Silent catalytic promiscuity in the high-fidelity terpene cyclase δ-cadinene synthase

Aza-analogues of carbocations inhibit δ-cadinene synthase: 1,6-cyclisation.

1. Materials and general methods. d-cadinene synthase (DCS) from Gossypium arboreum was produced as previously described 1 using competent cells from E.coli . Amorphadiene synthase (ADS) from Artemisia annua was produced as previously described by Demiray et al. 2 Aristolochene synthase (AS) from Penicillum roqueforti was produced as previously described in the literature. 3 Protein expression was induced by the use of isopropyl-thio-b-D-galactopyranoside (IPTG). Nickel affinity chromatography was used for protein purification of DCS and ADS. A diethylaminoethyl anion exchange column (DEAE, 75 mL) was used for AS purification. A prestained protein size marker (6.5-175kD) was used to identify protein by 10% SDS gel. An Amicon YM30 For synthetic procedures, all chemicals and solvents were obtained from commercial vendors and used without further purification unless otherwise noted. Anhydrous tetrahydrofuran (THF), diethyl ether, toluene and acetonitrile were obtained from a MBraun SPS800 solvent purification system. Dichloromethane (CH2Cl2) and trimethylamine (Et3N) were distilled under nitrogen over calcium hydride and potassium hydroxide, respectively. 1 H NMR, and 13 C NMR, spectra were measured on Bruker Avance III 600, Bruker Avance 500, Bruker Avance III HD 400, and Bruker Fourier 300 NMR spectrometers. The spectra are reported in order as chemical shifts in parts per million (ppm), downfield from tetramethylsilane ( 1 H and 13 C), integral, multiplicity, coupling and assignment, injection temperature 100 °C; with a split ratio of 5:1; initial pressure 1 kPa, scans in function: 1525 ; initial oven temperature 80 °C, ramp of 4 °C/min to 180 °C (2 min hold) , flow 1 mL/min, retention window 0 to 28 min; Method 2: injection port at 100 °C; split ratio 5:1; initial pressure 1 kPa; scans in function: 1449; initial temperature 80 °C hold, ramp of 4 °C/min to 180 °C (15 min hold), then 20 °C/min to 250 °C, flow 1 mL/min, retention window 0 to 28 min.
Thin layer chromatography (TLC) was performed on pre-coated aluminium plates of silica G/UV254. TLC visualisations were performed with 4.2% ammonium molybdate and 0.2% ceric sulfate in 5% sulfuric acid (Hanessian's stain), or by UV light. The optical rotations were determinated using a UNiPol L-serie L1000/L2000 Schmidt+Haesch polarimeter. All measurements were performed at room temperature using a 5 cm long cell path and a wavelength of 269 nm.
Infrared (IR) spectra were obtained using a Jasco V-660 spectrophotometer.
Ion-exchange chromatography was performed using ion-exchange resin (Amberlyst 131 wet, H+ form) pre-equilibrated with ion-exchange buffer (25 mM NH4HCO3 containing 2% isopropanol, 2 CV). Reverse phase HPLC was performed on a system comprising of a Dionex P680 pump and a Dionex UVD170U detector unit. The column used was a 150 x 21.2 mm Phenomenex Luna C-18 column.

Steady-state kinetics of DCS-His6, ADS and AS.
Kinetic assays were carried out according to the standard, linear range, micro-assay procedure previously described. 5 The reaction mixtures containing buffer, FDP and protein were prepared on ice in a total volume of 250 µL and were overlaid with ca. 0.8 mL of HPLC-grade hexane prior to incubation. The assay mixtures were initiated by addition of enzyme and incubated at room temp (30 °C) for 10 min. The reactions were immediately ice-cooled and quenched by addition of 200 µL of 100 mM EDTA (pH 8.5) and brief vortexing. The hexane overlay and two additional 1 mL hexane extracts were passed through a short pipette column containing silica gel. The column was washed with additional hexane (1 mL) and the combined filtrates were analysed by liquid scintillation counting using 15 mL of scintillation cocktail Ecoscint O. Steady-State kinetic parameters for DCS-His6 and mutants were obtained by direct fitting of the data to the Michaelis-Menten equation by nonlinear least squares regression in conjunction with the graphical procedures developed by Lineweaver-Burk using the commercial SigmaPlot package (Systat Software).