Monoterpene Cyclases Research Articles

Elucidation of iridodial formation mechanism - partial purification and characterization of the novel monoterpene cyclase from [formula omitted] cell suspension cultures

From the incubation system of 10-hydroxygeraniol and crude enzyme extracts from Rauwolfia serpentina cell suspension cultures, 10-oxogeraniol, 10-hydroxygeranial and 10-oxogeranial were isolated. A precursory role in the biosynthesis of iridodial has been demonstrated for these three compounds. This fact unequivocally substantiates our previous claim for the biosynthesis of iridodial. In addition, a novel monoterpene cyclase catalyzing the iridodial formation was partially purified and characterized.

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 α-fluorine substituent was shown to suppress the rate of cyclic product formation from both tritium-labeled analogs by at least two orders of magnitude. 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.

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.

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 (+)-α-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 (3 R)-linalyl pyrophosphate, the results suggest that the analog undergoes the normal pyrophosphate ionization-migration step, giving rise in this case to (3 R)-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.

The odorous constituents of peaches

(+)-Pinene cyclase (synthase) from Salvia officinalis leaf catalyzes the cyclization of geranyl pyrophosphate, via (3R)-linalyl pyrophosphate and the (4R)-α-terpinyl cation, to (+)-α-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)-α-terpinyl cation, to (−)-α-pinene and (−)-β-pinene and to lesser amounts of related olefins. Racemic thia analogs of the linalyl and α-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 α-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 α-terpinyl carbocation. Enantioselectivity in the enzymatic conversion of (RS)-α-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 discrimintae between enantiomers of α-terpinyl pyrophosphate in this unusual reaction. Either the α-terpinyl antipodes are too similar to allow differentiation by the pinene cyclases, or these enzymes lack an inherent requirment to distinguish the (4R)- and (4S)-forms because they encounter only one enantiomer in the course of the normal reaction from geranyl pyrophosphate.