Key points are not available for this paper at this time.
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. 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)ethylglycine; 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. Streptomyces The for the of S. lividans was grown in a YEME medium containing at 28 °C for the of For blue pigment S. lavendulae ATCC11924 was at 28 °C in medium M. T. J. Scholar). carrying the gene was used as a for S. lividans Escherichia coli and were used for DNA and E. coli and were used for protein is from the was used for the E. coli cells were grown in an medium J. T. A Scholar). When necessary, antibiotics were at the and DNA and plasmid of Streptomyces were a (11Kieser T. Buttner M.J. Chater K.F. Hopwood D.A. Streptomyces The Scholar). The plasmid DNA in E. coli was using the DNA system hybridization was using a and were with the and system to the by the and of hybridization between S. lavendulae ATCC11924 as a and S. lavendulae JCM4055 as a was using a to the that the hybridization was to The secondary which were for the S. lavendulae DNA were into and into E. coli were and plasmid DNA was from each To each plasmid DNA was on and was by hybridization at In this from the and were used as carrying a DNA to ATCC11924 were The DNA were to their sequences by a and analysis J. K. FEMS Microbiol. Lett. 1999; PubMed Google Scholar). were by the A DNA from one of designated was used for the of a DNA of a DNA the from S. lavendulae clone the DNA containing the from S. lavendulae hybridization was DNA using the as a The with a single The DNA were from to the and into E. coli which were by were by of the designated was to its DNA DNA and of the was with and DNA using the The DNA and protein sequences were with for The open reading were using a J. K. FEMS Microbiol. Lett. 1999; PubMed Google Scholar). A was with the at The domain structure of the protein was using the and at of bpsA in S. lividans DNA containing bpsA which was from the clone by with and was to with and to the plasmid was into S. lividans by the the was on an medium (11Kieser T. Buttner M.J. Chater K.F. Hopwood D.A. Streptomyces The Scholar). The was into a YEME medium containing and grown at 28 of bpsA in E. coli with the a PPTase from S. gene bpsA was by with using a is and a is The bpsA was into the of to The bpsA was from by with and and into the same of to The BPSA was as a with a at the and The gene which encodes a PPTase from S. C. L. B. Chem. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), was by from the S. using a is and a is The gene was into the of to The gene was from by with and and into the same of to The protein was as a with a at the A DNA which contains the of the was from by with and and into the same of to The and were into E. coli of bpsA with in E. coli was at and of the of the blue pigment in S. lavendulae ATCC11924 was by A of the at by was to the and the was to the blue pigment. The pigment was with and and in The pigment was in and a and of were the blue pigment was with with and in of blue pigment. For the purified blue pigment was in and with The were on a For the analysis of the by the blue pigment was in and to a For the same analysis by the pigment was in and to was on a using as an The blue pigment was in The of the blue pigment, as a showed the and in in E. coli and of BPSA and the of which the TE-domain, the expression for was using in the same as the bpsA, The used were a which was used in bpsA and a is The protein was produced with the at the and E. coli cells were grown in of an medium with at was for were in a binding and and by and was by at for The was on a and with a and was with a of The containing the BPSA were and a binding containing and subsequently a binding The was on a The was with and was with a Each protein purified to was at °C in the of in E. coli and of coli cells were grown in of an medium with at was for The cells were by and was by at for The was on a the was with was with The purified protein was at °C in the of the of NRPS, amino were as L. M. B. PubMed Scopus Google Scholar). The amino and of at the was by addition of of a containing activated and The was with of and in of addition of the was The the amino acid was used as a To the kinetic were in various of l-Gln for in which was at l-Gln a of the used in the kinetic for l-Gln is by the was by a using the The of of and BPSA. For the of the to the holoform, BPSA was with at °C for l-Gln and The were by the addition of l-Gln and and for at The of was with a with for of to the T-domain of of to the T-domain of was by The of in and BPSA and was for at °C to the of the T-domain to by the addition of addition of the was with of acid containing The protein was with acid and and to The was on an and by In of the BPSA, a containing BPSA, and which was in a was at °C for of the blue pigment was by addition of of and of acid The in vitro synthesis of the blue pigment was by the at The of the synthesized blue pigment was by as of BPSA, and was at °C for BPSA was to and the was from the of the protein in the by the peptide was with and using an A which to a peptide is containing the 4′-phosphopantetheinyl was used as a for the mutagenesis was using the mutagenesis to the The in bpsA was by DNA The of BPSA were overproduced in E. coli and purified in the same as BPSA. for the analysis of the BPSA cofactor was using a The was by a with a containing to and to for at the of sequence been in the the an NRPS in a DCS S. lavendulae clone biosynthesis genes for DCS, we DNA to DCS-producing S. lavendulae ATCC11924 using a subtractive hybridization the clone carrying the DCS biosynthesis we a clone a DNA designated a of the protein from the sequence a to a putative regulatory protein of S. (11Kieser T. Buttner M.J. Chater K.F. Hopwood D.A. Streptomyces The Scholar). that the protein in may some biosynthesis genes for secondary in S. lavendulae ATCC11924. the regulatory genes are with the biosynthesis genes for secondary (11Kieser T. Buttner M.J. Chater K.F. Hopwood D.A. Streptomyces The Scholar), a DNA was cloned from the of S. lavendulae ATCC11924 using the DNA as a in the sequence and J. K. FEMS Microbiol. Lett. 1999; PubMed Google of the DNA suggest the of and and and The of the A of a protein of amino acids showed to some The consists of A putative ribosome-binding was found of the The by consists of amino acids with a of and a to a large of In the protein a with NRPSs, designated S. C. D. J. 2002; PubMed Scopus Google from and from was found to a synthetase of a blue pigment, as we the gene as bpsA pigment gene The gene which consists of encodes a protein of amino acids with a of The of contains a of The gene shows a to the which the secondary in Streptomyces (2Wietzorrek A. Bibb M. Mol. Microbiol. 1997; 25: 1181-1184Crossref PubMed Scopus (249) Google Scholar). The was seen with the protein from S. M.A. Hopwood D.A. F. Full Text PDF PubMed Scopus Google Scholar), suggesting that the protein may as a transcriptional The gene of and the protein amino which is an of the shows to the synthetase from S. bpsA a the bpsA gene product, designated BPSA, a to a large of NRPS, the domain structural of BPSA was using the found that BPSA contains an A-domain, a T-domain, and a TE-domain at the the protein to be an NRPS of a single module is a putative Ox-domain integrated into the between the and sequences of the A-domain, and the is absent from the BPSA The sequences of each domain in BPSA are in The Ox-domain in BPSA with of B. C.T. PubMed Scopus Google and L. M. B. PubMed Scopus Google Scholar), which are in the biosynthesis of and S. C.T. Chem. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, L. M. C. B. FEMS Microbiol. Lett. PubMed Google conserved of each domain in in a The NRPS, BPSA, the to a on S. containing from the clone was into a Streptomyces The designated was into S. lividans The Streptomyces transformed with produced a blue pigment, with suggesting that bpsA encodes a single module type NRPS essential for the biosynthesis of the blue pigment. In the of the DNA containing bpsA and its was at the into the gene of suggesting that the cloned bpsA gene contains an BPSA in the of a Streptomyces to the blue ability of bpsA in E. coli cells. However, E. coli carrying bpsA produce the blue pigment suggesting that the recombinant BPSA is phosphopantetheinylated by a PPTase produced in E. coli cells. we used a gene a PPTase from S. designated C. L. B. Chem. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), of the E. coli the E. coli carrying bpsA and genes produced the blue pigment that the gene product, designated BPSA, and the holotype BPSA catalyze the synthesis of the blue pigment. However, the blue pigment in S. lividans was produced by bpsA that the PPTase from S. lividans catalyze the of BPSA from S. In Streptomyces strains a PPTase that to C. L. B. Chem. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). of the from S. found that S. lavendulae the blue pigment by the addition of The Streptomyces blue pigment was by and to the chemical The of the blue pigment the existence of a of and The of the blue pigment in various is in were very to of 5,5′-diamino-4,4′-dihydroxy-3,3′-diazadiphenoquinone-(2,2′), called as in (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). and a at as a which to the of indigoidine. The of the blue pigment The at and to the of and which for the structure of indigoidine. analysis was by the of the pigment, we that the chemical structure of the Streptomyces blue pigment is to that of of the blue pigment in various are from Kuhn are from Kuhn (9Kuhn R. Starr M.P. Kuhn D.A. Bauer H. Knackmuss H.J. Arch. Mikrobiol. 1965; 51: 71-84Crossref PubMed Scopus (41) Google in a In of the BPSA is an NRPS of a single module the blue pigment must be synthesized from only one amino In an NRPS amino acid and by with bound to the The which an amino acid as a is located between and in the T. Mootz H.D. Marahiel M.A. Chem. Biol. 1999; 6: Full Text PDF PubMed Scopus Google Scholar, G.L. J. Chem. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). The analysis of the in synthetase in with E. T. Marahiel M.A. J. 1997; PubMed Scopus Google that amino located between the and are responsible for the of the amino acid as a substrate. The between the amino acids and the been the be by the The suggests that the amino acids seen on the of BPSA are and and that the for the enzyme is To the for BPSA, we overproduced the protein and purified to which is in biosynthesis S. C.T. Chem. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), the purified BPSA also takes on a suggesting that FMN as a cofactor is bound to the Ox-domain of BPSA. In the of FMN in BPSA was by the of a cofactor and The of the amino acid by the purified BPSA was by the L. M. B. PubMed Scopus Google Scholar). and the of into was when l-Gln was used as a suggesting that the for BPSA is of BPSA of the conserved in the of various was with the PubMed Scopus Google Scholar). The are in and The conserved residues in sequences are by and with an the and are by the the of the purified BPSA of BPSA and The enzymatic kinetic of the in BPSA for l-Gln was determined by the The and of for l-Gln were and respectively. the for the of the and the holoform of BPSA was by a using the analogue C.T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The of the to the holoform of BPSA was by with the purified The of the was that of the holoform was A of the by the from the to the holoform, which been in C.T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), was in BPSA. the blue pigment synthesis by BPSA, l-Gln is by the to The is subsequently to the T-domain, is bound as a to the group of a To that the l-Gln be to the T-domain in BPSA, we an in vitro amino In this which the TE-domain in BPSA, was and used in addition to BPSA, the bound of l-Gln on the T-domain in the BPSA is to be released by the The enzyme kinetic parameters of were determined by the The of and and were to of BPSA. The which was bound to was by in the the to with were seen in the BPSA with suggest that l-Gln is on the activated T-domain of and that the TE-domain of BPSA functions to the of l-Gln on the we that in the T-domain of BPSA is with In of the by the BPSA and purified BPSA and we the blue pigment is synthesized in vitro using and as in the pigment was synthesized when l-Gln was used with The and were in the of Furthermore, analysis of the blue pigment, which was in the same as that produced by S. lavendulae ATCC11924. that the blue pigment is synthesized from l-Gln by the BPSA. of an Ox-domain integrated into the To the of the we into the Ox-domain of BPSA. of the putative of various NRPSs, which is in residues in the conserved these residues, we Lys598, Tyr601, Ser603, and by and to and respectively. The the purified BPSA a that each BPSA suggesting that these may FMN as a The analysis shows the of FMN in the type contains one of FMN The of FMN in was also suggesting that Lys598, Tyr601, Ser603, and residues may be essential for FMN A analysis of an from that and residues a group of FMN by the of H. H. T. S. K. M. J. Mol. Biol. PubMed Scopus Google Scholar). in the Ox-domain of BPSA may form a with the group of The in vitro experiment showed that each of an essential for adenylation of l-Gln the blue ability This may be a of the of FMN bound in the In this study, we that the on the in the BPSA is significantly that in the BPSA. BPSA the Ox-domain integrated into the A-domain, the adenylation of be FMN is from the of by we must the mechanism whereby the blue pigment is the binding of the l-Gln to a seen on the T-domain of BPSA, we a at the present transfer of l-Gln to the T-domain, the is released from the T-domain by cyclization with an The be catalyzed by the TE-domain, as by the amino analysis using when the oxidation of the by the Ox-domain from a report that the oxidation by the Ox-domain of when the is bound to the T-domain B. C.T. PubMed Scopus Google Scholar), the oxidation of l-Gln by the Ox-domain of BPSA may when bound to the However, we a that the oxidation l-Gln is and released from the have an that when the Ox-domain purified from the of E. coli the Ox-domain of bpsA is to the containing the protein and the blue pigment synthesis is This may a that l-Gln is and released from the T-domain, the oxidation of the released The may be between the and by to the blue pigment, the of from in the biosynthesis of have been found in E. S. C. D. J. 2002; PubMed Scopus Google Scholar), and and E. C. L. C. A. S. S. C. M. E. R. S. S. C. A. K. R. M. N. A. F. PubMed Scopus Google Scholar). been that the and genes in these The present study shows that only NRPS is responsible for the biosynthesis of indigoidine in these containing Streptomyces The gene is in A. E. L. R. L. S. K.L. M. 2002; PubMed Scopus Google Scholar). Interestingly, the structure of an protein from been determined R. D. E. T. J. H.J. A.M. M. M.A. A. C. S.K. J. E. J. M. L. C. E. A. Kuhn A. K. E. J. R. K. R. A. E. H. J. J. B. J. PubMed Scopus Google Scholar). In the been that the for may be a the gene is in the biosynthesis of which l-Gln as a substrate. The present study is the first report with to the of a single module type NRPS, which functions as a synthetase into with module Furthermore, using the technique of site-directed we first the amino acid residues essential for the on the from the that BPSA the blue pigment from only one amino acid as a the enzyme is an BPSA is a single module type NRPS, its analysis into the mechanism of The of BPSA is in T. and K. for their and for and S. for The of the was using a at the for and DNA sequence was with the of the for of
Takahashi et al. (Sat,) studied this question.