Cloning and Characterization of a Streptomyces Single Module Type Non-ribosomal Peptide Synthetase Catalyzing a Blue Pigment Synthesis
2007; Elsevier BV; Volume: 282; Issue: 12 Linguagem: Inglês
10.1074/jbc.m611319200
ISSN1083-351X
AutoresHitoshi Takahashi, Takanori Kumagai, Kyoko Kitani, Miwako Mori, Yasuyuki Matoba, Masanori Sugiyama,
Tópico(s)Click Chemistry and Applications
ResumoIn the present study, we cloned a gene, designated bpsA, which encodes a single module type non-ribosomal peptide synthetase (NRPS) from a d-cycloserine (DCS)-producing Streptomyces lavendulae ATCC11924. A putative oxidation domain is significantly integrated into the adenylation domain of the NRPS, and the condensation domain is absent from the module. When S. lividans was transformed with a plasmid carrying bpsA, the transformed cells produced a blue pigment, suggesting that bpsA is responsible for the blue pigment synthesis. However, to produce the blue pigment in Escherichia coli, the existence of the 4′-phosphopantetheinyl transferase (PPTase) gene from Streptomyces was necessary, in addition to bpsA. The chemical structure of the pigment was determined as 5,5′-diamino-4,4′-dihydroxy-3,3′-diazadiphenoquinone-(2,2′), called indigoidine. The bpsA gene product, designated BPSA, was overproduced in an E. coli host-vector system and purified to homogeneity, demonstrating that the recombinant enzyme prefers l-Gln as a substrate. The in vitro experiment using l-Gln also showed that the blue pigment was formed by the purified BPSA only when the enzyme was phosphopantetheinylated by adding a Streptomyces PPTase purified from E. coli cells. Each site-directed mutagenesis experiment of Lys598, Tyr601, Ser603, and Tyr608, which are seen in the oxidation domain of BPSA, suggests that these residues are essential for the binding of FMN to the protein and the synthesis of the blue pigment. In the present study, we cloned a gene, designated bpsA, which encodes a single module type non-ribosomal peptide synthetase (NRPS) from a d-cycloserine (DCS)-producing Streptomyces lavendulae ATCC11924. A putative oxidation domain is significantly integrated into the adenylation domain of the NRPS, and the condensation domain is absent from the module. When S. lividans was transformed with a plasmid carrying bpsA, the transformed cells produced a blue pigment, suggesting that bpsA is responsible for the blue pigment synthesis. However, to produce the blue pigment in Escherichia coli, the existence of the 4′-phosphopantetheinyl transferase (PPTase) gene from Streptomyces was necessary, in addition to bpsA. The chemical structure of the pigment was determined as 5,5′-diamino-4,4′-dihydroxy-3,3′-diazadiphenoquinone-(2,2′), called indigoidine. The bpsA gene product, designated BPSA, was overproduced in an E. coli host-vector system and purified to homogeneity, demonstrating that the recombinant enzyme prefers l-Gln as a substrate. The in vitro experiment using l-Gln also showed that the blue pigment was formed by the purified BPSA only when the enzyme was phosphopantetheinylated by adding a Streptomyces PPTase purified from E. coli cells. Each site-directed mutagenesis experiment of Lys598, Tyr601, Ser603, and Tyr608, which are seen in the oxidation domain of BPSA, suggests that these residues are essential for the binding of FMN to the protein and the synthesis of the blue pigment. The genus Streptomyces is well known for its ability to produce an enormous variety of bioactive secondary metabolites, including clinically useful antibiotics. For example, d-cycloserine (d-4-amino-3-isoxazolidone: DCS), 2The abbreviations used are: DCS, d-cycloserine; A-domain, adenylation domain; Ap, ampicillin; C-domain, condensation domain; Cm, chloramphenicol; Cy-domain, cyclization domain; DMF, dimethylformamide; Km, kanamycin; NMP, N-methylpyrrolidone; NRPS, non-ribosomal peptide synthetase; ORF, open reading frame; Ox-domain, oxidation domain; PPTase, 4′-phosphopantetheinyl transferase; RBS, ribosome-binding site; SARP, Streptomyces antibiotic regulatory protein; T-domain, thiolation domain; TE-domain, thioesterase domain; THF, tetrahydrofuran; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; BPSA, blue pigment synthetase A. which is a cyclic structural analogue of d-alanine and is produced by Streptomyces garyphalus and Streptomyces lavendulae, is a clinical medicine for the treatment of tuberculosis (1Pinsker K.L. Koerner S.K. Am. J. Hosp. Pharm. 1976; 33: 275-283PubMed Google Scholar). The biosynthesis genes for antibiotics, in general, form a cluster. In some cases, the final checkpoint in the transcriptional regulation of the cluster is controlled by a family of proteins called Streptomyces antibiotic regulatory proteins (SARPs), which have been characterized as transcriptional activators (2Wietzorrek A. Bibb M. Mol. Microbiol. 1997; 25: 1181-1184Crossref PubMed Scopus (249) Google Scholar). Many peptide antibiotics are known to be synthesized by non-ribosomal peptide synthetases (NRPSs). NRPSs, which are commonly found in microorganisms, are very large proteins containing sets of modules, each of which consists of various functional domains such as adenylation (A), condensation (C), cyclization (Cy), thiolation (T), and thioesterase (TE) domains (3Konz D. Marahiel M.A. Chem. Biol. 1999; 6: 39-48Abstract Full Text PDF PubMed Scopus (210) Google Scholar). The amino acid sequence of the peptide antibiotic, which is produced by each NRPS, is determined by the order of the modules. When an NRPS, which is formed as an apoform, takes the holoform, the T-domain of apo-NRPS must be phosphopantetheinylated (4Walsh C.T. Gehring A.M. Weinreb P.H. Quadri L.E.N. Flugel R.S. Curr. Opin. Chem. Biol. 1997; 1: 309-315Crossref PubMed Scopus (215) Google Scholar). This post-translational modification is catalyzed by a superfamily of enzymes known as 4′-phosphopantetheinyl transferases (PPTases), which transfer the phosphopantetheinyl group from CoA to a conserved serine residue of their T-domain (4Walsh C.T. Gehring A.M. Weinreb P.H. Quadri L.E.N. Flugel R.S. Curr. Opin. Chem. Biol. 1997; 1: 309-315Crossref PubMed Scopus (215) Google Scholar). In the process of peptide synthesis catalyzed by the holoform of NRPSs, individual amino acids are activated by the respective A-domains as amino acyl adenylates and subsequently are bound to the thiol group on the T-domains of the same modules. C-domains located downstream of each T-domain catalyze the condensation between the amino acid residues of adjacent modules so that a growing peptide chain moves from one module to the next until, finally, the completed peptide chain at the last module is released by the catalysis of the TE-domain (5Schwarzer D. Mootz H.D. Linne U. Marahiel M.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14083-14088Crossref PubMed Scopus (163) Google Scholar, 6Challis G.L. Naismith J.H. Curr. Opin. Struct. Biol. 2004; 14: 748-756Crossref PubMed Scopus (84) Google Scholar). In this study, during our attempt to clone DCS biosynthesis genes from a DCS-producing S. lavendulae ATCC11924 by the suppression subtractive hybridization method, which is a cost-effective and powerful technique for the isolation of species-specific DNA sequences from closely related microorganisms (7Diatchenko L. Lau Y.F. Campbell A.P. Chenchik A. Moqadam F. Huang B. Lukyanov S. Lukyanov K. Gurskaya N. Sverdlov E.D. Siebert P.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6025-6030Crossref PubMed Scopus (2736) Google Scholar, 8Hughes M.S. Beck L.A. Skuce R.A. Neill S.D. FEMS Microbiol. Lett. 1997; 156: 31-36Crossref PubMed Scopus (5) Google Scholar), we unexpectedly found that a DCS producer-originated gene, designated bpsA, encodes a protein classified into the NRPS family. Interestingly, this NRPS, designated BPSA, is a single module type enzyme and contains an oxidation (Ox)-domain. Heterologous expression of bpsA in S. lividans and E. coli demonstrated that BPSA functions as a synthetase for a blue pigment, which was identified as a water-insoluble blue 3,3′-bipyridyl pigment, indigoidine (9Kuhn R. Starr M.P. Kuhn D.A. Bauer H. Knackmuss H.J. Arch. Mikrobiol. 1965; 51: 71-84Crossref PubMed Scopus (41) Google Scholar, 10Mortimer P.S. Gladys C. Hans-Joachim K. Appl. Microbiol. 1966; 14: 870-872Crossref PubMed Google Scholar). An in vitro study shows that the holotype of BPSA, which was activated by in vitro phosphopantetheinylation, catalyzes the synthesis of the blue pigment using l-Gln as a substrate. The enzymatic kinetic parameters of BPSA were also determined. Furthermore, by the mutational analysis of the Ox-domain in BPSA, amino acid residues, which may be important for the binding of cofactor FMN, were suggested. Finally, we suggest a possible mechanism whereby the blue pigment is synthesized by BPSA. To the best of our knowledge, this is the first report that characterizes the single module type NRPS catalyzing a pigment synthesis. Bacterial Strains, Plasmids, and Growth Conditions—S. lavendulae ATCC11924 and S. lavendulae JCM4055 are a DCS producer and a DCS-non-producer, respectively. Both Streptomyces strains were grown at 28 °C in a YEME medium (11Kieser T. Buttner M.J. Chater K.F. Hopwood D.A. Practical Streptomyces Genetics. The John Innes Foundation, Norwich, UK2000Google Scholar) for the preparation of genomic DNA. S. lividans 66 was grown in a YEME medium containing 0.5% glycine at 28 °C for the preparation of protoplast. For blue pigment production, S. lavendulae ATCC11924 was cultured at 28 °C in medium B (12Yanagimoto M. Enatsu T. J. Ferment. Technol. 1983; 61: 545-550Google Scholar). Plasmid pIJ702 carrying the thiostrepton-resistance gene was used as a vector for S. lividans 66. Escherichia coli DH5α and pUC19 were used for DNA cloning and sequencing. E. coli BL21(DE3), pET-21a(+), and pET-28a(+) (Novagen) were used for protein expression. Plasmid pSTV28 (TaKaRa), whose replication origin is derived from the pACYC184 vector, was used for the co-expression experiment. E. coli cells were grown in an LB medium (13Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory Press, NY1989: A1Google Scholar). When necessary, appropriate antibiotics were added at the following concentrations: ampicillin (Ap), 100 μg/ml; kanamycin (Km), 30 μg/ml; and chloramphenicol (Cm), 34 μg/ml. DNA Manipulations—The genomic and plasmid DNAs of Streptomyces were isolated following a standard protocol (11Kieser T. Buttner M.J. Chater K.F. Hopwood D.A. Practical Streptomyces Genetics. The John Innes Foundation, Norwich, UK2000Google Scholar). The plasmid DNA in E. coli was isolated using the Wizard® Plus Minipreps DNA Purification system (Promega). Southern Hybridization—Southern hybridization was performed using a Hybond-N+ (Amersham Biosciences) membrane. Probe labeling, hybridization, and detection were performed with the AlkPhos Direct Labeling and Detection system (Amersham Biosciences) according to the protocol supplied by the manufacturer. Construction and Screening of Subtractive Hybridization Libraries—Subtractive hybridization between S. lavendulae ATCC11924 as a tester and S. lavendulae JCM4055 as a driver was carried out using a PCR-select bacterial genome subtraction kit (Clontech) according to the manufacturer's instruction manual, except that the hybridization temperature was set to 75 °C. The secondary PCR products, which were enriched for the S. lavendulae ATCC11924-specific DNA fragment, were subcloned into pGEM-T (Promega) and introduced into E. coli DH5α. Approximately 120 white colonies were randomly selected, and plasmid DNA was isolated from each clone. To eliminate nonspecific clones, each plasmid DNA was arrayed on Hybond-N+, and differential screening was conducted by dot-blot hybridization at 72 °C. In this case, AluI-digested genome DNAs from the tester and driver were used as probes. Ninety candidates carrying approximately a 0.5-kb DNA fragment specific to ATCC11924 were selected. The tester-specific DNA fragments obtained were analyzed to determine their nucleotide sequences followed by a BLAST search and Frame plot analysis (14Ishikawa J. Hotta K. FEMS Microbiol. Lett. 1999; 174: 251-253Crossref PubMed Google Scholar). Four clones were selected by the above screening. A DNA fragment (0.5-kb) from one of four clones, designated SA49, was used for the cloning of a larger DNA fragment (see below). Cloning of a 6-kb BamHI DNA Fragment Containing the Subtracted Region from S. lavendulae ATCC11924—To clone the DNA fragment containing the subtracted region (SA49) from S. lavendulae ATCC11924, Southern hybridization was performed against restriction enzyme-digested genomic DNA using the SA49 as a probe. The digestion with BamHI gave a single band having ∼6-kb. The DNA fragments were eluted from agarose gels, purified, ligated to the BamHI-digested pUC19, and introduced into E. coli DH5α. Positive clones, which were selected by colony hybridization, were finally confirmed by Southern hybridization. One of the positive clones, designated A49, was analyzed to determine its DNA sequence. DNA Sequencing and Homology Analysis of the Predicted Proteins—DNA sequencing was performed with ABI Prism 310 and 377 DNA sequencers (Applied Biosystems) using the BigDye terminator cycle sequencing ready reaction kit ver.1.1. The DNA and protein sequences were analyzed with GENETYX ver.7 for Windows (Software Development). The open reading frames (ORFs) were predicted using a FramePlot ver.2.3.2 (14Ishikawa J. Hotta K. FEMS Microbiol. Lett. 1999; 174: 251-253Crossref PubMed Google Scholar). A homology search was conducted with the FASTX program at DDBJ. The domain structure of the predicted protein was analyzed using the Conserved Domain data base and Search Service, v2.05, at NCBI. Expression of bpsA in S. lividans 66—A 4.8-kb DNA fragment containing bpsA (=orfB), which was obtained from the A49 clone by double digestion with BamHI and KpnI, was ligated to pIJ702-digested with BglII and KpnI to generate pIJA49/bpsA. After the chimeric plasmid was introduced into S. lividans 66 by the protoplast transformation, the resulting transformant was regenerated on an R5 medium (11Kieser T. Buttner M.J. Chater K.F. Hopwood D.A. Practical Streptomyces Genetics. The John Innes Foundation, Norwich, UK2000Google Scholar). The regenerated candidate was inoculated into a YEME medium containing 50 μgof thiostrepton/ml and grown at 28 °C. Co-expression of bpsA in E. coli with the Gene svp Encoding a PPTase from S. verticillus—The gene bpsA was amplified by PCR with KOD-Plus-polymerase (TOYOBO) using a 5′-phosphorylated sense primer, 5′-catatgactcttcaggagaccagcgtgctc-3′ (the NdeI site is underlined), and a 5′-phosphorylated antisense primer, 5′-aagcttctcgccgagcaggtagcggatgtg-3′ (the HindIII site is underlined). The amplified bpsA was inserted into the SmaI site of pUC19 to yield pUC/bpsA. The bpsA was cut off from pUC/bpsA by digestion with NdeI and HindIII and inserted into the same sites of pET-28a(+) to generate pET/bpsA. The resulting BPSA was obtained as a product with a His6 tag at the N and C termini. The gene svp, which encodes a PPTase from S. verticillus (15Sanchez C. Du L. Edwards D.J. Toney M.D. Shen B. Chem. Biol. 2001; 8: 725-738Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), was amplified by PCR from the S. verticillus genome using a 5′-phosphorylated sense primer, 5′-catatgatcgccgccctcctgccctcctg-3′ (the NdeI site is underlined), and a 5′-phosphorylated antisense primer, 5′-ctcgagcgggacggcggtccggtcgtccgc-3′ (the XhoI site is underlined). The amplified svp gene was inserted into the SmaI site of pUC19 to yield pUC/svp. The svp gene was removed from pUC/svp by digestion with NdeI and XhoI and inserted into the same sites of pET-21a(+) to give pET/svp. The Svp protein was obtained as a product with a His6 tag at the C terminus. A 2.2-kb DNA fragment, which contains svp under the control of the T7 promoter, was cut off from pET/svp by digestion with SphI and PvuI and inserted into the same sites of pSTV28 to yield pSTV/svp. The chimeric plasmids, pET/bpsA and pSTV/svp, were introduced into E. coli BL21(DE3). Co-expression of bpsA with svp in E. coli was performed at 18 °C. Purification and Chemical Properties of the Blue Pigment— Production of the blue pigment in S. lavendulae ATCC11924 was induced by γ-nonalactone. 3M. Sugiyama, unpublished data. A culture broth (2 liter) of the strain at 2 h after induction by γ-nonalactone was centrifuged (4,400 × g, 4 °C, 10 min) to remove the mycelium, and the resulting supernatant was further centrifuged (30,000 × g, 4 °C, 30 min) to collect the blue pigment. The pigment was washed twice with water and methanol and dried in vacuo. The pigment was dissolved in Me2SO under ultrasonication and filtered through a DISMIC-13JP filter (ADVANTEC), and 5 volumes of water were added. By centrifugation (30,000 × g, 4 °C, 30 min), the precipitated blue pigment was collected, washed ten times with water, twice with methanol, and dried in vacuo, yielding 11 mg of blue pigment. For visible spectral analysis, the purified blue pigment was dissolved in Me2SO, N-methylpyrrolidone (NMP), dimethylformamide (DMF), tetrahydrofuran (THF), and pyridine with sonication. The visible spectra were recorded on a JASCO V-550 spectrophotometer (JASCO). For the analysis of the molecular mass by electron impact (EI)-MS, the blue pigment was dissolved in THF and subjected to a JEOL JMS-SX102A spectrometer (JEOL). For the same analysis by MALDI-TOF, the pigment was dissolved in Me2SO and subjected to Voyager™ RP-3 (Applied Biosystems). 1H NMR was recorded on a JEOL-JNM-LA500 spectrometer (JEOL) using tetramethylsilane as an internal standard. The blue pigment was dissolved in deuterated Me2SO. The IR spectrum of the blue pigment, run as a potassium bromide tablet, showed the following frequencies: 3443, 3328, 3288, 3196, 3064, 2959, 2924, 2869, 2839, 2364, 1688, 1639, 1596, 1579, 1454, 1371, 1323, 1250, 1118, 1060, 1037, 956, 885, 824, 779, 752, 718, 689, 661, and 634 in wave number. Overexpression in E. coli and Purification of BPSA and BPSAΔTE—For the production of BPSAΔTE (1–1014), which lacks the TE-domain, the expression vector for bpsAΔTE was constructed using pET-28a(+) in the same way as the full-length bpsA, generating pET/bpsAΔTE. The PCR primers used were a 5′-phosphorylated sense primer, which was used in bpsA amplification, and a 5′-phosphorylated antisense primer, 5′-aagcttctgggcgacctcgcgctccag-3′ (the HindIII site is underlined). The BPSAΔTE protein was produced with the His6 tag at both the N and C termini. E. coli BL21(DE3) cells harboring pET/bpsA or pET/bpsAΔTE were grown in 2.5 liters of an LB medium supplemented with Km at 18 °C. Cultivation was performed without isopropyl-1-thio-β-d-galactopyranoside induction for 30 h. Cells were suspended in a binding buffer (20 mm Tris-HCl, pH 7.9, 500 mm NaCl, and 5 mm imidazole) and disrupted by sonication, and cell debris was removed by centrifugation at 27,000 × g for 20 min. The resulting supernatant was applied on a His-Bind resin column (1 × 10 cm, Novagen) and then washed with a wash buffer I (20 mm Tris-HCl, pH 7.9, 500 mm NaCl, and 30 mm imidazole). Elution was done with a linear gradient concentration of 30–500 mm imidazole. The fractions containing the BPSA or BPSAΔTE were collected and dialyzed against a binding buffer containing 1 mm EDTA (twice) and subsequently against a binding buffer (twice). The dialysate was reapplied on a flesh His-Bind resin column. The column was washed with wash buffer II (20 mm Tris-HCl, pH 7.9, 500 mm NaCl, and 60 mm imidazole). Elution was conducted with a 60–1000 mm imidazole. Each protein purified to homogeneity was stored at -20 °C in the presence of 50% glycerol until use. Overexpression in E. coli and Purification of Svp—E. coli BL21(DE3) cells harboring pET/svp were grown in 3 liters of an LB medium supplemented with Ap at 23 °C. Cultivation was performed without isopropyl-1-thio-β-d-galactopyranoside induction for 24 h. The cells were disrupted by sonication, and cell debris was removed by centrifugation at 27,000 × g for 20 min. The resulting supernatant fluid was applied on a His-Bind resin column (1 × 10 cm). After the column was washed with wash buffer II, elution was conducted with 60–500 mm imidazole. The purified Svp protein was stored at -20 °C in the presence of 50% glycerol until use. ATP/PPi Exchange Assay—To evaluate the acyl-adenylation of NRPS, amino acid-dependent ATP/PPi exchange assays were performed as described previously (16Du L. Chen M. Zhang Y. Shen B. Biochemistry. 2003; 42: 9731-9740Crossref PubMed Scopus (32) Google Scholar). The assay solution (100 μl) contained 300 nm His6-tagged protein, 5 mm ATP, 1.72 μm [32P]PPi (1 μCi, 60 Ci/mmol; PerkinElmer), 1 mm PPi, 1 mm MgCl2, 0.1 mm EDTA, 0.5 mm amino acid, and 20 mm Tris-HCl, pH 7.8. After 20 min of incubation at 30 °C, the reaction was terminated by addition of 0.9 ml of a charcoal suspension containing 0.25 m perchloric acid, 1.6% activated charcoal, and 0.1 m tetrasodium pyrophosphate. The charcoal was washed twice with 1 ml of distilled water and resuspended in 0.5 ml of distilled water. After addition of liquid scintillation fluid (ACSII; Amersham Biosciences), the radioactivity was measured. The reaction mixture without the amino acid substrate was used as a control. To determine the kinetic parameters, reactions were carried out in various concentrations of l-Gln for 6 min. Experiments in which ATP was varied (7.8 μm - 2 mm) at fixed l-Gln concentrations (3 mm) gave a Km value of 192 ± 13 μm. Thus, the ATP concentration (5 mm) used in the kinetic studies for l-Gln is saturating. PPi Release Assay—The PPi release rate by the A-domain was measured by a coupled continuous-spectrophotometric assay using the EnzChek Pyrophosphate Assay kit (Molecular Probes). The reactions contained 20 mm Tris-HCl, pH 7.8, 1 mm MgCl2, 0.1 mm EDTA, 0.2 mm MesG, 0.2 units of purine nucleoside phosphorylase, 0.2 units of inorganic pyrophosphatase, 5 mm ATP, 3 mm l-Gln, 100 nm Svp, 0.5 mm CoA, and 100 nm BPSA. For the conversion of the apoform to the holoform, BPSA was preincubated with Svp at 30 °C for 30 min without l-Gln and ATP. The reactions were started by the addition of l-Gln and ATP and monitored every 10 s for 6 min at 360 nm. The slope of 0–100 s was correlated with a standard curve created with PPi. Assays for Amino Acylation of l-[14C]Gln to the T-domain of BPSAΔTE—The loading of l-[14C]Gln to the T-domain of BPSAΔTE was investigated by autoradiography. The reaction of 100 μl in volume contained 20 mm Tris-HCl, pH 7.8, 1 mm MgCl2, 0.1 mm EDTA, 3 mm ATP, 10 μm l-[14C]Gln (0.2 μCi, 210 mCi/mmol; Moravek Biochemicals), 300 nm Svp, 0.5 mm CoA, and 300 nm BPSA or BPSAΔTE and was incubated for 30 min at 30 °C to allow the phosphopantetheinylation of the T-domain prior to initiation by the addition of l-[14C]Gln. At 15 min after addition of l-[14C]Gln, the reaction was quenched with 0.8 ml of 10% cold trichloroacetic acid containing 2% bovine serum albumin. The precipitated protein was washed with 10% cold trichloroacetic acid and acetone and subjected to 10% SDS-PAGE. The dried gel was exposed on an imaging plate and visualized by BAS-2000. In Vitro Synthesis of the Blue Pigment—To phosphopantetheinylate BPSA, a solution (1.4 ml) containing 660 nm BPSA, 810 nm Svp, 0.1 mm CoA, and 1 mm MgCl2, which was prepared in a 50 mm sodium phosphate buffer (pH 7.8), was incubated at 30 °C for 10 min. Synthesis of the blue pigment was initiated by addition of 200 μl of 10 mm ATP (final 1 mm) and 400 μl of 5 mm l-amino acid (final 1 mm). The in vitro synthesis of the blue pigment was monitored by measuring the absorbance at 590 nm. The molecular mass of the synthesized blue pigment was analyzed by MALDI-TOF as described above. MS/MS Analysis—A reaction mixture consisting of 20 mm Tris-HCl, pH 7.8, 1.5 μm BPSA, 2 μm Svp, 1 mm CoA, and 1 mm MgCl2 was incubated at 30 °C for 1 h. 4′-phosphopantetheinylated BPSA was subjected to SDS-PAGE, and the corresponding band was cut off from the gel. After digestion of the protein contained in the gel by trypsin (Promega), the resulting peptide preparation was desalted with ZipTip (Millipore) and analyzed using an AXIMA-QIT (Shimadzu) mass spectrometer. A peak (m/z = 2921), which corresponds to a peptide ENASVQDDFFESGGNSLIAVGLVR (Ser972 is underlined) containing the carbamidemethylated 4′-phosphopantetheinyl residue, was used as a precursor ion for the MS/MS analysis. Mutagenic Analysis—Site-directed mutagenesis was performed using the QuickChange II XL Site-directed mutagenesis kit (Stratagene) according to the supplier's instructions. The mutation in bpsA was confirmed by DNA sequencing. The mutants of BPSA were overproduced in E. coli BL21 (DE3) and purified in the same way as wild-type BPSA. HPLC Analysis—HPLC for the analysis of the BPSA cofactor was performed using a Hydrosphere C18 column (4.6 × 200 mm, YMC). The run was conducted by a gradient elution with a 20 mm potassium phosphate buffer (pH 6.9) containing 0.1% acetonitrile (from 90 to 30%) and methanol (from 10 to 70%) for 30 min at the flow rate of 1.5 ml/min. Nucleotide Sequence Accession Number—The sequence reported has been deposited in the DDBJ data base under the accession number AB240063. Finding an NRPS Gene in a DCS Producer S. lavendulae ATCC11924—To clone biosynthesis genes for DCS, we enriched DNA fragments specific to DCS-producing S. lavendulae ATCC11924 using a PCR-based subtractive hybridization method. We could not find the clone carrying the DCS biosynthesis genes. Instead, we noticed a clone harboring a 0.5-kb DNA fragment, designated SA49: a portion of the protein deduced from the nucleotide sequence had a 70% similarity to a putative regulatory protein of actinorhodin-producing S. coelicolor (11Kieser T. Buttner M.J. Chater K.F. Hopwood D.A. Practical Streptomyces Genetics. The John Innes Foundation, Norwich, UK2000Google Scholar). We assumed that the protein encoded in SA49 may control some biosynthesis genes for secondary metabolites in S. lavendulae ATCC11924. Because the regulatory genes are generally clustered with the biosynthesis genes for secondary metabolites (11Kieser T. Buttner M.J. Chater K.F. Hopwood D.A. Practical Streptomyces Genetics. The John Innes Foundation, Norwich, UK2000Google Scholar), a 6-kb BamHI-digested DNA fragment was newly cloned from the genome of S. lavendulae ATCC11924 using the SA49 DNA fragment as a probe. As shown in Fig. 1, the nucleotide sequence and FramePlot analyses (14Ishikawa J. Hotta K. FEMS Microbiol. Lett. 1999; 174: 251-253Crossref PubMed Google Scholar) of the 5913-bp DNA suggest the presence of two complete ORFs, named orfB and orfC, and two incomplete ones (orfA and orfD). The incomplete orfA starts out of the sequenced region. A portion of a deduced protein consisting of 193 amino acids showed high identity (60–80%) to some bacterial ribose phosphate pyrophosphokinases. The complete orfB consists of 3849 bp. A putative ribosome-binding site (RBS), AGGAAG, was found upstream of the start codon. The protein, encoded by orfB, consists of 1282 amino acids with a calculated molecular mass of 141 kDa and exhibits a significant similarity to a large number of NRPS. In particular, the protein displays a similarity with NRPSs, designated IndC (17Reverchon S. Rouanet C. Expert D. Nasser W. J. Bacteriol. 2002; 184: 654-665Crossref PubMed Scopus (151) Google Scholar) (GenBank™ accession number CAB87990) from Erwinia chrysanthemi (57% identity) and IgiD (GenBank™ accession number AAD54007) from Vogesella indigofera (46% identity). Because orfB was found to encode a synthetase of a blue pigment, as described below, we renamed the gene as bpsA (blue pigment synthetase-encoding gene A). The gene orfC, which consists of 789 bp, encodes a protein of 262 amino acids with a calculated molecular mass of 29.5 kDa. The upstream region of orfC contains a potent RBS of AGGAG. The orfC gene product shows a significant similarity to the SARP family, which controls the secondary metabolism in Streptomyces sp (2Wietzorrek A. Bibb M. Mol. Microbiol. 1997; 25: 1181-1184Crossref PubMed Scopus (249) Google Scholar). The highest similarity (59% identity) was seen with the actinorhodin operon activator protein (ActII-Orf4) from S. coelicolor (18Fernandez-Moreno M.A. Caballero J.L. Hopwood D.A. Malpartida F. Cell. 1991; 66: 769-780Abstract Full Text PDF PubMed Scopus (294) Google Scholar), suggesting that the orfC-encoded protein may act as a transcriptional activator. The orfD gene ends out of sequence, and the deduced protein (63 amino acids), which is an N-terminal region of the protein, shows 97% identity to the S-adenosylmethionine synthetase from S. spectabilis (GenBank™ accession number Q9X4Q2). bpsA Encodes a Single Module Type NRPS—Because the bpsA gene product, designated BPSA, exhibits a significant similarity to a large number of NRPS, the domain structural search of BPSA was carried out using the Conserved Domain Search Program (NCBI). We found that BPSA contains an A-domain, a T-domain, and a TE-domain at the C-terminal end; thus, the protein appears to be an NRPS of a single module type. Importantly, there is a putative Ox-domain integrated into the A-domain between the A8 and A9 signature sequences of the A-domain, and the C-domain is absent from the BPSA (Fig. 1). The motif sequences of each domain in BPSA are summarized in Table 1. The Ox-domain in BPSA exhibits similarity with those of EpoB (19Schneider T.L. Shen B. Walsh C.T. Biochemistry. 2003; 42: 9722-9730Crossref PubMed Scopus (89) Google Scholar) and BlmIII (16Du L. Chen M. Zhang Y. Shen B. Biochemistry. 2003; 42: 9731-9740Crossref PubMed Scopus (32) Google Scholar), which are involved in the biosynthesis of epothilone and bleomycin, respectively (20Chen H.W. O'Conner S. Cane D.E. Walsh C.T. Chem. Biol. 2001; 8: 899-912Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 21Du L. Chen M. Sanchez C. Shen B. FEMS Microbiol. Lett. 2000; 189: 171-175Crossref PubMed Google Scholar).TABLE 1Highly conserved core motifs of each domain
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