Supplements
Experimental Part
Strains and plasmids
E.coli BL21 (DE3)pLysS was purchased from Invitrogen (Karlsruhe, Germany). Plasmid pIU18 for overproduction of the 4-dimethylallyltryptophan synthase was constructed previously (Steffan et al. 2007). Cultivation was carried out in liquid TB-medium supplemented with 50 µg kanamycin per ml as described previously (Steffan et al. 2007).
Chemicals
Trisammonium salt of DMAPP was synthesised in analogy to trisammonium geranyl diphosphate synthesis reported by Woodside (Woodside et al. 1988). Cyclo-D-Trp-L-Pro and cyclo-L-Trp-L-Pro were synthesised previously (Grundmann and Li 2005). The other substrates were obtained from Bachem (Weil am Rhein, Germany).
Overproduction and purification of the His-tagged dimethylallyltryptophan synthase
The His-tagged protein was overproduced and purified to apparent homogeneity as described previously (Steffan et al. 2007).
Assay conditions
All of the enzyme assays contained 50 mM Tris/HCl (pH 7.5) and 5 mM CaCl2, differing from each other by incubation volumes, substrate concentrations, amounts of FgaPT2 and incubation times. The reaction mixtures of the standard assay for determination of the substrate specificity (100 µl) contained 1 mM tryptophan or cyclic dipeptides, 2 mM DMAPP and 1.8 µM of purified FgaPT2. After incubation for 16 h at 30 °C, the reactions were stopped with 100 µl of CH3OH. After removal of the protein by centrifugation (15,000 x g, 10 min, 4 °C), the enzymatic products were analysed on an HPLC system described below. Two independent assays were carried out for quantification. The assays for isolation of the enzymatic products for structural elucidation (4 ml) contained 1 mM cyclic dipeptide, 2 mM DMAPP and 18 µM of purified FgaPT2 and were incubated for 16 hours. The reaction mixtures were extracted with EtOAc, concentrated on a rotation evaporator at 30 °C and dissolved in 500 µl CH3OH before injection. The assays for determination of the kinetic parameters (100 µl) contained 0.05-1.5 mM of the cyclic dipeptide, 1 mM DMAPP and 0.88 µM of purified FgaPT2. The incubation time was 60 min.
HPLC analysis
Reaction mixtures were analysed on an Agilent HPLC Series 1100 by using an Eclipse XDB-C18 column (4.6 x 150 mm, 5 µm, Agilent) at a flow rate of 1 ml min-1. H2O (solvent A) and CH3CN (solvent B), each containing 0.5 % (V/V) CF3COOH, were used as solvents. A gradient was run from 15 % to 70 % B in 25 min. After washing with 100 % solvent B for 5 min, the column was equilibrated with 85 % solvent A for 5 min. The substances were detected with a Photo Diode Array detector and illustrated at 277 nm.
Spectroscopic analysis
The isolated products (50 - 200 µg) were analyzed by 1H-NMR spectroscopy, 1H-1H-COSY as well as by positive and negative electrospray ionization (ESI) mass spectrometry with a ThermoFinnigan TSQ Quantum. The mass spectrometer was coupled with an Agilent HPLC series 1100 equipped with a RP18-column (2 x 250 mm, 5 µm). For separation, the column was run with 10 % solvent B (CH3OH) in solvent A (H2O, each containing 0.1 % HCOOH) for 5 min, followed by a gradient from 10 % to 100 % B over 30 min. After washing with 100 % B, the column was equilibrated with 10 % B for 10 min. The flow rate was at 0.2 ml min-1.
NMR spectra were taken on an Avance DRX 500 spectrometer (Bruker) using DMSO or CDCl3 as solvents. The δ values are given in ppm and coupling constants in Hz. The solvent signals at 7.26 ppm in CDCl3 and 2.50 ppm in DMSO were used as reference, respectively.
Reference List
Grundmann A, Li S-M (2005) Overproduction, purification and characterization of FtmPT1, a brevianamide F prenyltransferase from Aspergillus fumigatus. Microbiology 151:2199-2207
Steffan N, Unsöld IA, Li S-M (2007) Chemoenzymatic synthesis of prenylated indole derivatives by using a 4-dimethylallyltryptophan synthase from Aspergillus fumigatus. Chembiochem 8:1298-1307
Woodside AB, Huang Z, Poulter CD (1988) Triammonium germanyl diphosphate. Org Synth 66:211-215
Tables and Figures:
Table 1: MS-data of the enzymatic products of FgaPT2
enzymatic product / Mr / ESI (+) m/z / ESI (-) m/z[M+1]+ / [2M+1]+ / ms2[M+1]+
(intensitiy) / [M-1]- / [2M-1]- / ms2[M-1]-
(intensity)
cyclo-L-4-DMAT-Gly / 311.16 / 312.1 / 623.0 / - / 310.4 / - / -
cyclo-L-4-DMAT-L-Leu / 367.23 / 368.2 / 735.0 / - / 366.4 / 733.1 / 366.0 (100), 169.0 (83)
cyclo-L-4-DMAT-L-Phe / 401.21 / 402.2 / 803.0 / - / 400.4 / 800.8 / 203.0 (100), 158.1 (4)
cyclo-L-4-DMAT-L-Pro / 351.19 / 352.2 / 703.0 / - / 350.3 / - / 350.0 (98), 153.0 (100), 125.0 (8)
cyclo-L-4-DMAT-L-Trp / 440.22 / 441.2 / 881.1 / 385.1 (23), 312.1 (5), 310.1 (7), 242.0 (17), 238.1 (51), 198.2 (100), 183.1 (7) / 439.4 / 879.1 / 439.0 (100), 310.0 (38), 242.0 (33)
cyclo-L-DMAT-L-DMAT / 508.28 / 509.3 / - / 451.9 (5), 438.2 (9), 437.2 (100), 423.2 (7), 406.1 (5), 405.1 (8), 377.2 (8) / 507.5 / - / 311.0 (8), 310.0 (100)
cyclo-L-DMAT-L-Tyr / 417.21 / 418.2 / 835.0 / 362.1 (28), 238.1 (7), 200.1 (7), 198.1 (100), 144.1 (4) / 416.4 / 833.1 / 416.0 (100), 358.7 (4), 310.0 (4), 218.9 (42)
cyclo-D-DMAT-L-Tyr / 417.21 / 418.3 / - / - / 416.5 / - / 416.0 (100), 310.0 (5), 219.0 (22)
Fig. 1: Substrate promiscuity of FgaPT2 towards tryptophan-containing cyclic dipeptides. Two assays were carried out for quantification and analysed by HPLC.