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Genome sequencing of Streptomyces species has highlighted numerous potential genes of secondary metabolite biosynthesis. The mining of cryptic genes is important for exploring chemical diversity. Here we report the metabolite-guided genome mining and functional characterization of a cryptic gene by biochemical studies. Based on systematic purification of metabolites from Streptomyces sp. SN-593, we isolated a novel compound, 6-dimethylallylindole (DMAI)-3-carbaldehyde. Although many 6-DMAI compounds have been isolated from a variety of organisms, an enzyme catalyzing the transfer of a dimethylallyl group to the C-6 indole ring has not been reported so far. A homology search using known prenyltransferase sequences against the draft sequence of the Streptomyces sp. SN-593 genome revealed the iptA gene. The IptA protein showed 27% amino acid identity to cyanobacterial LtxC, which catalyzes the transfer of a geranyl group to (−)-indolactam V. A BLAST search against IptA revealed much-more-similar homologs at the amino acid level than LtxC, namely, SAML0654 (60%) from Streptomyces ambofaciens ATCC 23877 and SCO7467 (58%) from S. coelicolor A3(2). Phylogenetic analysis showed that IptA was distinct from bacterial aromatic prenyltransferases and fungal indole prenyltransferases. Detailed kinetic analyses of IptA showed the highest catalytic efficiency (6.13 min−1 μM−1) for l-Trp in the presence of dimethylallyl pyrophosphate (DMAPP), suggesting that the enzyme is a 6-dimethylallyl-l-Trp synthase (6-DMATS). Substrate specificity analyses of IptA revealed promiscuity for indole derivatives, and its reaction products were identified as novel 6-DMAI compounds. Moreover, ΔiptA mutants abolished the production of 6-DMAI-3-carbaldehyde as well as 6-dimethylallyl-l-Trp, suggesting that the iptA gene is involved in the production of 6-DMAI-3-carbaldehyde.
Natural products have been an important resource for drug discovery and development. Actinomycetes have been a rich source of natural products, and a wide variety of these chemicals have been used as medicinal drugs (7, 40) and as bioprobes (56) for the elucidation of biological functions. Recently, the screening of bioactive compounds from microorganisms has often resulted in the identification of previously isolated compounds. The decreasing hit rate for new chemicals has reduced the advantage of natural product screening. However, genome sequencing of Streptomyces species highlighted numerous potential areas with metabolic diversity (4, 25, 42). The number of cryptic gene clusters was much larger than that of secondary metabolites identified from each strain. In addition, the cryptic gene clusters contained genes encoding plenty of unique modification enzymes that had the potential to expand the chemical diversity in drug seeds.
To uncover cryptic gene clusters that might code for biosynthesis of secondary metabolites, genome sequence-guided metabolite identification has been performed in combination with heterologous expression, gene knockout, and complementation analyses and silent gene activation studies. Many microbial metabolites have been discovered through genome mining approaches (5, 8, 26, 29, 34, 41, 50). On the other hand, predictions of protein function are not always successful from BLAST searches, because the substrates or products of unknown enzyme reactions cannot be predicted correctly. Only the type of protein function can be annotated by a homology search. The major difficulty for the identification of cryptic gene clusters is a lack of chemical information. Most gene clusters remain dormant or less active if there are no specific chemicals or physiological signals. Therefore, the discovery of secondary metabolites that are normally expressed at very low levels opens up a strategy for addressing the functions of cryptic gene clusters or unique genes. We performed a metabolite profiling and genome draft sequence analysis of a reveromycin A-producing strain, Streptomyces sp. SN-593 (43). Based on systematic isolation of secondary metabolites, we isolated a novel compound, 6-dimethylallylindole (DMAI)-3-carbaldehyde. There are many isolation reports on 6-DMAI derivatives from Streptomyces sp. (39, 46, 48), fungi (17, 24, 49), and plants (2, 3). However, the gene responsible for dimethylallyl transfer to the C-6 indole ring has not been identified for all living organisms. Because the unique modification enzyme retains a high potential to expand the diversity of natural products, we started a homology search and cloning of the target gene. Here we report the heterologous expression and biochemical characterization of a novel indole prenyltransferase (IptA) catalyzing the transfer of a dimethylallyl group to the C-6 indole ring.
Ammonium salts of dimethylallyl pyrophosphate (DMAPP), geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), and geranylgeranyl pyrophosphate (GGPP) were purchased from Sigma. Ampicillin, kanamycin, and chloramphenicol were purchased from Nacalai (Kyoto, Japan). The indole derivatives with the highest purity were purchased from Wako, Sigma, Aldrich, Fluka, Alfa Aesar, and MP Biochemicals. Brevianamide F and tryprostatin B were isolated from Aspergillus fumigatus (27).
The bacterial strains and plasmids used in this study are listed in Table Table1.1. Streptomyces sp. SN-593 cultures (43) were routinely grown at 28°C in an SY (0.1% yeast extract, 0.1% NZ-amine, 2% starch, and 1.5% agar) slant. For analysis of metabolites, one slant culture was inoculated into a 500-ml cylindrical flask containing 70 ml of YMG medium (0.5% malt extract [Difco], 0.5% yeast extract [Difco], 1.0% glucose, pH 6, before sterilization) for 3 days at 28°C on a rotary shaker at 150 rpm. One milliliter of preculture was inoculated into 500-ml cylindrical flasks containing 70 ml of the producing medium (2% potato dextrose [Difco], 1% malt extract [Difco], 1% dried yeast [Asahi beer], 5% tomato juice [Table Land; Maruzen Foods], 0.1% K2HPO4, 0.1% NaCl, 0.03% MgSO4·7H2O, 0.01% NaNO3, 0.005% ZnSO4·7H2O, and 0.005% CuSO4·5H2O, pH 6.5, before autoclaving) and was cultured for 5 days at 28°C on the same rotary shaker. Escherichia coli strains were grown at 37°C in LB broth (1% tryptone, 0.5% yeast extract, and 1% NaCl), LB Miller agar (Nacalai Tesque, Inc., Japan), or Terrific broth (1.2% Bacto tryptone, 2.4% yeast extract, 0.4% glycerol, 0.231% KH2PO4, and 1.254% K2HPO4). Ampicillin (100 μg ml−1), kanamycin (50 μg ml−1), and chloramphenicol (15 μg ml−1) were added for selection of plasmid-containing E. coli cells.
Ten liters of culture broth from producing medium was extracted with an equal volume of acetone and concentrated to remove acetone. The pH of the aqueous extract was adjusted to 4 by adding acetic acid, and the extract was extracted twice with an equal volume of ethyl acetate. The organic layer was concentrated in vacuo to give 6 g of dark red residue. The concentrated extract was applied to a silica gel column (120-g silica-packed column [ISCO Combi Flash Companion]), equilibrated with chloroform, and eluted with a 97:3 to 95:5 mixture of chloroform-methanol. Subsequently, the fraction was separated by high-performance liquid chromatography (HPLC) on a Senshu Pak Pegasil ODS column with methanol-0.05% formic acid in an H2O gradient system (70 to 100% methanol in 40 min) to obtain 70 subfractions. Subfraction 20 was purified on a reversed-phase HPLC (RP-HPLC) with a Senshu Pak Pegasil ODS column by the elution of acetonitrile-0.05% HCOOH in an H2O gradient system (70 to 90% acetonitrile in 30 min). It gave 1.3 mg of pale yellow powder.
The molecular formula was established by high-resolution past atom bombardment mass spectrometry (HRFAB-MS) on a JEOL JMS-700 instrument as C14H15NO (m/z 214.1245 [M + H]+; calculated value for C14H16NO, 214.1232): the UV (methanol) λmax (log ) values were 215 (3.95), 246 (3.91), 265 (3.93), and 295 (3.82) nm. The nuclear magnetic resonance (NMR) spectra were recorded with CDCl3 at 500 MHz on a JEOL ECP500 spectrometer. The NMR data suggested that the compound had a dimethylallyl group, as follows: δC 17.7 (C-4′, δH 1.73, s, 3H), 25.8 (C-5′, δH 1.75, s, 3H), 132.7 (C-3′), 123.2 (C-2′, δH 5.35, t, 1H, J = 7 Hz), and 34.5 (C-1′, δH 3.45, d, 2H, J = 7 Hz) ppm. The structure was confirmed by analysis of the two-dimensional (2D) NMR spectra. The HSQC spectrum revealed connections between protons and carbons. The DQF-COSY spectra showed the connectivity between H-4 and H-5 and between H-1′ and H-2′, which was supported by the coupling constants in the 1H NMR spectrum. The observed long-range correlations in the HMBC spectrum are shown in Fig. Fig.1.1. In addition, H-2 and H-7 were observed as singlet protons in the 1H NMR spectrum. These data allowed the identification of the 6-DMAI-3-carbaldehyde structure. Assuming that the H-7 was attached to C-4, the dimethylallyl group might be located at C-5. In that case, however, the observed long-range correlation from the proton at 7.14 ppm (H-7) to C-3a was unlikely, because it was a four-bond distance. Thus, the dimethylallyl group was located at C-6, and the structure was determined to be 6-DMAI-3-carbaldehyde.
The draft sequence of the Streptomyces sp. SN-593 genome was determined by a whole-genome shotgun strategy. Plasmid libraries with an average insert size of 2 to 5 kb were constructed. A fosmid library of Streptomyces sp. SN-593 was created using a copy control fosmid library production kit (Epicentre Biotechnology). The fosmid clones harbored about 40 kb of DNA fragments in the vector. End sequencing was carried out using a BigDye Terminator ver3.1 kit (Applied Biosystems). Sequencing products were run on automated model 3730xl capillary sequencers (Applied Biosystems). The Sanger data were assembled with the KB/Phrap/Consed system (15, 16, 19). A homology search was then carried out against the generated contig data, using BLASTP (1). Low-quality regions in the selected contig sequence were resequenced by PCR, primer walking, and shattered insert libraries (37).
DNA isolation and manipulation were performed as described previously (45). Genomic DNA was isolated from Streptomyces sp. SN-593 as described in Practical Streptomyces Genetics (28). DNA fragments were isolated from agarose by use of a gel extraction kit (Qiagen). Plasmids were purified using Wizard Plus SV miniprep kits (Promega). PCR amplification was carried out using a DNAEngine Peltier thermal cycler (Bio-Rad).
A PCR fragment of 1,158 bp containing iptA was amplified from fosmid clone 4C04, using PrimeSTAR DNA polymerase (TaKaRa) and the primers listed in Table Table1.1. After overnight LB-kanamycin (50 μg ml−1) culture of E. coli BL21 Star (DE3) that harbored pET28b(+)-iptA, cells were inoculated into 3 liters of Terrific broth-kanamycin (50 μg ml−1) and cultured at 37°C. Preculture was added to reach an initial optical density at 600 nm (OD600) of 0.1. When the absorbance at 600 nm reached a value of 0.5, IPTG (isopropyl-β-d-thiogalactopyranoside) was added to a final concentration of 0.5 mM. After growth for 6 h at 28°C, cells were harvested by centrifugation and frozen at −80°C. All subsequent steps were carried out at 4°C. After thawing on ice, cells were suspended in 100 ml buffer A (50 mM Tris-HCl [pH 7.5], 500 mM NaCl, 5 mM imidazole, and 10% glycerol) including 0.5 mg ml−1 lysozyme and 500 U Benzonase (Sigma). The cell suspension was sonicated 10 times on ice for 10 s each, with 1-min intervals between each sonication (Tomy UD-200 sonicator). Cellular debris was removed by centrifugation (10,000 × g for 30 min) (Tomy SRX-201 instrument). The supernatant was applied to a Ni-nitrilotriacetic acid (Ni-NTA) column (2 × 8 cm) (Qiagen) that was equilibrated with buffer A and washed with 100 ml of buffer A containing 0.2% Tween 20. After being washed with 100 ml of buffer A, the column was further washed with 100 ml of buffer A containing 40 mM imidazole. His-tag-binding protein (33 mg) was eluted with 85 ml of buffer A containing 200 mM imidazole. Protein (30 mg) from the Ni-NTA fraction was used for large-scale reactions for product identification. The remaining His-tagged IptA was treated with 100 units of thrombin and dialyzed against 1 liter of buffer B (50 mM Tris-HCl [pH 7.5], 500 mM NaCl, and 10% glycerol) for 3 h and against 2 liters of buffer B for 10 h. The pass-through fraction from the Ni-NTA column (2 × 8 cm) was applied directly to a benzamidine Sepharose column (1.5 × 3 cm) to remove thrombin. The purity of His-tag-free IptA (1.27 mg) was analyzed by SDS-PAGE (33).
To investigate its multimeric status, IptA was dialyzed against buffer C (50 mM Tris-HCl [pH 7.5], 200 mM NaCl, and 10% glycerol) and applied to a Superdex 200 column (GE Healthcare), previously equilibrated with buffer C, at a flow rate of 0.8 ml min−1. The column was calibrated with ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), RNase A (13.7 kDa), and aprotinin (6.5 kDa). The elution was established over 80 min, and the peak corresponding to a molecular mass of 40 kDa was concentrated to 0.915 mg ml−1 with Amicon Ultracel-30K microconcentrators (Millipore Corporation) and stored at −80°C for enzyme characterization. The highly purified His-tag-free IptA protein was used for biochemical characterization.
Standard assays were performed in 1.5-ml tubes with a final reaction volume of 100 μl containing 50 mM Tris-HCl (pH 7.5), 0.2 mM DMAPP, 0.5 mM substrate, and 16 to 60 pmol purified enzyme. For kinetic analyses, the substrate (prenyl acceptor) concentration was varied from 10 to 1,000 μM for l-Trp, 2 to 1,000 μM for l-abrine, 2 to 1,000 μM for α-methyl-dl-Trp, 2 to 500 μM for 5-hydroxy-l-Trp, and 1 to 1,000 μM for indole-3-carbaldehyde. The prenyl donor (DMAPP) concentration was varied from 6 to 240 μM. After preincubation of the reaction mixture at 30°C for 3 min, the reactions were initiated by the addition of enzyme and allowed to proceed for 0.5 min (l-Trp), 1 min (l-abrine), 0.5 min (α-methyl-dl-Trp), 1 min (5-hydroxy-l-Trp), or 90 min (indole-3-carbaldehyde). The reaction was terminated by the rapid addition of 0.5 μl formic acid and mixing by vortexing. After the addition of 10 μl acetonitrile, the mixture was centrifuged at 20,000 × g for 10 min (Kubota 3700 machine) at 4°C to remove protein. The supernatant was subjected to liquid chromatography-mass spectrometry (LC-MS), and the area under the prenylated product peak was measured. The amount of the prenylated product peak was calculated with a standard curve, which was obtained by using the maximum UV wavelength of each substrate. All of the substrates used were subjected to HPLC at a concentration range of 0.01 to 1 nmol for their calibration. The enzyme-specific activity (nmol product formed min−1 nmol enzyme−1) for various indole substrates was calculated by time-dependent product formation. The kinetic constants were calculated by nonlinear regression fit to the Michaelis-Menten equation. Kinetic constants calculated from Lineweaver-Burk, Eadie-Hofstee, and Hanes-Woolf plots were not significantly different from those derived from nonlinear regression.
An electrospray ionization (ESI)-MS analysis was carried out by using a Waters Alliance HPLC system equipped with a mass spectrometer (Q-Trap; Applied Biosystems). The HPLC conditions were as follows: column, XTerraMSC18 5-μm (2.1 × 150 mm) column; flow rate, 0.2 ml min−1; solvent A, H2O containing 0.05% formic acid; and solvent B, acetonitrile. After injection of the sample into a column equilibrated with 10% solvent B, the column was developed with a linear gradient of 10% to 100% solvent B over 30 min. Mass spectra were collected in either ESI-negative or ESI-positive mode.
For the identification of enzyme reaction products, 10 ml of reaction mixture containing 0.2 to 1.8 mg purified His-tagged IptA was incubated for 12 h with Trp derivatives, such as l-Trp, 5-hydroxy-l-Trp, 4-methyl-dl-Trp, 5-methoxy-l-Trp, 6-methyl-dl-Trp, and l-abrine. The solutions of reactants were concentrated by an evaporator to a volume of 2 ml, and 400 μl was injected into a Senshu Pak Pegasil ODS column (10 × 250 mm; SSC). The isolation was carried out using a Waters 600 HPLC system with a photodiode array detector (model 2996 PDA detector), and conditions of the preparations were as follows: flow rate, 4.5 ml min−1; solvent A, water containing 0.05% formic acid; and solvent B, acetonitrile. After injection of the sample into a column equilibrated with 35% acetonitrile, the column was developed by same-solvent isocratic elution for 60 min.
The molecular formula was established as C16H20N2O2 by HRFAB-MS (m/z 273.1637 [M + H]+; calculated value for C16H21N2O2, 273.1603): the UV (methanol) λmax (log ) values were 226 (4.14), 250 (3.75), and 288 (3.55) nm; [α]D24, +19.4 (c 0.07, methanol). The NMR spectra were recorded with CD3OD, and the structural assignments were confirmed by the 1H, 13C, COSY, HSQC, and HMBC NMR spectra. A dimethylallyl group was confirmed by the NMR data as follows: δC 17.8 (C-4′, δH 1.73, s, 3H), 25.9 (C-5′, δH 1.73, s, 3H), 132.5 (C-3′), 125.5 (C-2′, δH 5.34, t, 1H, J = 7.3 Hz), and 35.5 (C-1′, δH 3.40, d, 2H, J = 7.3 Hz). The indole ring system was confirmed by the comparison of 13C NMR chemical shifts with those of 6-DMAI-3-carbaldehyde and long-range correlations in the HMBC spectra (Fig. (Fig.1B).1B). The assignments of the indole ring were as follows: δC 124.6 (C-2, δH 7.11, s 1H), 109.4 (C-3), 126.6 (C-3a), 119.2 (C-4, δH 7.58, d, 1H, J = 7.8 Hz), 121.3 (C-5, δH 6.88, dd, 1H, J = 8.0, 1.2 Hz), 136.8 (C-6), and 111.6 (C-7, δH 7.14, br s, 1H) ppm. In addition, the long-range correlation from H-1′ to C-5, C-6, and C-7 suggested that the dimethylallyl group was attached to the C-6 position of the indole ring. Thus, the structure was determined to be 6-dimethylallyl-l-Trp.
The molecular formula was established as C16H20N2O3 by HRESI-TOF-MS (m/z 287.1421 [M − H]−; calculated value for C16H19N2O3, 287.1395); [α]D26, +90.5° (c 0.081, methanol). UV (methanol) λmax (log ) values were 226 (4.32) and 285 (3.74) nm. The NMR spectra were recorded with CD3OD, and the 1H and 13C chemical shift assignments were confirmed by 1H, 13C, DQF-COSY, HSQC, and HMBC spectra, as follows: δC 174.6 (s, C-12), 150.0 (s, C-5), 133.5 (s, C-7a), 132.6 (s, C-3′), 126.9 (s, C-3a), 126.0 (s, C-6), 124.8* (d, C-2), 124.7* (d, C-2′), 112.5 (d, C-7), 108.6 (s, C-3), 103.3 (d, C-4), 56.5 (d, C-11), 29.9 (t, C-1′), 28.9 (t, C-10), 26.0 (q, C-5′), and 17.8 (q, C-4′). *, 13C chemical shifts were exchangeable at C-2 and C-2′, as follows: δH 7.05 (1H, s, H-2), 7.03 (1H, s, H-7), 6.99 (1H, s, H-4), 5.36 (1H, t, J = 7.3 Hz, H-2′), 3.80 (1H, br dd, J = 9.2, 3.7 Hz, H-11), 3.43 (1H, dd, J = 15.1, 3.7 Hz, H-10a), 3.36 (2H, d, J = 7.3 Hz,H-1′), 3.02 (1H, dd, J = 15.1, 10.1 Hz H-10b), 1.74 (3H, s, Me-5′), and 1.71 (3H, s, Me-4′). The position of the C-6 substituent was confirmed by the long-range correlations from H-1′ to C-5, C-6, and C-7 in the HMBC spectrum.
The molecular formula was established as C17H22N2O2 by HRESI-TOF-MS (m/z 287.1770 [M + H]+; calculated value for C17H23N2O2, 287.1759). The proton chemical shifts in CD3OD were assigned as follows: δH 7.07 (1H, s, H-2), 6.96 (1H, s, H-5), 6.60 (1H, s, H-7), 5.32 (1H, t, J = 7.3 Hz, H-2′), 3.77 (1H, br s, H-11), 3.76 (1H, br s, H-10b), 3.07 (1H, dd, J = 15.1, 10.5 Hz, H-10), 2.68 (3H, s, 4-Me), 1.73 (6H, s, Me-4′, 5′); [α]D26, −40.8° (c 0.047, methanol). The UV (methanol) λmax (log ) values were 226 (3.26) and 272 (2.45) nm. The confirmation of the 6-dimethylallyl substituent was established by the observation of two signals, at δH 6.96 (H-5, s, 1H) and 6.60 (H-7, s, 1H) ppm, as singlets in the 1H NMR spectrum.
The molecular formula was established as C17H22N2O2 by HRFAB-MS (m/z 303.1775 [M + H]+; calculated value for C17H23N2O2, 303.1708); [α]D24, +4.6 (c 0.065, methanol). The UV (methanol) λmax (log ) values were 226 (3.79), 274 (3.33), and 298 (3.39) nm. The 1H and 13C chemical shifts were assigned as follows: δC 174.7 (s, C-12), 153.7 (s, C-5), 133.4 (s, C-7a), 132.5 (s, C-3′), 127.1 (s, C-6), 126.7 (d, C-3a), 124.73 (d, C-2), 124.69 (d, C-2′), 112.7 (d, C-7), 109.3 (s, C-3), 100.0 (d, C-4), 56.6 (d, C-11), 56.4 (q, 5-OMe), 30.1 (t, C-1′), 26.0 (q, C-5′), 28.8 (t, C-10), and 17.8 (q, C-4′); and δH 7.19 (1H, s, H-4), 7.09 (1H, s, H-7), 7.07 (1H, s, H-2), 5.31 (1H, t, J = 7.3 Hz, H-2′), 3.88 (3H, s, 5-OMe), 3.84 (1H, br dd, J = 3.7, 9.2 Hz, H-11), 3.47 (1H, dd, J = 3.7, 15.1 Hz, H-10b), 3.34 (2H, d, J = 6.9 Hz, H-1′), 3.08 (1H, dd, J = 9.2, 15.1 Hz, H-10b), 1.72 (3H, s, Me-5′), and 1.70 (3H, s, Me-4′). The position of the dimethylallyl substituent was assigned at C-6 by the observation of two signals of H-4 and H-7 as a singlet in the 1H NMR spectrum. It was confirmed by the long-range correlations of H-1′ with C-6 and C-7 in the HMBC spectrum.
[α]D26, −155.6° (c 0.03, methanol). The UV (methanol) λmax (log ) values were 221 (4.17) and 281 (3.46) nm. HRESI-TOF-MS showed m/z 287.17374 [M + H]+ (calculated value for C17H23N2O2, 287.17595). 1H and 13C NMR chemical shifts were as follows: δC 137.9 (s, C-7a), 132.4 (s, C-3′), 129.8 (s, C-6), 127.2 (s, C-3a), 124.5 (d, C-2), 123.8 (d, C-2′), 123.6 (d, C-5), 123.4 (s, C-7), 116.9 (d, C-4), 110.5 (s, C-3), 57.1 (d, C-11), 29.5 (t, C-10), 27.9 (t, C-1′), 25.8 (q, C-5′), 19.2 (q, 6-Me), and 18.1 (q, C-4′); and δH 7.41 (1H, d, J = 7.8 Hz, H-4), 7.10 (1H, s, H-2), 6.86 (1H, d, J = 7.8 Hz, H-5), 5.09 (1H, t, J = 6.4 Hz, H-2′), 3.76 (1H, br dd, J = 7.3, 4.1 Hz, H-11), 3.54 (2H, br d, J = 6.4Hz, H-1′), 3.41 (1H, br s, H-10a), 3.05 (1H, br s, H-10b), 2.33 (3H, s, 6-Me), 1.82 (3H, s, Me-4′), and 1.67 (3H, s, Me-5′). These data were found to be identical to those for 6-methyl-7-dimethylallyl-Trp.
The molecular formula was established as C17H22N2O2 by HRESI-TOF-MS as follows: m/z 285.1585 [M − H]−; calculated value for C17H21N2O2, 285.1603; [α]D26, −73.9° (c 0.044, methanol). The UV (methanol) λmax (log ) values were 225 (4.57), 271 (3.87), and 292 (3.80) nm. The NMR spectra were recorded with CD3OD. The structure was supported by comparison of the 1H and 13C NMR chemical shifts with those of 6-DMAI-3-carbaldehyde and also confirmed by the analysis of HMBC correlations. The 1H and 13C NMR chemical shifts were assigned as follows: δC 173.5 (s, C-12), 138.8 (s, C-7a), 136.8 (s, C-6), 132.5 (s, C-3′), 126.7 (s, C-3a), 125.5 (d, C-2′), 124.7 (d, C-2), 121.3 (d, C-5), 119.2 (d, C-4), 111.6 (d, C-7), 108.7 (s, C-3), 65.3 (d, C-11), 35.5 (t, C-1′), 33.1 (q, 11-NH-Me), 27.7 (t, C-10), 25.9 (q, C-5′), and 17.8 (q, C-4′); and δH 7.57 (1H, d, J = 8.2 Hz, H-4), 7.14 (2H, br s, H-2, 7), 6.88 (1H, dd, J = 8.2, 1.4 Hz, H-5), 5.34 (1H, t, J = 7.3 Hz, H-2′), 3.75 (1H, br dd, J = 7.3, 4.1 Hz, H-11), 3.44 (1H, dd, J = 15.6, 4.1 Hz, H-10a), 3.39 (2H, br d, J = 7.3 Hz, H-1′), 3.24 (1H, dd, J = 15.6, 7.3 Hz, H-10b), 2.55 (3H, s, 11-NH-Me), and 1.73 (6H, s, Me-4′,5′). The dimethylallyl group was attached at the C-6 position by the observation of the H-5 signal as a double doublet, with coupling constants of 8.2 and 1.4 Hz, in the 1H NMR spectrum. It was also supported by the long-range correlations of H-1′ with C-5, C-6, and C-7 in the HMBC spectrum.
The fosmid clone 4C04 was digested with BamHI and BglII. The 7.89-kb BamHI fragment containing the iptA gene fragment was separated by 0.8% agarose gel electrophoresis and ligated into a pIM vector to construct pIM-iptA (Table (Table1).1). The iptA gene was inactivated by PCR-targeted gene replacement according to the literature protocol (22). Plasmids (pKD46 and pKD13) and E. coli BW25113 were supplied by Hirotada Mori (Nara Institute of Science and Technology). Chloramphenicol-resistant (Cmr) pKD46, containing λ Red-mediated recombination functions, was used with ampicillin-resistant pIM-iptA. The pIM-iptA plasmid was transformed into E. coli BW25113 harboring pKD46. The iptA gene was then replaced with the aphII gene cassette to construct an iptA gene disruption vector (pIM-ΔiptA). Plasmid pKD13 was used as the template for the aphII gene cassette (12). The primer pairs listed in Table Table11 were used to construct pIM-ΔiptA.
Conjugation was performed according to a standard procedure (28), with the following modifications. Streptomyces sp. SN-593 spores were suspended in SY medium and incubated at 28°C for 4 h. The spores were mixed with E. coli GM2929 hsdS::Tn10 (pUB307::Tn7) (26) harboring pIM-ΔiptA and spread onto a modified MS [3% soy flour, 2% d-(−)-mannitol, 2% agar] plate (20 ml) containing 25 mM MgCl2. After incubation at 28°C for 20 h, each plate was overlaid with 1.5 ml of sterilized water to give a final concentration of 20 μg ml−1 thiostrepton and 5 μg ml−1 carumonum. An SY plate containing 0.5 μg ml−1 ribostamycin (Sigma) was used for second selection. After SY culture (70 ml) and sporulation on the MS plate, four iptA gene disruptants were selected by the ribostamycin-resistant and thiostrepton-sensitive phenotype. Gene replacement was confirmed by Southern blot analysis.
DNA probes for Southern analysis were amplified from pIM-ΔiptA by use of PrimeSTAR DNA polymerase under the following conditions: 98°C for 10 s and 25 cycles of 98°C for 10 s, 66°C for 5 s, and 68°C for 3.5 min. The primers used are listed in Table Table1.1. The amplified DNA was purified by gel extraction and labeled with AlkPhos Direct labeling reagents (GE Healthcare).
The wild type and the iptA gene disruptant were inoculated into a 500-ml cylindrical flask containing 70 ml of SY medium for 2 days at 28°C. One milliliter of the preculture was inoculated into a 500-ml cylindrical flask containing 70 ml of the producing medium. Five days after inoculation at 28°C, a total of 280 ml of culture broth was extracted with an equal volume of acetone and concentrated to remove acetone. The pH of the aqueous extract was adjusted to 4 by adding acetic acid and then extracted twice with a half-volume of ethyl acetate. After sodium sulfate treatment, the ethyl acetate layer was concentrated in vacuo to give about 170 mg of red residue. The concentrated extract was applied to an MPLC system (ISCO) equipped with a RediSep column (12-g silica gel column; ISCO). To collect 6-DMAI-3-carbaldehyde reproducibly, stepwise elution (100 ml) was performed at a flow rate of 30 ml min−1, using a chloroform-methanol solvent system (100:0, 100:1, 100:5, 100:10, 100:20, 100:50, 50:50, and 0:100). The chloroform-methanol (100:1) fraction (39 mg) containing 6-DMAI-3-carbaldehyde was concentrated and dissolved in 20 ml of methanol. The sample (5 μl) was applied to LC-MS analysis. To detect 6-dimethylallyl-l-Trp, the water layer after ethyl acetate extraction was extracted twice with a half-volume of n-butanol. The n-butanol layer was concentrated and dissolved in 2 ml of methanol, and the sample (1 μl) was applied to LC-MS analysis. LC/ESI-MS was performed as described in “Analyses of enzyme reaction products.”
The GenBank accession number for the analyzed sequence is AB512764.
Based on a UV- and LC-MS-guided approach, we isolated a novel natural product, 6-DMAI-3-carbaldehyde, from Streptomyces sp. SN-593 (Fig. (Fig.1A).1A). Although 6-DMAI-related compounds are widely distributed in living organisms (Table (Table2),2), the biosynthetic gene has not been reported. Therefore, the finding of a unique 6-DMAI metabolite prompted us to clone the gene responsible for the dimethylallyl transfer to the C-6 indole ring. The contig information obtained from genome draft sequencing of Streptomyces sp. SN-593 was utilized to search for the gene. BLAST searches against all known prenyltransferases revealed one putative prenyltransferase, designated IptA in the genetic organizations shown in Fig. Fig.22 and Table Table3.3. The genomic DNA sequence of iptA consists of 1,158 bp, and the predicted gene product comprises 385 amino acids with a molecular mass of 41.3 kDa. IptA showed weak homology (E value of 9e−16, 27% amino acid identity) to LtxC, which was found in the lyngbyatoxin A biosynthetic gene cluster from the cyanobacterium Lyngbya majuscule (14). A BLAST search against IptA revealed much-more-similar homologs than LtxC, i.e., SAML0654 (60%) from Streptomyces ambofaciens ATCC 23877 and SCO7467 (58%) from Streptomyces coelicolor A3(2), at the amino acid level (Fig. (Fig.3A).3A). Phylogenetic analysis showed three distinct groups: bacterial aromatic prenyltransferases, fungal indole prenyltransferases, and a new group of prenyltransferases which includes IptA, SAML0654, and SCO7467 (Fig. (Fig.3B).3B). These new prenyltransferases from Streptomyces are positioned between bacterial aromatic prenyltransferases and fungal indole prenyltransferases.
For biochemical characterization, the iptA gene was amplified and cloned into the pET28b(+) expression vector. The construct was introduced into E. coli BL21 Star (DE3). Induction with IPTG, NTA column purification, thrombin digestion, removal of the His tag by a second Ni-NTA column, and benzamidine Sepharose and Superdex 200 column chromatographies resulted in purified His-tag-free IptA, with a molecular mass of 41 kDa (Fig. (Fig.4A).4A). By Superdex 200 gel filtration experiments, IptA was eluted in the fraction corresponding to a molecular mass of 40.0 kDa, suggesting a monomeric property.
Because of weak homology to LtxC, which catalyzes the transfer of a geranyl group to (−)-indolactam V, and the isolation of 6-DMAI-3-carbaldehyde, the function of IptA was speculated to be 6-dimethylallyl transfer to l-Trp or directly to indole-3-carbaldehyde. LC-MS analyses of the IptA reaction product with l-Trp and DMAPP clearly showed a prenylated product peak with a retention time of 13.1 min (Fig. (Fig.4B).4B). The reaction product was identified as 6-dimethylallyl-l-Trp by NMR and MS analyses (Fig. (Fig.1B).1B). We also tested the reaction with GPP, because LtxC utilized GPP as a prenyl donor. However, geranylated l-Trp was not detected by LC-MS (Fig. (Fig.4C).4C). In addition, when IPP, FPP, or GGPP was mixed with l-Trp instead of DMAPP, no prenylated product was detected, indicating that IptA specifically utilizes DMAPP as a prenyl donor. When IptA was incubated with (−)-indolactam V in the presence of DMAPP or GPP, no prenylated product was detected.
To evaluate the formation of 6-DMAI-3-carbaldehyde, IptA was incubated with indole-3-carbaldehyde in the presence of DMAPP. The reaction product analyses by LC-MS showed the same retention time, UV spectrum, and molecular ion (m/z 212 [M − H]−) as the authentic reference of purified 6-DMAI-3-carbaldehyde (Fig. (Fig.5).5). Comparison of specific activities between l-Trp and indole-3-carbaldehyde indicated that the reaction of IptA with indole-3-carbaldehyde was much less effective than that with l-Trp (Table (Table4).4). Consequently, the detailed enzymatic properties of IptA were characterized mainly for 6-dimethylallyl-l-Trp synthase (6-DMATS) activity.
Effects of divalent metal ions on 6-DMATS activity were tested at a concentration range of 1 to 20 mM (Fig. (Fig.6A).6A). The addition of 1 to 3 mM Ca2+, Mg2+, or Mn2+ did not induce an enhancement of 6-DMATS activity. The addition of 10 mM Co2+ or Ni2+ reduced 40 to 50% of the activity. The addition of 3 to 10 mM Fe2+, Cu2+, or Zn2+ strongly inhibited the enzyme activity. Moreover, the presence of 1 mM EDTA did not inhibit 6-DMATS activity, suggesting that a metal ion is not essential for its activity. When indole-3-carbaldehyde was used as the substrate, no metal dependency was observed.
The effect of pH on the 6-DMATS activity was relatively broad, and maximal product formation was observed at pH 7.0 to 9.5 (Fig. (Fig.6B).6B). Moreover, heat treatment at 50°C for 5 min abolished the 6-DMATS activity (Fig. (Fig.7A).7A). The effect of temperature on the enzyme activity was also tested. The activation energy was estimated to be 46 kJ mol−1 by an Arrhenius plot, whose curve was straight over the range of 10 to 42°C (Fig. (Fig.7B7B).
To address substrate specificity, IptA was incubated with 15 Trp derivatives with a modified R3 side chain (Table (Table4).4). α-Methylation of dl-Trp retained the relative activity (106%). However, the methylation of the α-amino moiety of l-Trp (l-abrine) reduced the relative activity (51.2%). Changes in the stereochemistry of the amino group (d-Trp) resulted in a reduction in relative activity (4.3%). The exchange of the α-amino group of Trp with a hydroxyl group (dl-indole-3-lactic acid) and the deamination of l-Trp (indole-3-propionic acid) significantly reduced relative activity (0.41 and 0.18%, respectively). Acetylation of the α-amino moiety (N-acetyl-l-Trp) led to a loss of activity, suggesting that the amino group is important for optimum substrate recognition by IptA. Moreover, the decarboxylation of l-Trp (tryptamine) and the insertion of the methylene group in the α-position (l-β-homotryptophan) resulted in a decrease of relative activity (27.5% and 16.8%, respectively). In addition, the methyl ester of the carboxyl group (l-Trp methyl ester) resulted in a reduction of relative activity (4.6%), suggesting that the carboxyl group is also important for optimum substrate recognition by IptA. When l-Trp-containing dipeptides, such as brevianamide F (cyclo-l-Trp-l-Pro) and tryprostatin B, were incubated with IptA, no prenylated product was observed, supporting the role of the carboxyl group and amino group for substrate recognition. Moreover, when l-Phe, l-Tyr, l-Pro, or l-His, instead of l-Trp, was mixed with DMAPP, no prenylated product was detected. These results also suggested the role of the indole ring in the IptA reaction.
To address substrate specificity in detail, IptA was incubated with 11 Trp derivatives with a modified indole ring (Table (Table5).5). The substrates, modified at C-1, C-4, and C-5 of the indole ring, did not significantly reduce relative activity. NMR analyses demonstrated that 5-hydroxy-l-Trp, 4-methyl-dl-Trp, and 5-methoxy-l-Trp were converted into 6-dimethylallyl products, all of which turned out to be novel compounds. Consistent with these results, IptA hardly reacted with 6-chloro- and 6-bromo-dl-Trp. However, methyl modification at C-6 of the indole ring (6-methyl-dl-Trp) resulted in a 7-dimethylallyl product with 9.3% relative activity. A cocrystal structure analysis using 6-methyl-dl-Trp would be indispensable to explain the shift of the dimethylallyl transfer site to C-7 of the indole ring.
Kinetic parameters of IptA were investigated for the prenyl acceptors l-Trp, l-abrine, α-methyl-dl-Trp, 5-hydroxy-l-Trp, and indole-3-carbaldehyde (Table (Table6).6). The IptA reaction apparently followed Michaelis-Menten kinetics. The Km and kcat values for l-Trp were 9.4 ± 0.9 μM and 57.6 ± 0.9 min−1, respectively, giving a catalytic efficiency of 6.13 min−1 μM−1. l-Abrine showed a higher Km value (16.9 ± 0.9 μM) and a lower kcat value (38.5 ± 0.5 min−1) than those for l-Trp. Although α-methyl-dl-Trp and 5-hydroxy-l-Trp showed higher kcat values (67.4 ± 1.4 and 75.2 ± 2.8 min−1, respectively) than that for l-Trp, their Km values (32.0 ± 2.8 and 41.0 ± 5.3 μM, respectively) were higher than that for l-Trp. Consequently, the catalytic efficiencies of l-abrine (2.28 min−1 μM−1), α-methyl-dl-Trp (2.11 min−1 μM−1), and 5-hydroxy-l-Trp (1.83 min−1 μM−1) were 2.7 to 3.3 times lower than that of l-Trp. In addition, the Km and kcat values for indole-3-carbaldehyde were 19.9 ± 0.8 μM and 0.095 ± 0.001 min−1, respectively. The catalytic efficiency of indole-3-carbaldehyde (0.0048 min−1 μM−1) was about 1,300 times lower than that of l-Trp.
Moreover, the kinetic parameters of IptA were also evaluated for the prenyl donor DMAPP. The 6-DMATS activity of IptA was slightly inhibited with increasing concentrations of DMAPP. The kcat value (70.7 ± 1.6 min−1) obtained from DMAPP kinetics was 1.2 times higher than that of l-Trp (57.9 ± 0.9 min−1), as measured in the presence of 0.2 mM DMAPP. Consistent with the results for l-Trp, the prenyl transfer to indole-3-carbaldehyde was also slightly inhibited with increasing concentrations of DMAPP. The kcat value (0.108 ± 0.001 min−1) obtained from DMAPP kinetics was 1.1 times higher than that for indole-3-carbaldehyde (0.095 ± 0.001 min−1), as measured in the presence of 0.2 mM DMAPP. These results are consistent with the report for MaPT (13).
To address the role of the iptA gene in the biosynthesis of 6-DMAI-3-carbaldehyde, we constructed an iptA-inactivated mutant (Fig. (Fig.8)8) and analyzed the metabolites by LC-MS. All of the ΔiptA mutants abolished the production of 6-DMAI-3-carbaldehyde (Fig. (Fig.9C),9C), suggesting that the iptA gene is involved in the production of 6-DMAI-3-carbaldehyde. In addition, the ΔiptA mutants also abolished the production of 6-dimethylallyl-l-Trp (Fig. (Fig.9F9F).
Indole prenyltransferases have been characterized from fungal strains by S. M. Li (35, 36). The ABBA prenyltransferase family has also been characterized extensively (23, 32, 38, 44, 52). However, there has been no report of the gene responsible for indole 6-prenyltransferase. Metabolite profiling and genome mining using Streptomyces sp. SN-593 successfully shed light on the missing gene iptA (Fig. (Fig.2;2; Table Table3),3), which was phylogenetically distinct from other prenyltransferases (Fig. (Fig.3B).3B). Consistent with the isolation reports for prenylindole compounds from Streptomyces, highly homologous genes [SAML0654 from S. ambofaciens ATCC 23877 and SCO7467 from S. coelicolor A3(2)] were also found in the Streptomyces genome database. Interestingly, the gene organization of S. ambofaciens differs from that of Streptomyces sp. SN-593 by lacking a homolog of orf7 and orf8. The gene organization of S. coelicolor A3(2) differs from that of Streptomyces sp. SN-593 by lacking a homolog of orf5, orf7, and orf8. Each Streptomyces strain does not seem to produce a fixed secondary metabolite.
We speculated that the biosynthesis of 6-DMAI-3-carbaldehyde might start from prenylation of l-Trp, because the catalytic efficiency of l-Trp was about 1,300 times higher than that of indole-3-carbaldehyde (Table (Table6).6). Based on this speculation, 6-dimethylallyl-l-Trp might accumulate to a detectable amount in Streptomyces sp. SN-593 culture broth. If independent pathways exist for the biosynthesis of 6-dimethylallyl-l-Trp and 6-DMAI-3-carbaldehyde, the amounts of the products should be correlated with the catalytic efficiency of IptA. Therefore, we examined the presence of 6-dimethylallyl-l-Trp, as well as 6-DMAI-3-carbaldehyde, by LC-MS analysis (see Fig. S1 in the supplemental material). Surprisingly, we identified that the amount of 6-dimethylallyl-l-Trp (1.3 mg from a 10-liter culture) was three times lower than that of 6-DMAI-3-carbaldehyde (4.2 mg from a 10-liter culture). Because the gene annotation (Table (Table3)3) indicated the presence of tryptophanase, the low yield of 6-dimethylallyl-l-Trp might be explained by the conversion into 6-DMAI-3-carbaldehyde. However, we must await a final conclusion until the demonstration of incorporation of labeled 6-dimethylallyl-l-Trp into 6-DMAI-3-carbaldehyde. At present, we cannot rule out the possibility that 6-DMAI-3-carbaldehyde was produced in part from indole-3-carbaldehyde, because it was isolated from Streptomyces sp. SN-593 culture broth. Recently, a genome-minimized Streptomyces avermitilis host for the heterologous expression of various secondary metabolite gene clusters was reported (29). Because the S. avermitilis host lacks an iptA homolog, the heterologous expression of the whole gene organization (Fig. (Fig.2)2) will give us detailed information on the biosynthesis of the secondary metabolite.
In this study, detailed biochemical properties of IptA were compared with those of fungal indole prenyltransferases (see Table S1 in the supplemental material). To characterize the multimeric property of IptA, gel filtration analysis was performed using His-tag-free enzyme. IptA showed a monomeric feature, which is similar to the case for FtmPT1 (21) and 7-DMATS (30) from A. fumigatus. On the other hand, indole prenyltransferases such as FtmPT2 (20), FgaPT1 (54), and FgaPT2 (53) from A. fumigatus and DMATS from Claviceps purpurea (18) have been reported to be dimer proteins, indicating that the multimeric status of indole prenyltransferases has variation.
It was known that the activity of indole prenyltransferase is affected by the presence of metals, especially Mg2+ or Ca2+. Therefore, the 6-DMATS activity of IptA was measured in the presence of different metal ions at concentrations ranging from 1 to 20 mM (Fig. (Fig.6A).6A). The results clearly showed that the 6-DMATS activity was independent of the presence of metal ions. The independency of divalent metals was similar to that of FtmPT2 (20) and FgaPT1 (54) from A. fumigatus and MaPT (13) from Malbranchea aurantiaca. In contrast, other indole prenyltransferases, such as CdpNTP (58), FtmPT1 (21), FgaPT2 (53), and 7-DMATS (31) from A. fumigatus and 4-DMATS from C. purpurea (18), had enhanced activity in the presence of Mg2+ or Ca2+. This indicates that enhancement of enzyme activity by divalent cations is not conserved among the indole prenyltransferases (see Table S1 in the supplemental material).
The Km and kcat values for l-Trp and DMAPP (Table (Table6)6) are comparable to those for other indole prenyltransferases, such as FgaPT2 (51, 53), MaPT (13), and 4-DMATS (18). Substrate specificity analyses (Tables (Tables44 and and5)5) and kinetic parameters for indole substrates (Table (Table6)6) strongly suggested that the natural function of IptA was as a 6-DMATS. IptA is the first example of an indole prenyltransferase identified from Streptomyces. Moreover, it should be emphasized that IptA revealed substrate promiscuity for indole compounds and produced novel 6-DMAI products, such as 6-dimethylallyl-l-Trp, 5-hydroxy-6-dimethylallyl-l-Trp, 4-methyl-6-dimethylallyl-Trp, 5-methoxy-6-dimethylallyl-l-Trp, and 6-dimethylallyl-l-abrine. For these substrate specificity analyses, we utilized commercially available dl- mixtures of substrate. Although we could not determine an exact conversion ratio of l- to d- reaction products, it can be speculated that l-Trp derivatives react better than the d- forms. The significant preference for l- over d-Trp is shown in Table Table44.
Prenylated indole derivatives are widely distributed in nature and show diverse biological and pharmacological activities (57). Prenylation is important for the order of biosynthesis and the formation of bioactive compounds (6, 9-11, 27, 47, 55, 57, 59). Here we demonstrated that IptA widely accepted indole substrates and produced novel 6-DMAI compounds. Therefore, chemoenzymatic synthesis or pathway engineering of secondary metabolites by use of IptA might allow us to derivatize natural products with improved biological activities.
We are grateful to T. Kuzuyama and N. Kato for their precious advice. We thank J. Ishikawa for gene annotation, E. Oowada for gene annotation and disruption, and K. Aramaki for the isolation of secondary metabolites.
This work was supported in part by a Creative Scientific Research grant and a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
Published ahead of print on 26 March 2010.
†Supplemental material for this article may be found at http://jb.asm.org/.