The Biosynthesis of the Aromatic Myxobacterial Electron Transport Inhibitor Stigmatellin Is Directed by a Novel Type of Modular Polyketide Synthase
2002; Elsevier BV; Volume: 277; Issue: 15 Linguagem: Inglês
10.1074/jbc.m111738200
ISSN1083-351X
AutoresNikolaos Gaitatzis, Barbara Silakowski, Brigitte Kunze, Gabriele Nordsiek, Helmut Blöcker, Gerhard Höfle, Rolf Müller,
Tópico(s)Genomics and Phylogenetic Studies
ResumoDeductions from the molecular analysis of the 65,000-bp stigmatellin biosynthetic gene cluster are reported. The biosynthetic genes (stiA–J) encode an unusual bacterial modular type I polyketide synthase (PKS) responsible for the formation of this aromatic electron transport inhibitor produced by the myxobacterium Stigmatella aurantiaca. Involvement of the PKS gene cluster in stigmatellin biosynthesis is shown using site-directed mutagenesis. One module of the PKS is assumed to be used iteratively during the biosynthetic process, which seems to involve an unusual transacylation of the biosynthetic intermediate from an acyl carrier protein domain back to the preceding ketosynthase domain. Finally, the polyketide chain which is presumably catalyzed by a novel C-terminal domain in StiJ that does not resemble thioesterases, is cyclized and aromatized. The presented results of feeding experiments are in good agreement with the proposed biosynthetic scheme. In contrast to all other PKS type I systems reported to date, each module of StiA–J is encoded on a separate gene. The gene cluster contains a "stand alone" O-methyltransferase and two unusual O-methyltransferase domains embedded in the PKS. In addition, inactivation of a cytochrome P450 monooxygenase-encoding gene involved in post-PKS hydroxylation of the aromatic ring leads to the formation of two novel stigmatellin derivatives. Deductions from the molecular analysis of the 65,000-bp stigmatellin biosynthetic gene cluster are reported. The biosynthetic genes (stiA–J) encode an unusual bacterial modular type I polyketide synthase (PKS) responsible for the formation of this aromatic electron transport inhibitor produced by the myxobacterium Stigmatella aurantiaca. Involvement of the PKS gene cluster in stigmatellin biosynthesis is shown using site-directed mutagenesis. One module of the PKS is assumed to be used iteratively during the biosynthetic process, which seems to involve an unusual transacylation of the biosynthetic intermediate from an acyl carrier protein domain back to the preceding ketosynthase domain. Finally, the polyketide chain which is presumably catalyzed by a novel C-terminal domain in StiJ that does not resemble thioesterases, is cyclized and aromatized. The presented results of feeding experiments are in good agreement with the proposed biosynthetic scheme. In contrast to all other PKS type I systems reported to date, each module of StiA–J is encoded on a separate gene. The gene cluster contains a "stand alone" O-methyltransferase and two unusual O-methyltransferase domains embedded in the PKS. In addition, inactivation of a cytochrome P450 monooxygenase-encoding gene involved in post-PKS hydroxylation of the aromatic ring leads to the formation of two novel stigmatellin derivatives. polyketide synthase acyl carrier protein acyltransferase base pairs cyclization Dalton β-hydroxy-acyl-thioester dehydratase β-ketoacyl-ACP reductase β-ketoacyl-ACP synthase O-methyltransferase PK, polyketide spacer region high pressure liquid chromatography Microbial polyketides are well known as a structurally diverse family of natural products, many of which are clinically valuable drugs. They are derived from short chain carboxylic acids such as malonate in sequential decarboxylative condensations, mechanistically resembling fatty acid biosynthesis. Bacteria produce a vast variety of such structures using different types of polyketide synthases (PKSs)1; type I systems utilize multifunctional enzymes organized in modules, which harbor several enzymatically active domains per chain extension step to finally generate non-aromatic structures (1.Staunton J. Wilkinson B. Meijere A. Houk K. Kessler H. Lehn J. Ley S. Schreiber S. Thiem J. Topics in Current Chemistry. 195. Springer-Verlag, Berlin1998: 50-89Google Scholar, 2.Cane D.E. Chem. Rev. 1997; 97: 2463-2706Crossref PubMed Google Scholar). The structural diversity that can be observed in the resultant polyketides (PKs) is afforded by the variation of these domains. After the last chain extension step and subsequent modifications, most PKs are released from the PKS by a thioesterase domain, usually resulting in the formation of a free carboxylic acid or a lactone structure. Type II PKSs use polypeptides with single activities iteratively, resulting in polyketo acids that are cyclized resulting in aromatic moieties (2.Cane D.E. Chem. Rev. 1997; 97: 2463-2706Crossref PubMed Google Scholar, 3.Shen B. Meijere A. Houk K. Kessler H. Lehn J. Ley S. Schreiber S. Thiem J. Topics in Current Chemistry. 209. Springer-Verlag, Berlin2000: 1-53Google Scholar). Recently, a series of genes similar to plant chalcone synthases has been detected in bacteria. These were introduced as bacterial type III PKS systems into the nomenclature (4.Moore B.S. Höpke J. ChemBioChem. 2001; 2: 35-38Crossref PubMed Scopus (99) Google Scholar). In contrast to bacterial systems, fungal type I PKSs use modules iteratively and are capable of generating both aromatic and non-aromatic molecules (2.Cane D.E. Chem. Rev. 1997; 97: 2463-2706Crossref PubMed Google Scholar,3.Shen B. Meijere A. Houk K. Kessler H. Lehn J. Ley S. Schreiber S. Thiem J. Topics in Current Chemistry. 209. Springer-Verlag, Berlin2000: 1-53Google Scholar). The antifungal natural product stigmatellin A (Fig. 1) inhibits the electron flow in the respiratory chain of beef heart submitochondrial particles within the cytochrome bc1 complex (5.Thierbach G. Kunze B. Reichenbach H. Höfle G. Biochim. Biophys. Acta. 1984; 765: 227-235Crossref Scopus (84) Google Scholar) and has been shown to be a powerful inhibitor of the photosynthetic electron transport (6.Oettmeier W. Godde D. Kunze B. Höfle G. Biochim. Biophys. Acta. 1985; 807: 216-219Crossref Scopus (77) Google Scholar). The compound has been used widely in studies dealing with the characterization of electron transport processes (e.g. Ref. 7.Matsuno-Yagi A. Hatefi Y. J. Biol. Chem. 2001; 276: 19006-19011Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Several types of stigmatellins are produced by different myxobacterial species, including Stigmatella aurantiaca (8.Kunze B. Kemmer T. Höfle G. Reichenbach H. J. Antibiot. (Tokyo). 1984; 37: 454-461Crossref PubMed Scopus (71) Google Scholar, 9.Höfle G. Kunze B. Zorzin C. Reichenbach H. Liebigs Ann. Chem. 1984; 8: 1883-1904Crossref Scopus (41) Google Scholar). This bacterium was developed as a model organism to study secondary metabolism in myxobacteria (10.Beyer S. Kunze B. Silakowski B. Müller R. Biochim. Biophys. Acta. 1999; 1445: 185-195Crossref PubMed Scopus (95) Google Scholar, 11.Silakowski B. Schairer H.U. Ehret H. Kunze B. Weinig S. Nordsiek G. Brandt P. Blöcker H. Höfle G. Beyer S. Müller R. J. Biol. Chem. 1999; 274: 37391-37399Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 12.Silakowski B. Kunze B. Müller R. Arch. Microbiol. 2000; 173: 403-411Crossref PubMed Scopus (29) Google Scholar, 13.Silakowski B. Kunze B. Nordsiek G. Blöcker H. Höfle G. Müller R. Eur. J. Biochem. 2000; 267: 6476-6485Crossref PubMed Scopus (95) Google Scholar, 14.Silakowski B. Nordsiek G. Kunze B. Blöcker H. Müller R. Chem. Biol. 2001; 8: 59-69Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 15.Gaitatzis N. Kunze B. Müller R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11136-11141Crossref PubMed Scopus (109) Google Scholar), because this class of microorganisms has been established as potent producers of natural products with biological activity over the last decades (16.Reichenbach H. Höfle G. Grabley S. Thieriecke R. Drug Discovery from Nature. Springer-Verlag, Berlin1999: 149-179Google Scholar, 17.Reichenbach H. Höfle G. Biotechnol. Adv. 1993; 11: 219-277Crossref PubMed Scopus (94) Google Scholar). Nevertheless, there still is only limited knowledge of the basis of secondary metabolism in these fascinating Gram-negative bacteria, because most studies on PKS systems have focused on actinomycetes or fungi (1.Staunton J. Wilkinson B. Meijere A. Houk K. Kessler H. Lehn J. Ley S. Schreiber S. Thiem J. Topics in Current Chemistry. 195. Springer-Verlag, Berlin1998: 50-89Google Scholar, 3.Shen B. Meijere A. Houk K. Kessler H. Lehn J. Ley S. Schreiber S. Thiem J. Topics in Current Chemistry. 209. Springer-Verlag, Berlin2000: 1-53Google Scholar). Given the fact that myxobacteria have the largest of all known bacterial chromosomes (18.Neumann B. Pospiech A. Schairer H.U. J. Bacteriol. 1992; 174: 6307-6310Crossref PubMed Google Scholar) and a very complex life cycle (19.Reichenbach H. Dworkin M. Kaiser D. Myxobacteria II. American Society for Microbiology, Washington, D. C.1993: 13-62Google Scholar), one can imagine secondary metabolites having diverse functions during morphological and physiological differentiation. A multitude of secondary metabolic gene sets has been located in the chromosome of S. aurantiaca, and structure-gene relationships have been established using gene inactivation studies (20.Silakowski B. Kunze B. Müller R. Gene (Amst.). 2001; 275: 233-240Crossref PubMed Scopus (65) Google Scholar). The focus of these studies has thus far been to elucidate novel mechanisms of secondary metabolite formation and to find new natural products. We report here a set of genes responsible for stigmatellin formation in S. aurantiaca. Surprisingly, a bacterial type I PKS is found to be responsible for the formation of an aromatic compound and seems to use one module iteratively. Several other novel features are deduced from the molecular analysis of the gene cluster and from feeding studies using labeled precursors. Novel stigmatellin derivatives (Fig. 1) are generated because of the inactivation of a P450 monoxygenase gene via site-directed mutagenesis. Escherichia coli strains and S. aurantiaca Sg a15 and its descendants were cultured as described previously (10.Beyer S. Kunze B. Silakowski B. Müller R. Biochim. Biophys. Acta. 1999; 1445: 185-195Crossref PubMed Scopus (95) Google Scholar, 14.Silakowski B. Nordsiek G. Kunze B. Blöcker H. Müller R. Chem. Biol. 2001; 8: 59-69Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The cultivation of the strains, the preparation of the culture extracts, and the conditions for the analysis of the spectrum of secondary metabolites using diode array-detected HPLC analysis were described previously (14.Silakowski B. Nordsiek G. Kunze B. Blöcker H. Müller R. Chem. Biol. 2001; 8: 59-69Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). A solvent gradient of 0.2% aqueous acetic acid (A)/acetonitrile (B) from 50% B at 0 min to 75% B at 20 min was used (detection was at 252 nm). To 500-ml cultures of S. aurantiaca Sg a15, 0.2 g of [1-13C]acetate or [1-13C]propionate was added at day 1 after inoculation. After 4 days of continued cultivation, the cell mass was harvested and extracted with acetone. The extract was evaporated, and the residue was extracted with ethyl acetate to give ∼200 mg of crude extract. This was separated by chromatography on silica gel (CH2Cl2/MeOH, 97:3) and reverse phase chromatography on a C-18 column (Macherey-Nagel, Germany) (acetonitrile, 30 mm ammonium acetate buffer, 65:35) to give 1.5–2.0 mg of 13C-labeled stigmatellins. Similarly, 1 g of [1,2-13C]acetate was fed in five portions to a 2-liter culture for 4 days. Isolation as described above yielded 11 mg of stigmatellin A. As judged from the13C-satellites in the 1H NMR spectra, feeding of acetate and propionate resulted in isotope enrichments of 20 and 30%, respectively. For production of secondary metabolites, the mutant was cultured in 5 liters of Zein liquid medium containing 0,8% zein, 0,1% peptone (from casein tryptically digested), 0.1% MgSO4·7H2O, 50 mmHepes buffer, pH 7.2, and 1% of the adsorber resin XAD16 (Rohm & Haas). Incubation was done at 30 °C on a gyratory shaker at 160 rpm for about 3 days. Cell mass and adsorber resin were collected and extracted twice with acetone. The extract was concentrated to the water phase and extracted with ethyl acetate. The organic extract was dried with Na2SO4 and evaporated to give 2.7 g of a dark oil. This was dissolved in dichloromethane and applied to a silica gel column (35 g). Elution with dichloromethane/methanol, 99:1, afforded crude stigmatellin X (0.25 g), and elution with dichloromethane/methanol, 96:4, resulted in crude stigmatellin Y (0.21 g). Repeated RP 18 chromatography (acetonitrile, 50 mmammonium acetate buffer, pH 5.5, 75:25) afforded pure stigmatellin X (18 mg) and stigmatellin Y (12 mg). DC (silica gel, dichloromethane/methanol, 95:5), RF = 0.5; UV (methanol), 258, 268, 279, and 320 nm; 1H NMR (CD3OD), δ 6.20 (d, J = 2.2 Hz, 6H), 6.28 (d, J = 2.2 Hz, 8H), 12.98 (s, 5OH); electrospray ionization-mass spectrometry (negative ions), m/z469 (M − H)−. DC RF = 0.36; UV (methanol), 238, 256, 269, 279, and 305 nm; 1H NMR (CD3OD), δ 6.36 (d, J = 2.0 Hz, 8H, 6.37 (d, J = 2.0 Hz, 6H), 3.90 (s, 5-OCH3);13C NMR (CD3OD), δ 163.6 (C-1a), 107.4 (C-4a), 161.9 (C-5), 96.8 (C-6), 160.5 (C-7), 95.7 (C-8); nuclear Overhauser effect, irradiation of 5-OCH3 at δ 3.90 enhanced only 6-H at δ 6.37, electrospray ionization-mass spectrometry (negative ions), m/z 483 (M − H)−. The antibiotic activity of stigmatellins against Saccharomyces cerevisiae cultured in glucose-free N3 medium (glycerol 2%, casein peptone 1%, yeast extract 1%, phosphate 50 mm, pH 6.3) was determined using the agar diffusion assay with paper discs (8.Kunze B. Kemmer T. Höfle G. Reichenbach H. J. Antibiot. (Tokyo). 1984; 37: 454-461Crossref PubMed Scopus (71) Google Scholar). The assay used to determine the inhibition of NADH oxidation in beef heart submitochondrial particles was described previously (5.Thierbach G. Kunze B. Reichenbach H. Höfle G. Biochim. Biophys. Acta. 1984; 765: 227-235Crossref Scopus (84) Google Scholar). In this study, a UNICAM UV-visible spectrometer UV2 was employed to record the data. Chromosomal DNA from S. aurantiaca was prepared as described (21.Neumann B. Pospiech A. Schairer H.U. Trends Genet. 1992; 8: 332-333Abstract Full Text PDF PubMed Scopus (105) Google Scholar). Southern analysis of genomic DNA was performed using the standard protocol for homologous probes of the Digoxigenin DNA labeling and detection kit (Roche Molecular Biochemicals). PCR was carried out using Taq polymerase (Invitrogen) according to the manufacturer's protocol. 5% Me2SO was added to the mixture. Conditions for amplification with the Eppendorf mastercycler gradient were as follows: denaturation 30 s at 95 °C, annealing 30 s at 60 °C, and extension 45 s at 72 °C; 30 cycles and a final extension at 72 °C for 10 min. Sequencing of cosmids CS4 and CS4a was performed by a shotgun approach as follows. Sheared fragments of the two cosmids were subcloned separately into pTZ18R (Amersham Biosciences). At least 500 clones were selected from each cosmid library, and plasmid DNA was prepared (Qiagen) and sequenced using Big Dye RR Terminator Cycle Sequencing kit (PE Biosystems) and UPO/RPO primer (MWG Biotec). The gels were run on ABI-377 sequencers, and data were assembled and edited using the XGAP program (50.Bonfied J.K. Smith K. Staden R. Nucleic Acids Res. 1995; 23: 4992-4999Crossref PubMed Scopus (790) Google Scholar). All other DNA manipulations were performed according to standard protocols (22.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning. A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Amino acid and DNA alignments were done using the programs of the Lasergene software package (DNAstar Inc.) and ClustalW (23.Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55767) Google Scholar). The preparation of the cosmid library from S. aurantiaca Sg a15 has been described (10.Beyer S. Kunze B. Silakowski B. Müller R. Biochim. Biophys. Acta. 1999; 1445: 185-195Crossref PubMed Scopus (95) Google Scholar). Approximately 2200 cosmid harboring single colonies were picked into 96-well microtiter plates, grown in LB medium overnight, and replicated. To one copy of the library, 25% glycerol was added, and the plates were frozen at −80 °C. The second copy was transferred onto nylon membranes and used for colony hybridizations. Cosmid CS4 harboring a KS fragment encoding part of the stigmatellin biosynthetic gene cluster has been reported (10.Beyer S. Kunze B. Silakowski B. Müller R. Biochim. Biophys. Acta. 1999; 1445: 185-195Crossref PubMed Scopus (95) Google Scholar). After sequencing of this cosmid, a probe homologous to the end of CS4 could be generated using primers C22 (5′-CGCAGCCGTGGCCCGAGAGTGG-3′) and C23 (5′-CCTGGGAAGACGACGAAAACAA-3′). This probe was employed in a colony hybridization against the complete library. One of the resulting cosmids (CS4a) was chosen for sequencing of the rest of the gene cluster. For characterization of stiJ and stiLinsertional mutants, S. aurantiaca Sg a15 NGS12 and NGS910 were constructed. Therefore, internal fragments of stiJ and stiL were amplified as follows: using primers NGS1 (5′-GCGCCGCTGCCCTCTCC-3′) and NGS2 (5′-GCTGCGTGTACTCGGTCTCGTCAA-3′) a 505-bp DNA fragment encoding part of stiJ was amplified and subsequently inserted into pCR2.1TOPO (according to the manufacturer's protocol, Invitrogen) resulting in plasmid pNGS12. To amplify a 470-bp DNA-fragment of stiL, the primers NGS9 (5′-GAAGCAGGCCTCCGAGATGATGAT-3′) and NGS10 (5′-CCGGCAGGGGGATGTAGACCA-3′) were employed in a PCR. Plasmid pNGS910 was obtained and harbors the 470-bp DNA fragment in pCR2.1TOPO. The plasmids were transferred into S. aurantiaca Sg a15 wild type by electroporation (10.Beyer S. Kunze B. Silakowski B. Müller R. Biochim. Biophys. Acta. 1999; 1445: 185-195Crossref PubMed Scopus (95) Google Scholar). Verification of the mutant strains was performed in Southern experiments (data not shown). Several genes located upstream of the stigmatellin biosynthetic gene cluster were inactivated via insertional mutagenesis, because they seemed to encode proteins that might be involved in stigmatellin biosynthesis (see below). Therefore, 500 bp of ORF8 (cytochrome P450 monooxygenase-like protein) (a), 580 bp of ORF7 (serine/threonine kinase-like protein) (b), 430 bp of a ORF6 (methyltransferase-like protein) (c), and 403 bp of ORF2 (similar to a protein with unknown function) (d) were amplified via PCR as described above using the following primers: (a) C5, 5′-GCACCGCTACGCCGCCAATCT-3′, and C6, 5′-GACTTCGCCGCCGCCATCC-3′; (b) C7, 5′-GGAGAAGCGCACCCCTATGT-3′, and C8, 5′-GGCCAATTCTCCCGCTCTTCCTCT-3′; (c) C29, 5′-GCTCGCACGCTCCTTGAAA-3′, and C30, 5′-CGGGCCGGTCATCCACTC-3′; (d) C31, 5′-GGTGCCGTCCCCATCCTC-3′, and C32, 5′-GAACCAGCCGCTCCCACTCC-3′. The amplification products (a–d) were inserted into pCR2.1TOPO creating the plasmids pCBS18 (a), pCBS19 (b), pCBS23 (c), and pCBS24 (d). These constructs were electroporated into S. aurantiaca Sg a15 as described and resulting mutants (CBS18 (a), CBS19 (b), CBS23 (c), and CBS24 (d)) were verified using Southern experiments (data not shown). All of these mutants were analyzed for stigmatellin production and found to be stigmatellin-positive. No differences in the amount of stigmatellin produced in comparison to wild type were obvious in these experiments. A cosmid library of S. aurantiaca Sg a15 was prepared. Subsequently, a variety of cosmids hybridizing to PKS probes were isolated and used to amplify specific KS fragments of the PKS genes encoded on the respective DNA locus. One of these fragments, amplified from cosmid CS4, was used for a gene inactivation experiment in S. aurantiaca Sg a15 and resulted in a stigmatellin-negative phenotype (10.Beyer S. Kunze B. Silakowski B. Müller R. Biochim. Biophys. Acta. 1999; 1445: 185-195Crossref PubMed Scopus (95) Google Scholar). During the course of this study, the sequence of cosmid CS4 was determined, revealing the presence of several open reading frames (ORFs) with similarity to bacterial type I PKSs, which were designated stiA–stiE (see Fig. 2). To verify the expected involvement of the PKS genes in stigmatellin biogenesis, the gene disruption mutant of the previous study was identified as a stiC insertion. Subsequently, an overlapping cosmid was isolated from the library (cosmid CS4a, see Fig. 2) as described under "Materials and Methods." The sequence of the insert was analyzed as described and found to overlap with stiD. Four further PKS genes oriented into the same direction as stiA–E were identified and designated stiF–J.Analysis of the modular structure of the stigmatellin megasynthetase revealed that stiJ most likely encodes the last module of the biosynthetic gene cluster (see below). Thus, stiJ was subjected to another gene inactivation experiment as described above, and the verified mutant NGS12 was found to be a stigmatellin non-producing strain (Fig. 3).Figure 3Phenotypic analysis of S. aurantiaca Sg a15 and mutants NGS12 and NGS910. HPLC diagrams of the secondary metabolite production spectra of the wild type strain S. aurantiaca Sg a15 and mutants NGS12 and NGS910 are shown. S-A, stigmatellin A; S-X, stigmatellin X; S-Y, stigmatellin Y; A-A, aurachin A; A-C, aurachin C.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The codon bias (see Table I) of the genes reported is in accordance with other genes from myxobacteria (24.Shimkets L. Dworkin M. Kaiser D. Myxobacteria II. American Society for Microbiology, Washington, D. C.1993: 85-108Google Scholar). The overall G + C content of the sequenced sti region spanning ∼65 kbp is 66%.Table IDeduced functions of genes involved in stigmatellin biosynthesisGeneSize in bp, codon usage (% GC in 1st, 2nd, and 3rd position), size of deduced proteinDomains (location in the protein sequence)stiA7122 bp (70:50:75) 254,457 DaACPL-(0–84), KS-(85–508), ATL-(608–926), AT-(1049–1367), DH-(1378–1598), KR-(1962–2214), ACP-(2225–2327)stiB4758 bp (70:51:76) 170,213 DaKS-(31–456), AT-(565–880), KR-(1172–1427), ACP-(1436–1544)stiC5659 bp (69:51:75) 202,391 DaKS-(17–443), AT-(559–977), DH-(888–1109), KR-(1474–1722), ACP-(1740–1841)stiD5805 bp (69:49:76) 208,622 DaKS-(39–471), AT-(586–904),O-MT-(988–1263), KR-(1520–1778), ACP-(1793–1893)stiE5814 bp (68:49:79) 210,282 DaKS-(34–460), AT-(568–886), O-MT-(978–1252), KR-(1522–1769), ACP-(1785–1885)stiF6657 bp (68:51:78) 237,041 DaKS-(28–452), AT-(560–875), DH-(886–1104), ER1-aER, enoyl reductase.-(1485–1789), KR-(1813–2066), ACP-(2077–2177)stiG4197 bp (71:48:77) 150,204 DaKS-(37–463), AT-(580–898), DH-(908–1125), ACP-(1258–1358)stiH4815 bp (71:50:78) 172,349 DaKS-(37–462), AT-(572–891), KR-(1199–1445), ACP-(1466–1566)stiJ3780 bp (67:49:84) 136,343 DaKS-(34–460), AT-(566–885), ACP-(948–1048), CY-(1037–1259)stiK771 bp (58:40:84) 28,528 DaSimilar to methyltransferases (see text)stiL1533 bp (66:50:81) 55,958 DaSimilar to P450- monooxygenases (see text)1-a ER, enoyl reductase. Open table in a new tab The modular organization of PKSs involves activation and condensation of the following carboxylic acid onto the growing chain, catalyzed by an acyltransferase (AT) domain and a KS domain. The resulting β-keto acid may subsequently be processed by β-ketoacyl reductase (KR) domains, β-hydroxyacyl dehydratase (DH) domains, and enoyl reductase domains (reviewed in Ref. 2.Cane D.E. Chem. Rev. 1997; 97: 2463-2706Crossref PubMed Google Scholar). Sequence motifs typical for these domains in PKSs (25.Schwecke T. Aparicio J.F. Molnar I. König A. Khaw L.E. Haydock S.F. Oliynyke M. Caffrey P. Cortes J. Lester J.B. Bohm G.A. Staunton J. Leadlay P.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7839-7843Crossref PubMed Scopus (393) Google Scholar, 26.Donadino S. Staver M.J. McAlpine J.B. Swanson S.J. Katz L. Science. 1991; 252: 675-679Crossref PubMed Scopus (736) Google Scholar) were detected in StiA–StiJ as shown in Table I and Fig. 4. The acyl carrier protein (ACP) domains of the proteins contain the Prosite consensus signature of the putative binding site for the 4′-phosphopantetheine cofactor (Prosite signature numbers PS00012, R2082, and L2104). Additional domains within PKSs responsible for C-methylation (27.Gehring A.M. DeMoll E. Fetherston J.D. Mori I. Mayhew G.F. Blattner F.R. Walsh C.T. Perry R.D. Chem. Biol. 1998; 5: 573-586Abstract Full Text PDF PubMed Scopus (200) Google Scholar, 28.Kennedy J. Auclair K. Kendrew S.G. Park C. Vederas J.C. Hutchinson C.R. Science. 1999; 284: 1368-1372Crossref PubMed Scopus (533) Google Scholar) and O-methylation (11.Silakowski B. Schairer H.U. Ehret H. Kunze B. Weinig S. Nordsiek G. Brandt P. Blöcker H. Höfle G. Beyer S. Müller R. J. Biol. Chem. 1999; 274: 37391-37399Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar) of intermediates have recently been reported. The myxothiazol biosynthetic proteins MtaF and MtaG harbor such O-MT domains of different presumed activities; the O-MT domain of MtaE is responsible for the methylation of a hydroxyl, whereas the O-MT domain of MtaF is necessary for the methylation of the enol form of a keto group. Two O-MT domains were detected in StiD and StiE. They are very similar to each other (42% identity) and to the O-MT domain of MtaE (50 and 48% identity, respectively), whereas the O-MT domains of MtaF only show identities of 35, 29, and 36% to those of MtaE, StiD, and StiE, respectively. The C terminus of StiJ is 123 amino acids in size, located after the ACP of the protein, and does not show homology to any protein from the data bases. Large spacer regions (S) ∼300 amino acids in size are located between the AT and the KR domains of each module except for StiJ. These have been described for almost all PKS and fatty acid biosynthetic systems (2.Cane D.E. Chem. Rev. 1997; 97: 2463-2706Crossref PubMed Google Scholar, 29.Smith S. FASEB J. 1994; 8: 1248-1259Crossref PubMed Scopus (519) Google Scholar), but their function is not clear. These S regions can also be found in all myxobacterial PKS systems sequenced so far and show an identity of up to 35% on the amino acid level. Highly specific incorporation of label indicates that the carbon backbone and the C-methyl groups of stigmatellin are solely derived from acetate (after conversion to malonate) and propionate (after conversion to methylmalonate) (Table II and see Fig. 5). The C-2 of acetate is incorporated into propionate units at a significantly lower level, presumably caused by prior passing the citric acid cycle as described for other polyketides (30.Rawlings B.J. Nat. Prod. Rep. 2001; 18: 231-281Crossref PubMed Scopus (90) Google Scholar). Most significantly, doubly labeled acetate is incorporated in two different orientations into the phenyl ring. Due to different coupling constants to neighboring carbons, C-1a, C-4a, and C-7 show two sets of satellites of equal intensity (compare Refs. 31.Stoessl A. Stothers J. Can. J. Bot. 1978; 56: 2589-2593Crossref Google Scholarand 32.Birch A. Simpson T. Westerman P. Tetrahedron. 1975; 47: 4173-4177Crossref Scopus (13) Google Scholar, for example). This affords a free rotation of the acyl phloroglucinol intermediate, which in the biosynthetic sequence is first cyclized to a chromone and later hydroxylated at the 8-position (compare Fig. 5).Table II13C NMR spectral data of stigmatellin A in CD3OD at 100 MHz obtained from feeding of sodium [1-13C]propionate and sodium [1-13C]-, [2-13C]-, and [1,2-13C]acetate to S. aurantiaca Sg a15C atomδ[1-13C]Propionate[1-13C]Acetate[2-13C]Acetate[1,2-13C]AcetateCenter lineSatelite linesJC,CppmHz1a148.3—2-a—, Signals were very small or not detected.2210.31.664.9, 81.52165.4——1.51.65.351.03117.3———0.40.2—4179.6610.50.3—4°108.7——50.41.465.1, 71.75154.0—4.011.04.071.5694.2——112.710.570.47152.6—32—1.02.469.2, 78.88129.1——70.91.881.599.9——21.10.847.71′30.6——1.54.519.050.92′28.514161.53.21.734.93′35.6——1.51.80.8364′88.8132624.01.438.85′43.0——1.51.50.6396′82.7—12523.814.548.87′132.5——263.813.548.68′134.6—11014.716.056.69′126.3——263.011.856.710′138.9121512.00.8—11′135.9———1.01.0—12′128.4—7923.613.844.113′13.9——222.713.044.214′18.3—103.51.81.735.815′10.7——3.51.30.935.416′12.0—520.90.444.55-OMe56.8——11.0——7-OMe57.0——11.0——4′-OMe61.6——11.0——6′-Ome56.5——11.0——2-a —, Signals were very small or not detected. Open table in a new tab Upstream of stiA a gene encoding a putative cellulase (ORF1 which shows 31% identity and 47% homology to cellulase P23548 from Paenibacillus polymyxa) can be found. Further upstream, genes encoding ORF2–8 were detected. ORF2 is similar to a hypothetical 43.2-kDa protein from Streptomyces coelicolor(GenBankTM accession number AL049707, 37.9% identity on the amino acid level). ORF3 represents a putative ribosome binding factor with 30% identity to the ribosome binding factor A of S. aurantiaca (GenBankTM accession number CAB91142), whereas ORF4 is similar to several eucaryotic potassium channel β-chains, e.g. KB1 from Solanum tuberosum(GenBankTM accession number AJ000999, 41% identity). ORF5 is similar to eucaryotic acyl-CoA-binding proteins,e.g. from Rattus norvegicus(GenBankTM accession number X96560, 53% identity). ORF6 and ORF7 represent proteins with homology to methyltransferases and protein kinases, respectively. ORF6 is most similar to the probable methyltransferase MLL3908 from Rhizobium loti(GenBankTM accession number MLL3908, 64.9% identity), whereas ORF7 shows highest similarity to a protein kinase-like protein from S. coelicolor (GenBankTM accession numberQ53839, 32.9% identity). ORF8 is similar to a variety of cytochrome P450-dependent enzymes, e.g.GenBankTM access
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