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(Last modification: 10. April 2010)

 

Typ III Polyketidsynthasen (PKS) in Bakterien

 

Komplexe Ring-Faltung: 1,3,6,8-Tetrahydroxynaphthalen (T4HN)

 

a) Streptomyceten: Streptomyces griseus, Saccharopolyospora erythrea, Streptomyces  coelicolor

b) weiter unten besprochen: Myxobacteria: Eine 'Orphan PKS' aus Sorangium cellulosum ?

 

    - Abkürzung für das Protein:  RppA

 

Wichtige Publikationen:

  • Funa, N., Ohnishi, Y., Fujii, I., Shibuya, M., Ebizuka, Y. and Horinouchi, S.: A new pathway for polyketide synthesis in microorganisms. Nature 400, 897-899 (1999).       

  • Funa, N., Ohnishi, Y., Ebizuka, Y., Horinouchi, S.: Properties and substrate specificity of RppA, a chalcone synthase-related polyketide synthase in Streptomyces griseus. Journal of Biological Chemistry 277, 4628-4635 (2002).    

  • Funa, N., Ohnishi, Y., Ebizuka, Y., Horinouchi, S.: Alteration of reaction and substrate specificity of a bacterial type III polyketide synthase by site-directed mutagenesis. Biochemical Journal 367, 781-789 (2002).    

  • Cortés, J., Velasco, J., Foster, G., Blackaby, A. P., Rudd, B. A. M., Wilkinson, B., 2002. Identification and cloning of a type III polyketide synthase required for diffusible pigment biosynthesis in Saccharopolyspora erythraea. Molecular Microbiology 44, 1213-1224.

  • Izumikawa, M., Shipley, P. R., Hopke, J. N., O'Hare, T., Xiang, L., Noel, J. P., Moore, B. S., 2003. Expression and characterization of the type III polyketide synthase 1,3,6,8-tetrahydroxynaphthalene synthase from Streptomyces coelicolor A3(2). Journal of Industrial Microbiology and Biotechnology 30, 510-515.

 

Die Prototyp Reaktion

 

 

Was ist interessant und ungewöhnlich an dieser Reaktion, im Vergleich mit den Pflanzen-Enzymen?

  • Sie beginnt mit Malonyl-CoA 

  • Vier Kondensationen: Das war ungewöhnlich, wenigstens im Vergleich mit den Pflanzen-Enzymen, die zu der Zeit bekannt waren: Sie konnten nur bis drei zählen.
    Allerdings: Inzwischen gibt es seit 2004 mehrere hochinteressante Ausnahmen !!!

  • Die erste Ringfaltung ist vom STS-Typ (Aldolkondensation); die zweite entspricht der in CHS (Claisen-Kondensation),

  • Eine der terminalen Carboxyl-Gruppen wird abgespalten. Wichtig: Es ist die vom Starter Malonyl-Molekül, nicht die von der letzten Kondensationsreaktion. Das gleiche findet sich bei der Biosynthese von Phenylglycin durch eine Typ III PKS in Streptomyceten (mehr...), und in der Bildung von Phloroglucinol in Pseudomonas fluorescens (mehr...).  

Eine interessante Frage ist, ob dieses Enzym wohl den "aldol switch mechanism" verwendet, der mit der Stilbensynthase (STS) aus der Kiefer (Pinus sylvestris) entdeckt wurde: Mehr...

 

And es gibt noch andere interessante Eigenschaften von RppA:

  • RppA akzeptierte aliphatische Acyl-CoAs mit Längen von C4 bis C8 als Starter, und führte damit mehrfache Kondensationen durch, die zu Alpha-Pyronen und Phloroglucinolen (also CHS-Typ Ringschluss!) führten

  • Ausserdem gab es mit Octanoyl-CoA als Starter erstaunliche Resultate: Das Enzym führte fünf Kondensationen mit Malonyl-CoA durch! Dies wurde so interpretiert, dass die aktive Tasche von RppA grösser ist als in allen anderen Typ III PKS, die soweit aus Pflanzen und Bakterien bekannt waren (damals: Inzwischen gibt es bakterielle mit noch grösseren Substratbindungs-Taschen!)

  • Und auch das konnte RppA: Es benutzte Acetoacetyl-CoA und Methylmalonyl-CoA, und bildete ein C-methyliertes Pyron daraus: 3,6-Dimethyl-4-hydroxy-2-pyron

Warum ist dies potentiell interessant?

  • Es gibt RppA verwandte Proteine in anderen Bakterien, bei denen nicht so klar ist, was die physiologische Rolle ist. Die breite Substrat-Akzeptanz des Prototyps könnte bedeuten, dass die in vivo Funktion verwandter Proteine ganz anders sein könnte.  Möglicherweise sind sie an der Biosynthese von Substanzen beteiligt, die man noch gar nicht kennt?

2004: Die Kristall-Struktur von RppA liefert interessante neue Einsichten, z.B. eine plausible Erklärung für die Akzeptanz so vieler verschiedener Substrate

  • Austin, M. B., Izumikawa, M., Bowman, M. E., Udwary, D. W., Ferrer, J. L., Moore, B. S., Noel, J. P.: Crystal structure of a bacterial type III polyketide synthase and enzymatic control of reactive polyketide intermediates. Journal of Biological Chemistry 279, 45162-45174 (2004).

Noch ein interessanter Aspekt dieser Reaktion: Das gleiche komplexe Produkt kann durch ganz andere Enzyme auch gebildet werden, z.B. durch eine riesige "iterative Polyketidsynthase" aus einem Pilz: Mehr...

  • Fujii, I., Mori, Y., Watanabe, A., Kubo, Y., Tsuji, G. and Ebizuka, Y.:  Enzymatic synthesis of 1,3,6,8-tetrahydroxynaphthalene solely from malonyl coenzyme A by a fungal iterative type I polyketide synthase PKS1. Biochemistry 39, 8853-8858 (2000). 

Und es wird noch merkwürdiger:
Das Pentaketid 1,3,6,8-Tetrahydroxynaphthalen (T4HN), also das gleiche Produkt, kann in Pilzen auch noch anders gebildet werden: Durch eine Polyketidsynthase, die ein Heptaketid bildet (also sieben Kondensationen!), welches dann durch hydrolytische Abspaltung von Acetoacetat verkürzt wird, und damit T4HN  produziert!

  • Fujii, I., Yasuoka, Y., Tsai, H. F., Chang, Y. C., Kwon-Chung, K. J., Ebizuka, Y.: Hydrolytic polyketide shortening by ayg1p, a novel enzyme involved in fungal melanin biosynthesis. Journal of Biological Chemistry 279, 44613-44620 (2004)

  • Tsai, H. F., Fujii, I., Watanabe, A., Wheeler, A. H., Chang, Y. C., Yasuoka, Y., Ebizuka, Y., Kwon-Chung, K. J.: Pentaketide melanin biosynthesis in Aspergillus fumigatus requires chain-length shortening of a heptaketide precursor. Journal of Biological Chemistry 276, 29292-29298 (2001).

  • Watanabe, A., Fujii, I., Tsai, H. F., Chang, Y. C., Kwon-Chung, K. J., Ebizuka, Y.: Aspergillus fumigatus alb1 encodes naphthopyrone synthase when expressed in Aspergillus oryzae. FEMS Microbiology Letters 192, 39-44 (2000).

 

 

Mechanismus der Kettenverkürzung

 

Zum Seitenanfang

 

 

b) Myxobacteria: Eine 'Orphan PKS' aus einem Myxobacterium: Sorangium cellulosum

   Schlüsselpublikation: Gross et al. (2006)
 

   Die Typ III PKS SoceCHS1 ist sicher einer der ein bisschen rätselhaften Fälle. Sie wurde identifiziert bei Absuche des Genoms, zusammen mit etlichen anderen. Expression eines rekombinanten Proteins in E. coli schlug fehl, aber offenbar hatte ein Pseudomonas Stamm kein Problem damit, und es war nicht einmal notwendig, ein rekombinantes Protein zu zu isolieren: Die Kulturen wurden nach der Induktion des Proteins rot! Die Analyse des Pigmentes zeigte dann, dass es sich um 2,5,7-Trihydroxy-1,4-Naphthochinon handelte, das Autooxidationsprodukt von 1,3,6,8-Tetrahydroxynaphthalen. Sorangium cellulosum, wenigstens unter Laborbedingungen, exprimiert das Gen gar nicht, es gibt keine Beschreibung dieses Naturstoffes in diesen Bakterien, und ein 'Knock-out' des Gens hatte keinerlei detektierbare Effekte auf die Morphologie, Pigmentierung, oder andere bekannte Sekundärstoffwechselprodukte in diesen Bakterien. Eine typische 'Orphan PKS'?

    Trotzdem ist folgendes eine Überlegung wert: Das Prototyp Enzym RppA aus Streptomyceten hat einige ziemlich unerwartete Eigenschaften; es akzeptiert nicht nur Malonyl-CoA, sondern auch eine ganze Reihe anderer Substrate, und es synthetisiert ein breites Spektrum an  Pyronen und Phloroglucinolen (siehe oben). Ist es möglich, dass diese Myxobakterien eine oder mehrere Substanzen synthetisieren, die entweder noch gar nicht bekannt, oder nicht als mögliche Typ III  PKS Produkte erkannt wurden? Das kann noch spannend werden.

 

Links zu Beispielen von 'Orphan PKS'

Zum Seitenanfang

 

Links zu bakteriellen Typ III PKS

Zum Seitenanfang

 

Zitate

  • Funa, N., Ohnishi, Y., Fujii, I., Shibuya, M., Ebizuka, Y. and Horinouchi, S.: A new pathway for polyketide synthesis in microorganisms. Nature 400, 897-899 (1999).
                 Chalcone synthases, which biosynthesize chalcones (the starting materials for many flavonoids), have been believed to be specific to plants. However, the rppA gene from the Gram-positive, soil-living filamentous bacterium Streptomyces griseus encodes a 372-amino-acid protein that shows significant similarity to chalcone synthases. Several rppA-like genes are known, but their functions and catalytic properties have not been described. Here we show that a homodimer of RppA catalyses polyketide synthesis: it selects malonyl-coenzyme-A as the starter, carries out four successive extensions and releases the resulting pentaketide to cyclize to 1,3,6,8-tetrahydroxynaphthalene (T4HN). Site-directed mutagenesis revealed that, as in other chalcone synthases, a cysteine residue is essential for enzyme activity. Disruption of the chromosomal rppA gene in S. griseus abolished melanin production in hyphae, resulting in 'albino' mycelium. T4HN was readily oxidized to form 2,5,7-trihydroxy-1,4-naphthoquinone (flaviolin), which then randomly polymerized to form various coloured compounds. T4HN formed by RppA appears to be an intermediate in the biosynthetic pathways for not only melanins but also various secondary metabolites containing a naphthoquinone ring. Therefore, RppA is a chalcone-synthase- related synthase that synthesizes polyketides and is found in the Streptomyces and other bacteria.
    Protein accession number: BAA33495. Note: this entry replaced in 1999 the original entry which apparently contained some sequencing errors.
    Zum Seitenanfang

  • Funa, N., Ohnishi, Y., Ebizuka, Y., Horinouchi, S.: Properties and substrate specificity of RppA, a chalcone synthase-related polyketide synthase in Streptomyces griseus. Journal of Biological Chemistry 277, 4628-4635 (2002).
             RppA, a chalcone synthase-related polyketide synthase (type III polyketide synthase) in the bacterium Streptomyces griseus, catalyzes the formation of 1,3,6,8-tetrahydroxynaphthalene (T4HN) from five molecules of malonyl-CoA. The Km value for malonyl-CoA and the kcat value for T4HN synthesis were determined to be 0.93 +/- 0.1 µM and 0.77 +/- 0.04 min-1, respectively. RppA accepted aliphatic acyl-CoAs with the carbon lengths from C4 to C8 as starter substrates and catalyzed sequential condensation of malonyl-CoA to yield alpha-pyrones and phloroglucinols. In addition, RppA yielded a hexaketide, 4-hydroxy-6-(2',4',6'-trioxotridecyl)-2-pyrone, from octanoyl-CoA and five molecules of malonyl-CoA, suggesting that the size of the active site cavity of RppA is larger than any other chalcone synthase-related enzymes found so far in plants and bacteria. RppA was also found to synthesize a C-methylated pyrone, 3,6-dimethyl-4-hydroxy-2-pyrone, by using acetoacetyl-CoA as the starter and methylmalonyl-CoA as an extender. Thus, the broad substrate specificity of RppA yields a wide variety of products.
    Zum Seitenanfang

  • Funa, N., Ohnishi, Y., Ebizuka, Y., Horinouchi, S.: Alteration of reaction and substrate specificity of a bacterial type III polyketide synthase by site-directed mutagenesis. Biochemical Journal 367, 781-789 (2002).
              RppA, which belongs to the type III polyketide synthase family, catalyses the synthesis of 1,3,6,8-tetrahydroxynaphthalene (T4HN), which is the key intermediate of melanin biosynthesis in the bacterium Streptomyces griseus. The reaction of T4HN synthesis catalysed by RppA is unique in the type III polyketide synthase family, in that it selects malonyl-CoA as a starter substrate. The Cys-His-Asn catalytic triad is also present in RppA, as in plant chalcone synthases, as revealed by analyses of active-site mutants having amino acid replacements at Cys(138), His(270) and Asn(303) of RppA. Site-directed mutagenesis of the amino acid residues that are likely to form the active-site cavity revealed that the aromatic ring of Tyr(224) is essential for RppA to select malonyl-CoA as a starter substrate, since substitution of Tyr(224) by amino acids other than Phe and Trp abolished the ability of RppA to accept malonyl-CoA as a starter, whereas the mutant enzymes Y224F and Y224W were capable of synthesizing T4HN via the malonyl-CoA-primed reaction. Of the site-directed mutants generated, A305I was found to produce only a triketide pyrone from hexanoyl-CoA as starter substrate, although wild-type RppA synthesizes tetraketide and triketide pyrones in the hexanoyl-CoA-primed reaction. The kinetic parameters of Ala(305) mutants and identification of their products showed that the substitution of Ala(305) by bulky amino acid residues restricted the number of elongations of the growing polyketide chain. Both Tyr(224) (important for starter substrate selection) and Ala(305) (important for intermediate elongation) were found to be conserved in three other RppAs from Streptomyces antibioticus and Streptomyces lividans.
    Zum Seitenanfang

  • Gross, F., Luniak, N., Perlova, O., Gaitatzis, N., Jenke-Kodama, H., Gerth, K., Gottschalk, D., Dittmann, E., Müller, R., 2006. Bacterial type III polyketide synthases: phylogenetic analysis and potential for the production of novel secondary metabolites by heterologous expression in pseudomonads. Archives of Microbiology 185, 28-38.
         Type III polyketide synthases (PKS) were regarded as typical for plant secondary metabolism before they were found in microorganisms recently. Due to microbial genome sequencing efforts, more and more type III PKS are found, most of which of unknown function. In this manuscript, we report a comprehensive analysis of the phylogeny of bacterial type III PKS and report the expression of a type III PKS from the myxobacterium Sorangium cellulosum in pseudomonads. There is no precedent of a secondary metabolite that might be biosynthetically correlated to a type III PKS from any myxobacterium. Additionally, an inactivation mutant of the S. cellulosum gene shows no physiological difference compared to the wild-type strain which is why these type III PKS are assumed to be "silent" under the laboratory conditions administered. One type III PKS (SoceCHS1) was expressed in different Pseudomonas sp. after the heterologous expression in Escherichia coli failed. Cultures of recombinant Pseudomonas sp. harbouring SoceCHS1 turned red upon incubation and the diffusible pigment formed was identified as 2,5,7-trihydroxy-1,4-naphthoquinone, the autooxidation product of 1,3,6,8-tetrahydroxynaphthalene. The successful heterologous production of a secondary metabolite using a gene not expressed under administered laboratory conditions provides evidence for the usefulness of our approach to activate such secondary metabolite genes for the production of novel metabolites.
    Accession of the protein ('SoceCHS1'):     AAZ93633
     Zurück zum Text

  • Austin, M. B., Izumikawa, M., Bowman, M. E., Udwary, D. W., Ferrer, J. L., Moore, B. S., Noel, J. P.: Crystal structure of a bacterial type III polyketide synthase and enzymatic control of reactive polyketide intermediates. Journal of Biological Chemistry 279, 45162-45174 (2004).
    In bacteria, a structurally simple type III polyketide synthase (PKS) known as 1,3,6,8-tetrahydroxynaphthlene synthase (THNS) catalyzes the iterative condensation of five CoA-linked malonyl units to form a pentaketide intermediate. THNS subsequently catalyzes dual intramolecular Claisen and aldol condensations of this linear intermediate to produce the fused ring tetrahydroxynaphthalene (T4HN) skeleton. The type III PKS-catalyzed polyketide extension mechanism, utilizing a conserved Cys-His-Asn catalytic triad in an internal active site cavity, is fairly well understood. However, the mechanistic basis for the unusual production of T4HN and dual cyclization of its malonyl-primed pentaketide is obscure. Here we present the first bacterial type III PKS crystal structure, that of Streptomyces coelicolor THNS, and identify by mutagenesis, structural modeling, and chemical analysis the unexpected catalytic participation of an additional THNS-conserved cysteine residue in facilitating malonyl-primed polyketide extension beyond the triketide stage. The resulting new mechanistic model, involving the use of additional cysteines to alter and steer polyketide reactivity, may generally apply to other PKS reaction mechanisms, including those catalyzed by iterative type I and II PKS enzymes. Our crystal structure also reveals an unanticipated novel cavity extending into the "floor" of the traditional active site cavity, providing the first plausible structural and mechanistic explanation for yet another unusual THNS catalytic activity: its previously inexplicable extra polyketide extension step when primed with a long acyl starter. This tunnel allows for selective expansion of available active site cavity volume by sequestration of aliphatic starter-derived polyketide tails, and further suggests another distinct protection mechanism involving maintenance of a linear polyketide conformation.
    Zurück zum Text

  • Fujii, I., Yasuoka, Y., Tsai, H. F., Chang, Y. C., Kwon-Chung, K. J., Ebizuka, Y.: Hydrolytic polyketide shortening by ayg1p, a novel enzyme involved in fungal melanin biosynthesis. Journal of Biological Chemistry 279, 44613-44620 (2004)
            The pentaketide 1,3,6,8-tetrahydroxynaphthalene (T4HN) is a key precursor of 1,8-dihydroxynaphthalene-melanin, an important virulence factor in pathogenic fungi, where T4HN is believed to be the direct product of pentaketide synthases. We showed recently the involvement of a novel protein, Ayg1p, in the formation of T4HN from the heptaketide precursor YWA1 in Aspergillus fumigatus. To investigate the mechanism of its enzymatic function, Ayg1p was purified from an Aspergillus oryzae strain that overexpressed the ayg1 gene. The Ayg1p converted the naphthopyrone YWA1 to T4HN with a release of the acetoacetic acid. Although Ayg1p does not show significant homology with known enzymes, a serine protease-type hydrolytic motif is present in its sequence, and serine-specific inhibitors strongly inhibited the activity. To identify its catalytic residues, site-directed Ayg1p mutants were expressed in Escherichia coli, and their enzyme activities were examined. The single substitution mutations S257A, D352A, and H380A resulted in a complete loss of enzyme activity in Ayg1p. These results indicated that the catalytic triad Asp352-His380-Ser257 constituted the active-site of Ayg1p. From a Dixon plot analysis, 2-acetyl-1,3,6,8-tetrahydroxynaphthalene was found to be a strong mixed-type inhibitor, suggesting the involvement of an acyl-enzyme intermediate. These studies support the mechanism in which the Ser257 at the active site functions as a nucleophile to attack the YWA1 side-chain 1'-carbonyl and cleave the carbon-carbon bond between the naphthalene ring and the side chain. Acetoacetic acid is subsequently released from the Ser257-O-acetoacetylated Ayg1p by hydrolysis. An enzyme with activity similar to Ayg1p in melanin biosynthesis has not been reported in any other organism.
    Zurück zum Text

  • Tsai, H. F., Fujii, I., Watanabe, A., Wheeler, A. H., Chang, Y. C., Yasuoka, Y., Ebizuka, Y., Kwon-Chung, K. J.: Pentaketide melanin biosynthesis in Aspergillus fumigatus requires chain-length shortening of a heptaketide precursor. Journal of Biological Chemistry 276, 29292-29298 (2001).
         Chain lengths and cyclization patterns of microbial polyketides are generally determined by polyketide synthases alone. Fungal polyketide melanins are often derived from a pentaketide 1,8-dihydroxynaphthalene, and pentaketide synthases are used for synthesis of the upstream pentaketide precursor, 1,3,6,8-tetrahydroxynaphthalene (1,3,6,8-T4HN). However, Aspergillus fumigatus, a human fungal pathogen, uses a heptaketide synthase (Alb1p) to synthesize its conidial pigment through a pentaketide pathway similar to that which produces 1,8-dihydroxynaphthalene-melanin. In this study we demonstrate that a novel protein, Ayg1p, is involved in the formation of 1,3,6,8-T4HN by chain-length shortening of a heptaketide precursor in A. fumigatus. Deletion of the ayg1 gene prevented the accumulation of 1,3,6,8-T4HN suggesting the involvement of ayg1 in 1,3,6,8-T4HN production. Genetic analyses of double-gene deletants suggested that Ayg1p catalyzes a novel biosynthetic step downstream of Alb1p and upstream of Arp2p (1,3,6,8-T4HN reductase). Further genetic and biochemical analyses of the reconstituted strains carrying alb1, ayg1, or alb1 + ayg1 indicated that Ayg1p is essential for synthesis of 1,3,6,8-T4HN in addition to Alb1p. Cell-free enzyme assays, using the crude Ayg1p protein extract, revealed that Ayg1p enzymatically shortened the heptaketide product of Alb1p to 1,3,6,8-T4HN. Thus, the protein Ayg1p facilitates the participation of a heptaketide synthase in a pentaketide pathway via a novel polyketide-shortening mechanism in A. fumigatus.
    Zurück zum Text

  • Watanabe, A., Fujii, I., Tsai, H. F., Chang, Y. C., Kwon-Chung, K. J., Ebizuka, Y.: Aspergillus fumigatus alb1 encodes naphthopyrone synthase when expressed in Aspergillus oryzae. FEMS Microbiology Letters 192, 39-44 (2000).
            Conidial pigment biosynthesis is an important virulence factor in Aspergillus fumigatus, a human fungal pathogen. Involvement of DHN-melanin pathway in the biosynthesis of A. fumigatus conidial pigment implies that the Alb1p polyketide synthase (PKS) is a 1,3,6,8-tetrahydroxynaphthalene (T4HN) synthase. The Alb1p, however, shows higher sequence similarity to a naphthopyrone synthase than to a T4HN synthase. To clarify the function of Alb1p, the alb1 gene was overexpressed in a heterologous host Aspergillus oryzae. The Alb1p PKS product in this heterologous expression system was identified as heptaketide naphthopyrone instead of pentaketide T4HN. The data suggest that Alb1p is a naphthopyrone synthase.
     Zurück zum Text

  • Fujii, I., Mori, Y., Watanabe, A., Kubo, Y., Tsuji, G. and Ebizuka, Y.:  Enzymatic synthesis of 1,3,6,8-tetrahydroxynaphthalene solely from malonyl coenzyme A by a fungal iterative type I polyketide synthase PKS1. Biochemistry 39, 8853-8858 (2000).
         The Colletotrichum lagenarium PKS1 gene encoding iterative type I polyketide synthase of 1,3,6,8-tetrahydroxynaphthalene (T4HN) was overexpressed in Aspergillus oryzae. SDS-PAGE analysis of the cell-free extract prepared from the transformant showed an intense band of 230000 which corresponded to the molecular weight of the deduced PKS 1 protein. By using this cell-free extract, in vitro synthesis of T4HN was successfully confirmed as the first example of the fungal multi-aromatic ring polyketide synthase activity ever detected. To identify the starter unit for T4HN synthesis, C-14-labeled acetyl CoA and/or C-14-labeled malonyl CoA were used as substrates for T4HN synthase reaction. Observed was the incorporation of C-14 label into T4HN solely from malonyl CoA even in the absence of acetyl CoA and not from acetyl CoA. This in vitro result unambiguously identified that malonyl CoA serves as the starter as well as extender units in the formation of T4HN by fungal polyketide synthase PKS1.
    Zurück zum Text

  • Izumikawa, M., Shipley, P. R., Hopke, J. N., O'Hare, T., Xiang, L., Noel, J. P., Moore, B. S., 2003. Expression and characterization of the type III polyketide synthase 1,3,6,8-tetrahydroxynaphthalene synthase from Streptomyces coelicolor A3(2). Journal of Industrial Microbiology and Biotechnology 30, 510-515.
          Sequence analysis of the metabolically rich 8.7-Mbp genome of the model actinomycete Streptomyces coelicolor A3(2) revealed three genes encoding predicted type III polyketide synthases (PKSs). We report the inactivation, expression, and characterization of the type III PKS homologous y gene product as 1,3,6,8-tetrahydroxynaphthalene synthase (THNS). Incubation of recombinant THNS with malonyl-CoA showed T4HN production, as demonstrated by UV and HPLC analyses. The K(m) value for malonyl-CoA and the k(cat) value for T4HN synthesis were determined spectrophotometrically to be 3.58+/-0.85 micro M and 0.48+/-0.03 min(-1), respectively. The C-terminal region of S. coelicolor THNS, which is longer than most other bacterial and plant type III PKSs, was shortened by 25 amino acid residues and the resulting mutant was shown to be slightly more active (K(m)=1.97+/-0.19 micro M, k(cat)=0.75+/-0.04 min(-1)) than the wild-type enzyme.
    Protein Accession: NP_625495
    Zurück zum Text

  • Cortés, J., Velasco, J., Foster, G., Blackaby, A. P., Rudd, B. A. M., Wilkinson, B., 2002. Identification and cloning of a type III polyketide synthase required for diffusible pigment biosynthesis in Saccharopolyspora erythraea. Molecular Microbiology 44, 1213-1224.
          The soluble, diffusible red-brown pigment produced by a Saccharopolyspora erythraea 'red variant' has been shown to contain glycosylated and polymerized derivatives of 2,5,7-trihydroxy-1,4-naphthoquinone (flaviolin). Flaviolin is a spontaneous oxidation product of 1,3,6,8-tetrahydroxynaphthalene (T4HN), which is biosynthesized in bacteria by a chalcone synthase-like (CS-like) type III polyketide synthase (PKS). A fragment of the gene responsible for T4HN biosynthesis in S. erythraea E_8-7 was amplified by polymerase chain reaction (PCR) using degenerate primers based on conserved regions of known plant CS and bacterial CS-like genes. From the isolated fragment, a suicide vector was prepared, which was subsequently used to disrupt the red-brown pigment-producing (rpp ) locus in S. erythraea, generating a mutant that displayed an albino phenotype. Chromosomal DNA from the albino mutant was subsequently used in a vector-recapture protocol to isolate a plasmid that contained an insert spanning the entire rpp locus. Sequencing of the insert revealed that the disrupted open reading frame (ORF) encodes a CS-like protein displaying 69% sequence identity to the rppA gene of Streptomyces griseus. The S. griseus rppA gene encodes RppA, the first characterized bacterial CS-like protein, which is sufficient in vitro for the synthesis of T4HN from malonyl-CoA. The rppA disruption mutant and rppA sequence provided a means by which to address the mechanism of diffusible pigment biosynthesis, as well as to investigate any link between this and the modulation of erythromycin A titre, which has been observed for S. erythraea variants.
    Accession of protein = AAL78053.

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