A Novel Quinone-forming Monooxygenase Family Involved in Modification of Aromatic Polyketides
2005; Elsevier BV; Volume: 280; Issue: 15 Linguagem: Inglês
10.1074/jbc.m500190200
ISSN1083-351X
AutoresNobutaka Funa, Masanori Funabashi, Etsuro Yoshimura, Sueharu Horinouchi,
Tópico(s)Genomics and Phylogenetic Studies
ResumoRppA is a type III polyketide synthase (PKS) that catalyzes condensation of five molecules of malonyl-CoA to form 1,3,6,8-tetrahydroxynaphthalene (THN). In Streptomyces antibioticus IFO13271 and several other Streptomyces species, an open reading frame, named momA, is present as a neighbor of rppA. MomA belonged to the “cupin” superfamily because it contained a set of two motifs that is responsible for binding one equivalent of metal ions. MomA catalyzed monooxygenation of the THN produced from malonyl-CoA by the action of RppA to form flaviolin. In addition, it used several polyketides as substrates and formed the corresponding quinones. MomA required redox-active transition metal ions (Ni2+, Cu2+, Fe3+, Fe2+, Mn2+, and Co2+) for its activity, whereas it was inhibited by a redox-inert transition metal ion (Zn2+). MomA neither possessed any flavin prosthetic group nor required nicotinamide cofactors for monooxygenation, which shows that MomA as a member of the cupin superfamily is a novel monooxygenase. Consistent with the catalytic property of MomA, WhiE-ORFII showing similarity in amino acid sequence to MomA and containing a cupin domain also catalyzed monooxygenation of THN. whiE-ORFII is located immediately upstream of the “minimal PKS” gene within the whiE type II PKS gene cluster for biosynthesis of a gray spore pigment in Streptomyces coelicolor A3(2), and a number of whiE-ORFII homologues are present in the biosynthetic gene cluster for polyketides of type II in various Streptomyces species. These findings show that a novel class of quinone-forming monooxygenases is involved in modification of aromatic polyketides synthesized by PKSs of types II and III. RppA is a type III polyketide synthase (PKS) that catalyzes condensation of five molecules of malonyl-CoA to form 1,3,6,8-tetrahydroxynaphthalene (THN). In Streptomyces antibioticus IFO13271 and several other Streptomyces species, an open reading frame, named momA, is present as a neighbor of rppA. MomA belonged to the “cupin” superfamily because it contained a set of two motifs that is responsible for binding one equivalent of metal ions. MomA catalyzed monooxygenation of the THN produced from malonyl-CoA by the action of RppA to form flaviolin. In addition, it used several polyketides as substrates and formed the corresponding quinones. MomA required redox-active transition metal ions (Ni2+, Cu2+, Fe3+, Fe2+, Mn2+, and Co2+) for its activity, whereas it was inhibited by a redox-inert transition metal ion (Zn2+). MomA neither possessed any flavin prosthetic group nor required nicotinamide cofactors for monooxygenation, which shows that MomA as a member of the cupin superfamily is a novel monooxygenase. Consistent with the catalytic property of MomA, WhiE-ORFII showing similarity in amino acid sequence to MomA and containing a cupin domain also catalyzed monooxygenation of THN. whiE-ORFII is located immediately upstream of the “minimal PKS” gene within the whiE type II PKS gene cluster for biosynthesis of a gray spore pigment in Streptomyces coelicolor A3(2), and a number of whiE-ORFII homologues are present in the biosynthetic gene cluster for polyketides of type II in various Streptomyces species. These findings show that a novel class of quinone-forming monooxygenases is involved in modification of aromatic polyketides synthesized by PKSs of types II and III. Aromatic polyketides are widely distributed in bacteria, fungi, and plants, and their structural diversity reflects the variety of pharmacological and veterinary properties (1O'Hagen D. Mellor J. The Polyketide Metabolites. Ellis Horwood, NY1991Google Scholar). The synthesis of this class of natural products is comprised of the carbon skeleton synthesis by three distinct classes of PKSs 1The abbreviations used are: PKS, polyketide synthase; THN, 1,3,6,8-tetrahydroxynaphthalene; DHN, 1,3-dihydroxynaphthalene; EA, emodin anthrone; HPLC, high performance liquid chromatography; ICP-AES, inductively coupled plasma atomic emission spectroscopy; ARD, acireductone dioxygenase; IFO, Institute of Fermentation; BLAST, Basic Local Alignment Search Tool.1The abbreviations used are: PKS, polyketide synthase; THN, 1,3,6,8-tetrahydroxynaphthalene; DHN, 1,3-dihydroxynaphthalene; EA, emodin anthrone; HPLC, high performance liquid chromatography; ICP-AES, inductively coupled plasma atomic emission spectroscopy; ARD, acireductone dioxygenase; IFO, Institute of Fermentation; BLAST, Basic Local Alignment Search Tool. (types I–III) and post-PKS tailoring enzymes including oxidation, reduction, glycosylation, halogenation, and so on (2Rix U. Fischer C. Remsing L.L. Rohr J. Nat. Prod. Rep. 2002; 19: 542-580Crossref PubMed Scopus (224) Google Scholar). In bacteria, the genes for PKS and modification enzymes are usually clustered, forming a polyketide biosynthetic gene cluster. Type I PKSs are multifunctional enzymes that are organized into modules, each of which harbors a set of distinct catalytic domains (3Rawlings J.B. Nat. Prod. Rep. 2001; 18: 190-227Crossref PubMed Scopus (88) Google Scholar, 4Rawlings J.B. Nat. Prod. Rep. 2001; 18: 231-281Crossref PubMed Scopus (88) Google Scholar). AviM from Streptomyces viridochromogenes Tü57 is an unusual type I PKS, which uses its active site iteratively for the synthesis of the aromatic polyketide moiety of avilamycin A (5Gaisser S. Trefzer A. Stockert S. Kirschning A. Bechthold A. J. Bacteriol. 1997; 179: 6271-6278Crossref PubMed Google Scholar). Type II PKSs, exemplified by WhiE for the biosynthesis of a gray polyketide spore pigment of Streptomyces coelicolor A3(2) (6Davis N.K. Chater K.F. Mol. Microbiol. 1990; 4: 1679-1691Crossref PubMed Scopus (133) Google Scholar), contain a single set of iteratively used active sites that are carried on separate proteins. Type II PKSs consist of a “minimal PKS” and auxiliary subunits (7Carreras C.W. Khosla C. Biochemistry. 1998; 37: 2084-2088Crossref PubMed Scopus (80) Google Scholar, 8Bao W. Wendt-Pienkowski E. Hutchinson C.R. Biochemistry. 1998; 37: 8132-8138Crossref PubMed Scopus (75) Google Scholar). The pivotal component of the minimal PKS is a ketosynthase, which is responsible for condensation of acetate building blocks. Type III PKSs are ketosynthases with a homodimeric form, which act iteratively for polyketide chain extension (9Austin M.B. Noel J.P. Nat. Prod. Rep. 2003; 20: 79-110Crossref PubMed Scopus (721) Google Scholar). RppA, which catalyzes the condensation of five molecules of malonyl-CoA to synthesize THN and is responsible for melanin synthesis in Streptomyces griseus, is a member of type III PKSs (10Funa N. Ohnishi Y. Fujii I. Shibuya M. Ebizuka Y. Horinouchi S. Nature. 1999; 400: 897-899Crossref PubMed Scopus (236) Google Scholar). Within a gene cluster for a given polyketide, several enzymes involved in modification of the polyketide skeleton are in most cases encoded. These modification enzymes or post-PKS tailoring enzymes usually play a critical role in contributing to the biological properties exhibited by a variety of polyketide compounds (11Hutchinson C.R. Curr. Opin. Microbiol. 1998; 1: 319-329Crossref PubMed Scopus (112) Google Scholar). Therefore, studying the modifications by these enzymes of aromatic polyketides that are synthesized by types II and III PKSs is important and would sometimes lead to discoveries of their novel catalytic properties. These modification enzymes could be employed as a member in so-called combinatorial biosynthesis for the purpose of synthesizing “unnatural” natural compounds. We focused on an open reading frame (momA) that lies immediately downstream of rppA encoding a type III PKS in Streptomyces antibioticus IFO13271. Because RppA synthesizes THN from malonyl-CoA and because momA is located near rppA in several other Streptomyces species, we expected that MomA might be involved in modification of THN. In bacteria, functionally related genes are often organized as an operon or located as neighbors. As expected, MomA was found to catalyze monooxygenation of THN to yield flaviolin. MomA was also found to be necessary to convert flaviolin into mompain, a certain protein in the cell lysate of Streptomyces lividans that, together with MomA, converted flaviolin into mompain. We also characterized WhiE-ORFII, which shows similarity in amino acid sequence to MomA and is encoded within the whiE gene cluster responsible for the synthesis of a gray spore pigment in S. coelicolor A3(2) (6Davis N.K. Chater K.F. Mol. Microbiol. 1990; 4: 1679-1691Crossref PubMed Scopus (133) Google Scholar). The spore pigment is synthesized by a “minimal PKS” (type II PKS) encoded within the gene cluster. In agreement with the sequence similarity between the two enzymes, WhiE-ORFII catalyzed monooxygenation of THN and several other aromatic polyketides to yield the corresponding quinones. MomA and WhiE-ORFII contained a sequence conserved in “cupin” proteins and required redox-active transition metal ions. We therefore propose that MomA and WhiE-ORFII are members of a novel monooxygenase family that is responsible for post-PKS tailoring reactions in polyketide synthesis of both types II and III. Materials—Emodin, naphthalene, 2-hydroxy-1,4-naphthoquinone, and 1,3-dihydroxynaphthalene (DHN) were purchased from Aldrich. 1-Naphthol was purchased from Sigma. 2-Naphthol, resorcinol, phloroglucinol, 8-quinolinol, tiron (1,2-dihydroxy-3,5-benzene-disulfonic acid), and 1,10-phenanthroline were purchased from Wako. 5-Methylresorcinol was purchased from Tokyo Kasei. THN and emodin anthrone (EA) were synthesized according to the method of Ichinose et al. (12Ichinose K. Ebizuka Y. Sankawa U. Chem. Pharm. Bull. 2001; 49: 192-196Crossref PubMed Scopus (19) Google Scholar) and Falk et al. (13Falk H. Meyer J. Oberreiter M. Monatsh. Chem. 1993; 124: 339-341Crossref Scopus (133) Google Scholar), respectively. Bacterial Strains, Plasmids, and Media—S. griseus IFO13350 and S. antibioticus IFO13271 were obtained from the Institute of Fermentation (IFO), Osaka, Japan. S. coelicolor A3(2) and S. lividans TK21 were obtained from D. A. Hopwood (see Ref. 14Hopwood D.A. Bibb M.J. Chater K.F. Kieser T. Bruton C.J. Kieser H.M. Lydiate D.J. Smith C.P. Ward J.M. Schrempf H. Genetic Manipulation in Streptomyces: A Laboratory Manual. John Innes Foundation, Norwich, UK1985: 1-356Google Scholar). Escherichia coli JM109 and plasmids pUC19 and pKf19, used for DNA manipulation, were purchased from Takara Biochemicals. An rppA homologue from S. antibioticus on pET16b-Sa-rppA (15Funa N. Ohnishi M. Ebizuka Y. Horinouchi S. Biochem. J. 2002; 367: 781-789Crossref PubMed Google Scholar) was used as the hybridization probe for cloning a longer DNA fragment including rppA. For expression of RppA and MomA in Streptomyces, pIJ6021 containing a thiostrepton-inducible tipA promoter (16Takano E. White J. Thompson C.J. Bibb M.J. Gene (Amst.). 1995; 166: 133-137Crossref PubMed Scopus (127) Google Scholar) was used. For expression of His-tagged proteins in E. coli BL21 (DE3), pET26b and pET16b (Novagen) were used. Media and growth conditions for E. coli were described by Maniatis et al. (17Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1982Google Scholar). S. coelicolor A3(2) and S. lividans were routinely cultured at 30 °C on yeast extract malt extract medium (14Hopwood D.A. Bibb M.J. Chater K.F. Kieser T. Bruton C.J. Kieser H.M. Lydiate D.J. Smith C.P. Ward J.M. Schrempf H. Genetic Manipulation in Streptomyces: A Laboratory Manual. John Innes Foundation, Norwich, UK1985: 1-356Google Scholar), which was supplemented with 5 μg/ml of kanamycin or thiostrepton, when necessary. Construction of Plasmids—Restriction enzymes and other DNA-modifying enzymes were purchased from Takara Biochemicals. [α-32P]dCTP for DNA labeling with the Takara BcaBest DNA labeling system was purchased from Amersham Biosciences. General recombinant DNA techniques, including Southern hybridization and colony hybridization, were described by Maniatis et al. (17Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1982Google Scholar). Nucleotide sequences were determined with the ThermoSequenase fluorescence-labeled primer cycle sequencing kit on an automated DNA sequencer (Amersham Biosciences). The nucleotide sequence reported in this paper has been deposited in the GenBank™ data base under accession number AB198053. Construction of pMF1—Southern hybridization against the chromosomal DNA from S. antibioticus IFO13271 with the rppA sequence on pET16b-Sa-rppA as the 32P-labeled probe revealed the presence of a 3.2-kb SalI-SphI DNA fragment giving a positive signal. The 3.2-kb fragment was cloned into pUC19, resulting in pUC19-MF0 (see Fig. 1B). The 1.3-kb SacI-KpnI fragment, which was prepared from pUC19-MF0 by partial digestion with SacI followed by KpnI digestion (a KpnI site is present in the multicloning site of pUC19-MF0), together with a 0.9-kb NdeI-SacI fragment excised from pET16b-Sa-rppA were cloned between the NdeI and KpnI site of pKf19 to construct pKf19-MF1. The 2.2-kb NdeI-EcoRI fragment (an EcoRI site is present in the multicloning site of pKf19-MF1), excised from pKf19-MF1, was cloned between the NdeI and EcoRI sites of pIJ6021, resulting in pMF1. Construction of pMF2—The 1.1-kb NdeI-BamHI fragment, excised from pET16b-Sa-rppA, was cloned between the NdeI and BamHI sites of pIJ6021, resulting in pMF2. Construction of pMF3—The nucleotide sequence (CGTATG) covering the ATG start codon of MomA was changed to CATATG to create an NdeI site by PCR with primer I, 5′-GCGAAGCTTAGCGGAGGAAACCCATATGACC-3′ (the italic letters indicate nucleotides to be changed; the underlined letters indicate a HindIII site), and primer II, 5′-GCGGGATCCATATCGGTTCCATCTCGGGT-3′(the underlined letters indicate a BamHI site). After amplification by PCR under the standard conditions, the HindIII-BamHI fragment was prepared and cloned between the HindIII and BamHI sites of pUC19, resulting in pUC19-MF3. The absence of undesired alterations was checked by nucleotide sequencing. The NdeI-BamHI fragment, excised from pUC19-MF3, was cloned between the NdeI and BamHI sites of pIJ6021, resulting in pMF3. Construction of pET16b-MomA— The NdeI-BamHI fragment excised from pUC19-MF3 was cloned between the NdeI and BamHI sites of pET16b (Novagen), resulting in pET16b-MomA. Construction of pET26b-MomA—The nucleotide sequence (TGACTC) covering the TGA stop codon of MomA was changed to CTCGAG to create an XhoI site by PCR with primer I and primer III, 5′-GCGGAATTCCTCGAGGCTCCCACTCTGGTTCGCGT-3′ (the italic letters indicate nucleotides to be changed; the underlined letters indicate an EcoRI site). The amplified 0.6-kb momA fragment was cloned between the HindIII and EcoRI sites of pUC19, resulting in pUC19-C-MomA. The absence of undesired alterations was checked by nucleotide sequencing. The NdeI-XhoI fragment excised from pUC19-C-MomA was cloned between the NdeI and XhoI sites of pET26b, resulting in pET26b-MomA. Construction of pET26b-WhiE-ORFII—The whiE-ORFII gene was amplified by PCR with primer IV, 5′-GCGGAATTCCATATGACAGACCAGCAGGTACGCATCG-3′ (the italic letters indicate nucleotides to be changed; the underlined letters indicate an EcoRI site), and primer V, 5′-GCGAAGCTTGGATCCCATGACACCACCTCGGCCGCGGG-3′ (the italic letters indicate nucleotides to be changed; the underlined letters indicate a HindIII site). The template was the chromosomal DNA of S. coelicolor A3(2) M130. The sense primer IV was designed to change the ATG start codon of whiE-ORFII to an NdeI site, and the antisense primer V was designed to replace the stop codon with a BamHI site. The amplified product was cloned between the EcoRI and HindIII sites of pUC19, and the absence of undesired alterations was checked by nucleotide sequencing. The whiE-ORFII sequence was then excised as a NdeI-BamHI fragment and inserted between the NdeI and BamHI sites of pET-26b, resulting in pET26b-WhiE-ORFII. Isolation and Identification of Polyketides Produced by S. lividans Harboring pMF1—S. lividans TK21 harboring pMF1, pMF2, or pMF3 was inoculated to 2 liters of yeast extract malt extract liquid medium containing 5 μg/ml kanamycin and grown at 30 °C. After 17 h, 5 μg/ml of thiostrepton was added to induce the tipA promoter, and the culture was continued for further 24 h. The culture broth was adjusted to pH 1.0 with 6 m HCl, extracted with ethyl acetate, and then passed through a pad of Celite. The Celite was washed with ethyl acetate. The organic layers were combined, washed with brine, and dried with Na2SO4. After evaporation to dryness, the crude material was dissolved in methanol and passed through a Sep-Pak column (Waters) for removal of undesired aliphatic compounds. The sample was lyophilized and dissolved in Me2SO for reverse-phase high performance liquid chromatography (HPLC) analysis. Conditions of HPLC are as follows: ODS-80Ts column (Tosoh) was eluted with a linear gradient from 5 to 40% CH3CN in water (each containing 2% acetic acid) over 30 min and then 100% CH3CN within 10 min at a flow rate of 0.8 ml/min. UV absorbance was detected at 330 nm. Mompain in the crude sample was purified by reverse phase preparative HPLC (Docosil B, C22, 20 × 250 mm) by a linear gradient from 10 to 40% CH3CN in water (each containing 1% acetic acid) within 60 min at a flow rate of 5 ml/min. The collected fractions were lyophilized to give 27 mg of mompain as a purple solid. 1H NMR (500 MHz, CD3OD) δ 6.31 (s, 2H, ArH); 13C NMR (125 MHz, CD3OD) δ 105.1 (C-4a), 112.2 (C-3, C-6), 112.9 (C-8a), 159.1 (C-2, C-7), 168.4 (C-4, C-5), 174.7 (C-1, C-8); high resolution electrospray ionization-time-of-flight mass spectrum was m/z 221.00913 (calculated for C10H5O6, 0.52 mmu error). Production of Recombinant MomA and WhiE-ORFII in E. coli—For production of MomA with a His tag at its N terminus, E. coli BL21 (DE3) harboring pET16b-MomA was grown at 26 °C overnight in LB medium containing 100 μg/ml ampicillin, and the cells were collected by centrifugation. For expression of WhiE-ORFII with a His tag at its C terminus, E. coli BL21 (DE3) harboring pET26b-WhiE-ORFII was grown at 37 °C for 3 h in LB medium containing 25 μg/ml kanamycin, followed by induction of the T7 promoter with 1 mm isopropyl β-d-thiogalactopyranoside. After further growth at 37 °C for 2 h, the cells were collected by centrifugation. The cleared cell lysates containing MomA and WhiE-ORFII were prepared by sonication, and removal of cell debris by centrifugation at 10,000 × g was for 30 min. A nickel-nitrilotriacetic acid resin (Qiagen) was used for purification of MomA and WhiE-ORFII from the cleared cell lysates. Protein concentrations were measured with a Bio-Rad protein assay kit using bovine serum albumin as a standard. Gel Filtration and SDS-PAGE Analysis—SDS-PAGE was carried out using the buffer system of Laemmli in 15% polyacrylamide gel, and protein bands were visualized by staining with Coomassie Brilliant Blue R-250. The molecular sizes of recombinant MomA and WhiE-ORFII were determined by gel filtration on a PROTEIN KW-802.5 column (Shodex) that had been equilibrated with 10 mm sodium phosphate buffer (pH 7.0) containing 150 mm K2SO4. The molecular size standards used are as follows: albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen (25 kDa), and ribonuclease (13.7 kDa). Glutaraldehyde Cross-coupling Analysis—MomA or WhiE-ORFII (10 μg each) was mixed with glutaraldehyde (final concentration, 0.01–0.1%) in 50 mm NaH2PO4 (pH 8.0) in a total volume of 50 μl and incubated for 20 min at room temperature. The reactions were stopped by adding 4 μl of 2 m Tris-HCl (pH 8.0) and applied to SDS-PAGE. Prosthetic Group Investigation of MomA and WhiE-ORFII—For determination of the prosthetic group and metal ion, the standard reaction mixture contained THN (1 μm for MomA and 100 μm for WhiE-ORFII), 500 μm each of organic cofactors or 100 μm each of metal ions in 100 mm sodium phosphate (pH 7.5), and 0.59 μg of MomA or 56 μgof WhiE-ORFII in a total volume of 400 μl. After the reaction mixture had been preincubated at 30 °C for 5 min, the reaction was initiated by adding THN and incubated for 15 s for MomA and 1 min for WhiE-ORFII. Reactions were stopped by adding 80 μl of 6 m HCl, and the material in the mixture was extracted with 400 μl of ethyl acetate. The organic layer was evaporated, and the residual material was dissolved in 10 μl of dimethyl sulfoxide for HPLC analysis. Reverse phase HPLC conditions were as follows: ODS-80Ts (C18) column (4.6 × 150 mm; Tosoh), maintained at 40 °C, eluted with 25% CH3CN in H2O (each contained 2% acetic acid) with detection at 260 nm; flow rate, 1.0 ml/min. UV spectra were detected on a Waters 996 photodiode array detector. The amount of flaviolin was quantified with the standard curve established using authentic flaviolin, which was prepared by a large scale reaction of MomA using synthetic THN as the substrate. Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) Analysis—The metal contents of MomA and WhiE-ORFII were determined by using ICP-AES (Seiko). For preparation of metal ion-free enzymes, 1,10-phenanthroline (500 μm) or 8-quinolinol (500 μm) was added to MomA (5.5 nm in 10 mm Tris-HCl buffer (pH 8.0)) or WhiE-ORFII (24 nm in 10 mm Tris-HCl buffer (pH 8.0)). After 2 h of incubation, the residual chelators were removed by dialysis four times against 2 liters of 10 mm Tris-HCl buffer (pH 8.0). For preparation of metal ion-substituted MomAs, the 1,10-phenanthroline-treated MomAs (1.66 nm) were incubated with 125 μm of metal ions at 4 °C for 2 h, followed by desalting column chromatography (PD-10, Amersham Biosciences) for eliminating unbound metals. Binding of the metals was confirmed by analyzing ICP-AES for each sample. Determination of Kinetic Parameters of MomA and WhiE-ORFII— The reactions, containing 100 mm sodium phosphate (pH 7.5) and 0.59 μg of MomA or 56 μg of WhiE-ORFII, were performed in a total volume of 400 μl, except for the reaction of THN by MomA, which was performed with 0.29 μg of MomA in 200 μl. The concentrations of THN were 0.1–1.0 μm for MomA and 50–500 μm for WhiE-ORFII. The concentrations of EA and DHN were 10–100 μm. After the reaction mixture had been preincubated at 30 °C for 2–5 min (differed depending on the substrate), the reactions were initiated by adding the substrate and were continued for 15 s for THN, 1 min for DHN, and 5 min for EA for the MomA reactions, and 1 min for THN for the WhiE-ORFII reaction. The reactions were stopped with 40 or 80 μlof6 m HCl, and the material in the mixture was extracted with an equal volume of ethyl acetate. The organic layer was collected and evaporated. The residual material was dissolved in 10 μl of dimethyl sulfoxide for HPLC analysis. The products were separated and quantified by HPLC using the standard curve. Steady-state parameters were determined by the Hanes plot. Identification of the rppA-momA Operon in S. antibioticus—We reported previously (10Funa N. Ohnishi Y. Fujii I. Shibuya M. Ebizuka Y. Horinouchi S. Nature. 1999; 400: 897-899Crossref PubMed Scopus (236) Google Scholar) that rppA, a type III polyketide synthase catalyzing the synthesis of THN from malonyl-CoA, is responsible for melanin biosynthesis in S. griseus. Our preliminary data showed that a gene, named P-450mel, located just upstream of rppA was responsible for the biosynthesis of a dark brown, THN-derived melanin in this strain (Fig. 1A). On the other hand, introduction of a DNA fragment containing rppA and its downstream gene, later named momA, from S. antibioticus IFO13271 in S. lividans resulted in production of a diffusible red pigment. A similar red pigment was found in the culture broth of S. antibioticus IFO13271. The red pigment, which gave a UV spectrum similar to that of the chromophore of flaviolin (data not shown), was distinct from the THN-derived melanin. We therefore supposed that the THN produced by the action of RppA might be converted into melanin by the catalytic activity of P-450mel in S. griseus and into a flaviolin-like compound by the activity of MomA in S. antibioticus. We determined the nucleotide sequence of momA and deduced the amino acid sequence of MomA, as described below. In Saccharopolyspora erythraea, rppA is also required for the biosynthesis of a diffusible pigment (18Cortés J. Velasco J. Foster G. Blackaby A.P. Rudd B.A. Wilkinson B. Mol. Microbiol. 2002; 44: 1213-1224Crossref PubMed Scopus (57) Google Scholar). The gene organization in the downstream region of rppA in this strain revealed the presence of both P-450mel and momA homologues (Fig. 1A). A similar arrangement of the genes was also found in both S. coelicolor A3(2) (19Bently S.D. Chater K.F. Cerdeño-Tárraga A-M. Challis G.L. Thomson N.R. James K.D. Harris D.E. Quail M.A. Kieser H. Harper D. Bateman A. Brown S. Chandra G. Chen C.W. Collins M. Cronin A. Fraser A. Goble A. Hidalgo J. Hornsby T. Howarth S. Huang C.-H. Kieser T. Larke L. Murphy L. Oliver K. O'Neil S. Rabbinowitsch E. Rajandream M.-A. Rutherford K. Rutter S. Seeger K. Saunders D. Sharp S. Squares R. Squares S. Taylor K. Warren T. Wietzorrek A. Woodward J. Barrell B.G. Parkhill J. Hopwood D.A. Nature. 2002; 417: 141-147Crossref PubMed Scopus (2568) Google Scholar) and Streptomyces avermitilis (20Ômura S. Ikeda H. Ishikawa J. Hanamoto A. Takahashi C. Shinose M. Takahashi Y. Horikawa H. Nakazawa H. Osonoe T. Kikuchi H. Shiba T. Sakaki Y. Hattori M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12215-12220Crossref PubMed Scopus (675) Google Scholar), in which the complete genome sequences have been reported. A BLAST search predicted that the deduced amino acid sequence of momA from S. antibioticus shares overall similarity among the functionally uncharacterized gene products, SCO1208 in S. coelicolor A3(2) and SAV7129 in S. avermitilis (Fig. 1, A and C). The overall identity in the amino acid sequence of SCO1208, SAV7129, and ORF3 in S. erythraea to S. antibiotics MomA is 57, 58, and 58%, respectively. Another protein (SAV1496) of S. avermitilis was also found as a MomA homologue (43% identity), whereas SAV1496 is not located close to rppA. The gene organizations around momA in S. coelicolor A3(2) and S. avermitilis are the same; P-450mel and momA are present in this order downstream of rppA, as in S. erythraea, and three other genes, assigned as aldehyde dehydrogenase, unknown, and acyl-CoA dehydrogenase genes, are present in the same orientation. We therefore assumed that MomA and P-450mel were physiologically important for pigmentation and melanin synthesis in streptomycetes, although little was known about the functions of these RppA-associated proteins. Thus, we set out to characterize the catalytic properties of MomA in pigment biosynthesis in S. antibioticus, because no P-450mel homologues, which might hamper the characterization of the compound converted from THN by competitively converting it into melanin, were found near the rppA-momA operon. The nucleotide sequence of the 3.2-kb SalI-SphI DNA fragment from S. antibioticus IFO13271 was determined, and open reading frames were predicted by the FramePlot analysis (14Hopwood D.A. Bibb M.J. Chater K.F. Kieser T. Bruton C.J. Kieser H.M. Lydiate D.J. Smith C.P. Ward J.M. Schrempf H. Genetic Manipulation in Streptomyces: A Laboratory Manual. John Innes Foundation, Norwich, UK1985: 1-356Google Scholar) (Fig. 1, B and C). Within the fragment, three open reading frames, rppA, momA, and a gene for a σ factor of RNA polymerase, were encoded. Because the space between the termination codon of rppA and the initiation codon of momA was only 31 bp, these two genes appeared to be transcribed as a single transcriptional unit. The BLAST analysis of MomA toward the conserved domain data base predicted the presence of a barrel domain of the cupin superfamily (Fig. 1C). The characteristic conserved sequences of motif 1 and motif 2 of cupin proteins were designated as G(X)5HXH(X)3–4E(X)6G (X indicates any amino acid) and G(X)5PXG(X)2H(X)3N, respectively (21Dunwell J.M. Purvis A. Khuri S. Phytochemistry. 2004; 65: 7-17Crossref PubMed Scopus (398) Google Scholar). The two His residues and the Glu residue in motif 1, together with the His residue in motif 2, act as ligands for the active site metal ion (21Dunwell J.M. Purvis A. Khuri S. Phytochemistry. 2004; 65: 7-17Crossref PubMed Scopus (398) Google Scholar). These residues were all conserved among the MomA sequences in Fig. 1C, suggesting that MomA is a metalloprotein. Although the cupin-fold proteins are widely distributed in Archaea, Eubacteria, and Eukaryota, we could not predict the catalytic function from the results of homology searching. Expression of MomA and RppA in S. lividans—We constructed three plasmids, pMF1 carrying both rppA and momA, pMF2 carrying rppA alone, and pMF3 carrying momA alone (Fig. 1B), by using pIJ6021, and we introduced them by transformation into S. lividans TK21. The genes were all under the control of the thiostrepton-inducible tipA promoter in pIJ6021. After induction of the tipA promoter with thiostrepton, cell extracts were prepared and analyzed by HPLC. S. lividans harboring pMF1 produced two polyketides, neither of which was observed in the control extract prepared from the S. lividans harboring the vector (Fig. 2A). Further fermentation of S. lividans harboring pMF1 resulted in the accumulation of a polar product, which was identified as mompain by proton and carbon NMR spectra, with the aid of heteronuclear multiple bond correlation analysis and a high resolution electrospray ionization-time-of-flight mass spectrum. Mompain was not detected in the extract of S. lividans harboring pMF2. Moreover, whereas THN is an unstable compound
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