Terpene Synthase Activity Assays.

Sesquiterpene synthase activity in crude protein extracts was determined with enzyme assays consisting of 50 µL of protein extract and 50 µL of assay buffer [10 mM Tris·HCl, 10% (vol/vol) glycerol, 1 mM DTT (pH 7) with 10 mM MgCl₂] and 10 µM (E,E)-FPP (Sigma), (Z,Z)-FPP (Echelon), or (Z,E)-FPP as substrate. (Z,E)-FPP was synthesized according to method of Cane et al. (44) and was kindly provided by Nathalie Gatto and Wilhelm Boland, Max Planck Institute for Chemical Ecology, Jena, Germany. Heterologously expressed TPS enzymes (50 µL) were incubated with geranyl diphosphate [(E)-GPP] (Sigma), all available FPP substrates, as well as geranylgeranyl diphosphate [(E,E,E)-GGPP] (Sigma) as described above. Assays were performed in 1-mL glass vials, and enzyme products were collected using solid-phase microextraction (SPME). After penetrating the PTFE-lined silicone septum of the cap with a SPME fiber holder, a SPME fiber coated with 100-µm polydimethylsiloxane (Supelco) was exposed to the headspace in the vial for 2 h at 30 °C. Afterward, the SPME fiber was directly inserted into the inlet of a gas chromatograph. For collection and analysis of (E,E,E)-GGPP–derived diterpene products, assays were overlaid with 100 µL of hexane and incubated for 2 h at 30 °C. After mixing for 60 s, 1 µL of the organic phase was removed and directly injected into a gas chromatograph.

For analyzing the influence of different divalent metal cofactors on PsTPS1 activity and product specificity, enzyme assays with (Z,E)-FPP as substrate and three different concentrations (0.1, 1, and 10 mM) of each MgCl₂ and MnCl₂ were performed as described above. Assays were overlaid with 100 µL of hexane and incubated for 2.5 h at 30 °C. After mixing for 60 s, 1 µL of the organic phase was removed and directly injected into a gas chromatograph.