Protonation of a Neutral (S)-β-Bisabolene Intermediate Is Involved in (S)-β-Macrocarpene Formation by the Maize Sesquiterpene Synthases TPS6 and TPS11

Tobias G Köllner,Christiane Schnee,Shenghong Li,Aleš Svatoš,Bernd Schneider,Jonathan Gershenzon,Jörg Degenhardt

doi:10.1074/jbc.m802682200

Tobias G Köllner, Christiane Schnee + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m802682200

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Abstract

Terpene synthases are responsible for the large diversity of terpene carbon skeletons found in plants. The unique, carbocationic reaction mechanism of these enzymes can form multiple products from a single prenyl diphosphate substrate. Two maize genes were isolated that encode very similar sesquiterpene synthases, TPS6 and TPS11, which both produce beta-bisabolene, a common monocyclic sesquiterpene, and beta-macrocarpene, an uncommon bicyclic olefin. Investigation of the reaction mechanism showed that the formation of beta-macrocarpene proceeds via a neutral beta-bisabolene intermediate and requires reprotonation by a proton that may ultimately be abstracted from water. This reprotonation is dependent on the pH and the presence of a Mg(2+) cofactor. Mutational analysis of the enzyme demonstrated that a highly conserved tyrosine residue in the active center of the enzymes is important for the protonation process. TPS6 and TPS11 are transcribed both in leaves and roots of maize, but the respective terpene products were only detected in roots. The expression in roots was up-regulated by herbivore damage to the leaves, suggesting a long distance signal transduction cascade between leaves and roots.

Highlights

EXPERIMENTAL PROCEDURESPlant and Insect Material—Plants of the maize (Zea mays L.) variety B73 (KWS seeds, Einbeck, Germany) were grown in commercially available potting soil in a climate-controlled chamber with a 16-h photoperiod, 1 mmol (m2)Ϫ1 sϪ1 of photosynthetically active radiation, a temperature cycle of 22 °C/ 18 °C (day/night), and 65% relative humidity. 12–15-day-old plants (20 –30 cm high, 4 –5 expanded leaves) were used in all experiments
Synthase from Abies grandis synthesize 34 and 52 different sesquiterpenes from their farnesyl diphosphate substrate, respectively [4]. This unusual behavior is due to an electrophilic reaction mechanism common to all terpene synthases, which is initiated by elimination of the allylic diphosphate from the prenyl diphosphate substrate
A proton, which is abstracted from carbon atom 13 of the germacrenyl cation to form the neutral intermediate germacrene A, is transferred via the carboxyl group of aspartate 525 and hydroxyl group of tyrosine 520 back to a different position (C6) on the same carbon skeleton to form a second germacrenyl cation [8]

Summary

EXPERIMENTAL PROCEDURES

Plant and Insect Material—Plants of the maize (Zea mays L.) variety B73 (KWS seeds, Einbeck, Germany) were grown in commercially available potting soil in a climate-controlled chamber with a 16-h photoperiod, 1 mmol (m2)Ϫ1 sϪ1 of photosynthetically active radiation, a temperature cycle of 22 °C/ 18 °C (day/night), and 65% relative humidity. 12–15-day-old plants (20 –30 cm high, 4 –5 expanded leaves) were used in all experiments. To determine the catalytic activity of the recombinant proteins, enzyme assays containing 20 ␮l of the bacterial extract and 80 ␮l of assay buffer with 10 ␮M substrate (either GPP, (E,E)-FPP, (Z,E)-FPP, or GGPP), 10 mM MgCl2, 0.2 mM NaWO4, and 0.1 mM NaF in a Teflon-sealed, screw-capped 1-ml GC glass vial were performed. For product identification by NMR, four assays each containing 300 ␮l of bacterial extract, 218 ␮g of (E,E)-FPP, 10 mM MgCl2, 0.2 mM NaWO4, and 0.1 mM NaF in a total volume of 600 ␮l of assay buffer were overlaid with 500 ␮l of n-pentane and incubated for 5 h at 25 °C. The n-pentane was concentrated under a the putative terpene synthases TPS6 and TPS11, we expressed stream of nitrogen to a volume of 200 ␮l and stored at both ORFs in E. coli.

RESULTS

DISCUSSION

B Sesquiterpene hydrocarbons