Limonene cyclase

A cell-free system from Salvia officinalis (Lamiaceae) that synthesizes 1,8-cineole (10), limonene (11), terpinolene (12), and a-terpineol (13) from GPP (NPP also serves as a precursor) has been established. These cyclic monoterpenes were formed by independent routes from the precursor and not as free intermediates of a common reaction sequence (Loomis and Croteau, 1980). Fractionation of the soluble preparation allowed separation of 1,8-cineole, a-terpineol, and limonene cyclase activities. Thus, a series of competing, but distinct, cyclases exist in relatively crude preparations with the ability to synthesize cyclic monoterpenes in this plant. A single protein seemed to be involved in the formation of 1,8-cineole (10) from an acyclic precursor and there was no evidence for any free intermediates between GPP and 1,8-cineole. It is possible that some sort of channeled process occurs that precludes entry of exogenous precursors to the enzyme of the cell-free system or release of mtermedi-... 

The PEG precipitate (30- 0%) obtained from the 100,000 g supernatant of the epicarp of immature fruits of calamondin was submitted to chromatography on a column of DEAE-Sephacel and developed with a linear gradient of NaCl (0.1-0.2M). As shown in fig. 1, the limonene and selinene cyclase activities were resolved. The electrophoresis of the fractions involved in mono- and sesquiterpene biosynthesis showed the purification obtained after the DEAE-Sephacel column. Incubations of small gel pieces with [ H]-GPP and [ H]-FPP led to the localization of the cyclases involved in both limonene and selinene synthesis and indicated that the two cyclases are well separated on the gel. In order to improve the purification of these cyclases, the active fractions eluted from the DEAE-Sephacel column were submitted to affinity chromatography using Sepharose APP. This procedure contributed to a 90-fold and 70-fold purification for limonene and selinene cyclases respectively. An isoelectric point of 5A 0.2 was found for limonene-cyclase whereas 3-selinene-cy-clase presented a value of 6 0.2. After gel permeation with Sephadex G 200, the same molecular weight of 67 3 kD was attributed to the two cyclases. In addition, prenyltransferases obtained from a DEAE-Sephacel column developed with a step gradient (0.1, 0.2, 0.3 M NaCl) were also isolated on the same affinity column. These prenyltransferases were 160 fold purified. Their molecular weight was about 70 kD and they exhibited an isoelectric point of 3.2. 

Fig. 1 - Separation on DEAE-Sephacel chromatography column of cyclase activities involved in biosynthesis of mono- and sesquiterpene hydrocarbons, with a linear gradient of NaCl (0.1 - 0.2 M). Limonene-cyclase and selinene-cyclase were detected after incubation with pH] GPP and [ H] FPP respectively.

Rajaonarivony, J.I.M., Gershenzon, J. and Croteau, R. (1992) Characterization and mechanism of (4S)-limonene synthase, a monoterpene cyclase from the glandular trichomes of peppermint (Mentha x piperita). Arch. Biochem. Biophys., 296, 49-57. 

Hyatt DC, Youn B, Zhao Y, Santhamma B, Coates RM, Croteau RB, Kang C. Structure of limonene synthase, a simple model for terpenoid cyclase catalysis. Proc. Natl. Acad. Sci. U.S.A. 2007 104 5360-5365. 

Two pinene cyclases have been isolated from sage (19,35). Electrophoretically pure pinene cyclase I converts geranyl pyrophosphate to (+)-a-pinene and to lesser quantities of (+)-camphene and (+)-limonene, whereas pinene cyclase II, of lower molecular weight, converts the acyclic precursor to (-)-B-pinene and to lesser quantities of (-)-a-pinene, (-)-camphene and (-)-limonene. Both purified enzymes also utilize neryl and linalyl pyrophosphate as alternate substrates for olefin synthesis. The availability of enzyme systems catalyzing formation of enantiomeric products from a common, achiral substrate has provided an unusual opportunity to examine the stereochemistry of cyclization. 

The pinene cyclases convert the anomalous linalyl enantiomer to abnormal levels of acyclic (e.g. myrcene) and monocyclic (e.g. limonene) terpenes, these aberrant products perhaps arising via ionization of the tertiary substrate in the transoid or other partially extended (exo) form (see below). In any event, for all "normal" cyclizations examined thus far, the configuration of the cyclizlng linalyl Intermediate has been confirmed to be that which would be expected on the basis of an anti-endo conformation. Scattered attempts at intercalating the cyclization cascade with analogs of proposed cyclic intermediates (e.g. a-terpinyl and 2-pinyl pyrophosphate) have been unsuccessful (20,35,36). 

In a subsequent examination of the cyclases that formed ( 4-)- and ( — )-pinene, cyclase 1,96,000 MW, converted geranyl pyrophosphate to (-1-)-a-pinene (d-a-pimm) (14), but produced smaller amounts of (-b)- or if-camphene (15) and (+)- or /-limonene (11) as side products (Johnson and Croteau, 1987). Cyclase II, 55,000 MW, transformed geranyl pyrophosphate into ( —)-p-pinene (/- -pinene) (16) along with smaller amounts of /-a- or (— )-a-pinene, (-)- or /-camphene, ( —)- or /-limonene, and myrcene (17) as coproducts (Fig. 19.7) (Croteau, 1984 Johnson and Croteau, 1987). Extensive purification of each enzyme and differential inactivation studies ensured that each set of stereochemi-cally related products was synthesized by a single, distinct enzyme. 

Preparations from Mentha piperita (peppermint, La-miaceae) convert the acyclic precursors geranyl and neryl pyrophosphate into limonene (11). The bulk of the cyclase activity is in the soluble-enzyme fraction (Croteau, 1984). 

The nearly complete catalytic intolerance for glutamyl and alanyl substitutions in the DDxxD motif of limonene synthase is novel and unlike the much less pronounced effects of comparable substitutions in the sesquiterpene cyclase trichodiene synthase [97, 98]. However, pre-steady state kinetic analysis of trichodiene synthase [101] and several other sesquiterpene synthases [102] has recently shown that product release is rate limiting in these cases, and thus can mask the kinetic influence of the aspartate mutations on earlier steps in the catalytic cycle. In the instance of monoterpene cyclase catalysis, product release is not the slow step since comparison of k at values with GPP and LPP as substrate clearly reveals the initial ionization-isomerization to be rate limiting. Thus, perturbations that influence the first ionization step will be fully reflected in overall rate suppression for limonene synthase. This kinetic sensitivity at the initial steps of the reaction cycle does not, however, explain the near complete intolerance of limonene synthase to aspartate substitution in the DDxxD motif and it is thus tempting to speculate a more specific, but presently unidentified, influence on the requisite isomerization of GPP. 

The sequence of reactions catalyzed by the type A limonene synthase (monoterpene cyclase) (Fig. 4) is initiated by ionization-isomerization... 

Williams, D.C., McGarvey, D.J., Katahira, E.J. and Croteau, R. (1998) Truncation of limonene synthase preprotein provides a fuUy active pseudomature form of this monoterpene cyclase and reveals the function ofthe amino-terminal arginine pair. Biochemistry, 37,12213-12220. 

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