Biochemical characterization of purified sesquiterpene synthases

Utilization of “cluster tubes” in strips of 8 with accompanying lids in strips of 8 in a 96-well plate format is key to this method. Each strip represents one of the statistically necessary triplicates such that it includes negative and positive controls in addition to the substrate concentrations or other variables to be assayed.

Cluster tubes are normally used for extracting nucleic acids and protein from plants and fungi using a bead-beating principle. They are chemically and physically robust, regularly available at little expense, compatible with the 96-well form factor, and finally, they are more convenient than glass vials.

Controls in the assay were buffer alone, enzyme alone, substrate alone, and finally, samples from expression and purification of a pellet that harbors an empty expression vector.

Varying enzyme and substrate concentrations were tested in a small pilot assay setup to establish the intervals where substrate and enzyme concentration exhibits an adequate measurable activity. For each pilot reaction, 0.1–5 μg enzyme was assayed against 1.625–200 mM FPP, final pH 7.5, spiked with tritium-labeled FPP. This method established the adequate enzyme and substrate concentrations for low reagent usage and short assay time. Each substrate concentration was assayed in mono- or duplicate for one minute, including controls (reaction mix only, boiled enzyme). The pilot assay was performed identically to the final assay as described below.

Following the establishment of optimal enzyme concentration, the final assay could be performed. The enzyme fraction (EF) for one reaction was 1 μg enzyme diluted to 20 μL using 5 mM Tris-HCl pH 7.5, including triplicates with boiled and denatured enzyme. The substrate fraction (SF) for one reaction (80 μL per reaction) was made for each final substrate concentration to be assayed: 200, 100, 50, 25, 12.5, 6.75, 3.375, and 0 μM FPP. Each SF was spiked with ³H-FPP to a final specific activity of 0.082 mCi/mmol. Serial dilution was applied, and everything was mixed and kept on ice, not omitting the additional denatured enzyme and any other controls necessary. EF was buffered with Tris-HCl pH 7.5, and to a final reaction concentration of 45–50 mM MgCl₂.

First, 20 μL EF was dispensed to each reaction tube. Then, the reaction was started by adding 80 μL SF. Reactions were rapidly overlaid with 200 μL hexane, and tubes were sealed with lids. At the chosen timepoint(s), reactions were stopped by addition of 100 μL of 0.25 M EDTA, together with 0.5 M KOH or NaOH. The tubes were sealed again and were shaken thoroughly while holding the lids secure for at least one minute in order to ensure maximal extraction of product from the reaction phase. No subsequent extractions were performed. Then, the tubes were placed in a storage cassette, centrifuged for 1 minute at 1500 RCF, and either frozen at −20°C for later analysis or analyzed directly.

For analysis, 50 μL hexane overlay or control was mixed with 200 μL Ecoscint A scintillation fluid in a 96-well scintillation plate and sealed with plastic adhesive film. Plates were counted on a Microbeta 1450 scintillation counter for one minute.

The results were analyzed using Microsoft Excel for calculations of raw data and SigmaPlot 12 or 13 for data analysis and results. The amount of substrate converted in each reaction was calculated, and the amount of enzyme was standardized to per minute per microgram. The reaction rate per microgram enzyme per second was established, and the reaction rate (1/second) was calculated. The triplicate data were processed using SigmaPlot to provide enzymatic parameters, graphs, and error bars.