75(P-61) 放線菌由来のタイプIII型ポリケタイド合成酵素の機能解析(ポスター発表の部)

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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 elucidated. 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 cyclizes to form 1,3,6,8-tetrahydroxynaphthalene (THN). Disruption of the chromosomal rppA gene in S. griseus abolished melanin production in hyphae, resulting in "albino" mycelium. RppA accepted C4 to C8 aliphatic acyl-CoAs as starter substrates and catalyzed sequential condensation of malonyl-CoA to yield α-pyrones and phloroglucinols. In addition, RppA yielded a hexaketide, 4-hydroxy-6-(2',4',6'-trioxo-tridecyl)-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 so far found from 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. Site-directed mutagenesis of the amino acid residues probably forming the active site cavity revealed that the aromatic ring of Tyr^<224> was 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 to accept malonyl-CoA as a starter. Of the site-directed mutants generated. A305I was found to produce only a triketide pyrone from hexanoyl-CoA as a starter, although RppA synthesizes tetraketide and triketide pyrones in the hexanoyl-CoA-primed reaction, showing that the substitution of Ala^<305> by bulky amino acid residues restricted the number of elongations of the growing polyketide chain.
天然有機化合物討論会の論文
2002-09-01

75(P-61) 放線菌由来のタイプIII型ポリケタイド合成酵素の機能解析(ポスター発表の部)

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