1Z-[1-3H]linalyl pyrophosphate to (+)-alpha-pinene and (-)-beta-pinene, respectively, and by the location of the tritium in the derived camphors as before. The summation of the results fully defines the overall stereochemistry of the coupled isomerization and cyclization of geranyl pyrophosphate to the antipodal pinenes.     

Biosynthesis of monoterpenes: stereochemistry of the coupled isomerization and cyclization of geranyl pyrophosphate to camphane and isocamphane monoterpenes

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.     

Pinene cyclases I and II. Two enzymes from sage (Salvia officinalis) which catalyze stereospecific cyclizations of geranyl pyrophosphate to monoterpene olefins of opposite configuration

A soluble enzyme preparation from immature sage (Salvia officinalis) leaves has been shown to catalyze the cation-dependent cyclization of geranyl pyrophosphate to the isomeric monoterpene olefins (+/-)-alpha-pinene and (-)-beta-pinene and to lesser amounts of camphene and limonene (Gambliel, H., and Croteau, R. (1982) J. Biol. Chem. 257, 2335-2342). This preparation was fractionated by gel filtration on Sephadex G-150 to afford two regions of enzymic activity termed geranyl pyrophosphate:pinene cyclase I (Mr approximately equal to 96,000), which catalyzed the conversion of geranyl pyrophosphate to the bicyclic olefin (+)-alpha-pinene, and to smaller quantities of the rearranged olefin (+)-camphene and the monocyclic olefin (+)-limonene, and geranyl pyrophosphate:pinene cyclase II (Mr approximately equal to 55,000), which transformed the acyclic precursor to (-)-alpha-pinene and (-)-beta-pinene, as well as to (-)-camphene, (-)-limonene, and the acyclic olefin myrcene. The multiple olefin biosynthetic activities co-purified with pinene cyclase I on four subsequent chromatographic and electrophoretic steps, and the ability to cyclize geranyl pyrophosphate and the related allylic pyrophosphates neryl pyrophosphate and linalyl pyrophosphate was likewise coincident throughout purification. Fractionation of pinene cyclase II by an identical sequence showed that the activities for the synthesis of the stereochemically related (-)-olefins co-purified, as did the ability to utilize the three acyclic precursors. The general properties of cyclase I and cyclase II were determined, and a scheme for the biosynthesis of the pinenes and related monoterpene olefins was proposed.     

Biosynthesis of monoterpenes. Enantioselectivity in the enzymatic cyclization of (+)- and (-)-linalyl pyrophosphate to (+)- and (-)-pinene and (+)- and (-)-camphene

Cyclase I from Salvia officinalis leaf catalyzes the conversion of geranyl pyrophosphate to the stereo-chemically related bicyclic monoterpenes (+)-alpha-pinene and (+)-camphene and to lesser quantities of monocyclic and acyclic olefins, whereas cyclase II from this plant tissue converts the same acyclic precursor to (-)-alpha-pinene, (-)-beta-pinene and (-)-camphene as well as to lesser amounts of monocyclics and acyclics. These antipodal cyclizations are considered to proceed by the initial isomerization of the substrate to the respective bound tertiary allylic intermediates (-)-(3R)- and (+)-(3S)-linalyl pyrophosphate. [(3R)-8,9-14C,(3RS)-1E-3H]Linalyl pyrophosphate (3H:14C = 5.14) was tested as a substrate with both cyclases to determine the configuration of the cyclizing intermediate. This substrate with cyclase I yielded alpha-pinene and camphene with 3H:14C ratios of 3.1 and 4.2, respectively, indicating preferential, but not exclusive, utilization of the (3R)-enantiomer. With cyclase II, the doubly labeled substrate gave bicyclic olefins with 3H:14C ratios of from 13 to 20, indicating preferential, but not exclusive, utilization of the (3S)-enantiomer in this case. (3R)- and (3S)-[1Z-3H]linalyl pyrophosphate were separately compared to the achiral precursors [1-3H]geranyl pyrophosphate and [1-3H]neryl pyrophosphate (cis-isomer) as substrates for the cyclizations. With cyclase I, geranyl, neryl, and (3R)-linalyl pyrophosphate gave rise exclusively to (+)-alpha-pinene and (+)-camphene, whereas (3S)-linayl pyrophosphate produced, at relatively low rates, the (-)-isomers. With cyclase II, geranyl, neryl, and (3S)-linalyl pyrophosphate yielded exclusively the (-)-isomer series, whereas (3R)-linalyl pyrophosphate afforded the (+)-isomers at low rates. These results are entirely consistent with the predicted stereochemistries and additionally revealed the unusual ability of these enzymes to catalyze antipodal cyclizations when presented with the unnatural linalyl enantiomer.     

Biosynthesis of monoterpenes: inhibition of (+)-pinene and (-)-pinene cyclases by thia and aza analogs of the 4R- and 4S-alpha-terpinyl carbocation.

(+)-Pinene cyclase (synthase) from Salvia officinalis leaf catalyzes the cyclization of geranyl pyrophosphate, via (3R)-linalyl pyrophosphate and the (4R)-alpha-terpinyl cation, to (+)-alpha-pinene and to lesser quantities of stereochemically related monoterpene olefins, whereas (-)-pinene cyclase converts the same achiral precursor, via (3S)-linalyl pyrophosphate and the (4S)-alpha-terpinyl cation, to (-)-alpha-pinene and (-)-beta-pinene and to lesser amounts of related olefins. Racemic thia analogs of the linalyl and alpha-terpinyl carbocation intermediates of the reaction sequence were previously shown to be good uncompetitive inhibitors of monoterpene cyclases, and inhibition was synergized by the presence of inorganic pyrophosphate. These results suggested that the normal reaction proceeds through a series of carbocation:pyrophosphate anion paired intermediates. Both the (4R)- and the (4S)-thia and -aza analogs of the alpha-terpinyl cation were prepared and tested as inhibitors with the antipodal pinene cyclases, both in the absence and in the presence of inorganic pyrophosphate. Although the inhibition kinetics were complex, cooperative binding of the analogs and inorganic pyrophosphate was demonstrated, consistent with ion pairing of intermediates in the course of the normal reaction. Based on the antipodal reactions catalyzed by the pinene cyclases, stereochemical differentiation between the (4R)- and the (4S)-analogs was anticipated; however, neither enzyme effectively distinguished between enantiomers of the thia and aza analogs of the alpha-terpinyl carbocation. Enantioselectivity in the enzymatic conversion of (RS)-alpha-terpinyl pyrophosphate to limonene by the pinene cyclases was also examined. Consistent with the results obtained with the thia and aza analogs, the pinene cyclases were unable to discriminate between enantiomers of alpha-terpinyl pyrophosphate in this unusual reaction. Either the alpha-terpinyl antipodes are too similar to allow differentiation by the pinene cyclases, or these enzymes lack an inherent requirement to distinguish the (4R)- and (4S)-forms because they encounter only one enantiomer in the course of the normal reaction from geranyl pyrophosphate.     

Monoterpene cyclases. Stereoelectronic requirements for substrate binding and ionization

Enzymes from Salvia officinalis, capable of catalyzing the electrophilic isomerization and subsequent cyclization of geranyl pyrophosphate (3,8-dimethylocta-2E,6-dienyl pyrophosphate) to the monoterpenes (+)-alpha-pinene and (+)-bornyl pyrophosphate, were examined with a series of substrate analogs modified in carbon chain length and in the geometric and electronic character of the C2-C3 and C6-C7 olefinic domains. Inhibition studies with these monoterpene cyclases indicated that the pyrophosphate ester function was the principal determinant of substrate recognition and that the C2-C3 olefin was recognized largely on the basis of geometry, whereas the primary basis of interaction with the C6-C7 olefin was electronic. A related group of allylic pyrophosphates was tested for the ability to undergo enzyme-catalyzed ionization to afford olefinic and/or alcoholic products. From the relative reaction rates it was deduced that the alignment of the allylic pi-system with the C1-OP bond was essential for ionization of the substrate and that specific interaction with the distal C6-C7 isopropylidene function served not only to optimize orbital alignment but also to exclude water from the active site, and thus determine the partitioning of cationic intermediates into olefins or alcohols. From the combination of results, the interrelationships of substrate functional groups within the active site could be approximated and the topology of geranyl pyrophosphate binding to the cyclase thereby formulated.     

Biosynthesis of monoterpenes. Enantioselectivity in the enzymatic cyclization of (+)- and (-)-linalyl pyrophosphate to (+)- and (-)-bornyl pyrophosphate

Enzymes from Salvia officinalis and Tanacetum vulgare leaf epidermis catalyze the conversion of the acyclic precursor geranyl pyrophosphate to the cyclic monoterpenes (+)- and (-)-bornyl pyrophosphate, respectively. The antipodal cyclizations are considered to proceed by the initial isomerization of the substrate to the respective bound tertiary allylic intermediates (-)-(3R)- and (+)-(3S)-linalyl pyrophosphate. [(3R)-8,9-14C,(3RS)-1E-3H] Linalyl pyrophosphate (3H:14C = 5.22) was tested as a substrate with the cyclases from both sources to determine the configuration of the cyclizing intermediate. This substrate yielded (-)-bornyl pyrophosphate with 3H:14C ratio greater than 31, indicating specific utilization of (+)-(3S)-linalyl pyrophosphate as predicted. With the (+)-bornyl pyrophosphate cyclase, the 3H:14C ratio of the product was about 4.16, indicating a preference for the (-)-(3R)-enantiomer, but the ability also to utilize (+)-(3S)-linalyl pyrophosphate. (3R)- and (3S)-[1Z-3H]Linalyl pyrophosphate were separately compared to the achiral precursors [1-3H] geranyl pyrophosphate and [1-3H]neryl pyrophosphate (cis-isomer) as substrates for the cyclizations. All functional precursors afforded optically pure (-)-(1S,4S)-bornyl pyrophosphate with the T. vulgare-derived cyclase (as determined by chromatographic separation of diastereomeric ketals of the derived ketone camphor), and (+)-(3S)-linalyl pyrophosphate was the preferred substrate. With the (+)-bornyl pyrophosphate cyclase from S. officinalis, geranyl, neryl, and (-)-(3R)-linalyl pyrophosphates gave the expected (+)-(1R,4R)-stereoisomer as the sole product, and (-)-(3R)-linalyl pyrophosphate was the preferred substrate. However, (3S)-linalyl pyrophosphate yielded (-)-(1S,4S)-bornyl pyrophosphate, albeit at lower rates, indicating the ability of this enzyme to catalyze the anomalous enantiomeric cyclization.     

Evidence for an essential histidine residue in 4S-limonene synthase and other terpene cyclases.

(4S)-Limonene synthase, isolated from glandular trichome secretory cell preparations of Mentha x piperita (peppermint) leaves, catalyzes the metal ion-dependent cyclization of geranyl pyrophosphate, via 3S-linalyl pyrophosphate, to (-)-(4S)-limonene as the principal product. Treatment of this terpene cyclase with the histidine-directed reagent diethyl pyrocarbonate at a concentration of 0.25 mM resulted in 50% loss of enzyme activity, and this activity could be completely restored by treatment of the preparation with 5 mM hydroxylamine. Inhibition with diethyl pyrocarbonate was distinguished from inhibition with thiol-directed reagents by protection studies with histidine and cysteine carried out at varying pH. Inactivation of the cyclase by dye-sensitized photooxidation in the presence of rose bengal gave further indication of the presence of a readily modified histidine residue. Protection of the enzyme against inhibition with diethyl pyrocarbonate was afforded by the substrate geranyl pyrophosphate in the presence of Mn2+, and by the sulfonium ion analog of the linalyl carbocation intermediate of the reaction in the presence of inorganic pyrophosphate plus Mn2+, suggesting that an essential histidine residue is located at or near the active site. Similar studies on the inhibition of other monoterpene and sesquiterpene cyclases with diethyl pyrocarbonate suggest that a histidine residue (or residues) may play an important role in catalysis by this class of enzymes.     

Studies of the cryptic allylic pyrophosphate isomerase activity of trichodiene synthase using the anomalous substrate 6,7-dihydrofarnesyl pyrophosphate

Two enantiomeric analogues of farnesyl pyrophosphate (1) were tested as inhibitors and anomalous substrates of trichodiene synthase, which catalyzes the cyclization of trans,trans-farnesyl pyrophosphate (1) to the sesquiterpene hydrocarbon trichodiene (2). The reaction has been shown to involve preliminary isomerization of 1 to the tertiary allylic isomer nerolidyl pyrophosphate (3) which is cyclized without detectable release of the intermediate from the active site of the cyclase. Both (7S)-trans-6,7-dihydrofarnesyl pyrophosphate (7a) and (7R)-trans-6,7-dihydrofarnesyl pyrophosphate (7b), prepared from (3R)- and (3S)- citronellol (9a and 9b), respectively, proved to be modest competitive inhibitors of trichodiene synthase. The values of Ki(7a), 395 nM, and Ki(7b), 220 nM, were 10-15 times the observed Km for 1 and half the Ki of inorganic pyrophosphate alone. Incubation of either 7a or 7b with trichodiene synthase resulted in formation of a mixture of products which by radio/gas-liquid chromatographic and GC/selected ion mass spectrometric analysis was shown to be composed of 80-85% isomeric trienes 19-21 and 15-20% allylic alcohols 12 and 18. Examination of the water-soluble products resulting from incubation of 7a also revealed the generation of 24% of the isomeric cis-6,7-dihydrofarnesyl pyrophosphate (26). The combined rate of formation of anomalous alcoholic and olefinic products was 10% the Vmax determined for the conversion of 1 to 2. The results can be explained by initial enzyme-catalyzed isomerization of dihydrofarnesyl pyrophosphate (7) to the corresponding tertiary allylic isomer dihydronerolidyl pyrophosphate (8). Since the latter intermediate is unable to cyclize due to the absence of the 6,7-double bond, ionization of 8 and quenching of the resulting ion pair by deprotonation, capture of water, or collapse to the isomeric primary pyrophosphate esters will generate the observed spectrum of anomalous products.