46(P-3) 微生物青紫色色素ビオラセインの生合成研究 : 生合成メカニズム及び仮想中間体の構造(ポスター発表の部)

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In this symposium, we present the biosynthetic studies of violacein, a bacterial pigment colored with purplish-blue, which shows antibiotic activity, the induction of apoptosis to V79 cell-line, cytotoxicity against tumor cells such as L929, M5076 and HeLa and so on. 1. Biosynthetic Mechanism. The feeding experiments using stable-isotopically labelled tryptophans have made clear the biosynthetic origins of all the constitutive carbon and hydrogen atoms. The labelled positions of violacein prepared by the feeding of [2-^<13>C]- and [3-^<13>C]tryptophans have showed that intramolecular 1,2-shift of indole ring occurred during the biosynthesis. The occurrence of indole shift during the biosyntheses of natural products has never been reported before. Incorporation experiment of the molecular oxygen ^<18>O_2 has revealed that all the three oxygen atoms of violacein skeleton originated from the atmospheric oxygen gas, showing the involvement of oxygenase. The nitrogen atom of 2-pyrrolidone moiety is derived from α-amino group of tryptophan. The mixed feeding experiment of [2-^<13>C]- and [α-^<15>N]tryptophans (1 to 1 ratio) has revealed that intact incorporation of tryptophan molecule occurs on the oxindole side (right side), taking into the consideration the results that no incorporation of tryptamine and oxindolylalanine was observed. 2. Metabolites by enzyme inhibitors and mutants. By using enzyme inhibitors and the mutants, prepared with N-nitrosoguanidine, we isolated interesting compounds. Metabolites 5 and 6 lack the oxygen atom at 16-position. If oxygen is incorporated at this position as shown in Fig. 4, violacein pigment will be produced, suggesting that this oxygenation is the final step of the biosynthesis process. The blocked mutant No 9 produced compound 9 and 10, whose structure resemble with those of staurosporin and rebeccamycin, but the indole shift does not occur. Dimeric structure of indole ring is also found in metabolites 11-13, which were produced by mutant H-25. These three compounds are closely related each other in respect of the biosyntheses, as illustrated in Fig. 5, suggesting that epoxide ring intermediate is responsible for the indole shift of violacein biosynthesis. 3. Metabolites produced by the cell-free extract. After many trials, we have established the cell-free system for violacein production from tryptophan. The most important thing for the cell-free biosynthesis was to add NADPH into the cell-free extract. No addition of NADPH gave green-colored incubation mixture, which was composed mainly of green and orange-colored materials (5 and 6). The green compounds were novel metabolites, named as deoxychromoviridans 16 and chromoviridans 17. However, sufficient supplementation of NADPH led to the production of 1,2,5,6, 16 and 17, as shown in Fig. 6. All the structures of metabolites are closely related each other, although 15 and 16 have an extra C_1 unit at the dipyrromethene moiety and were not oxygenated at any position. The oxygenation at 11-position of 16 and 17 would lead to the formation of 1 or 2. 4. Molecular cloning of the biosynthetic gene cluster. We have succeeded in obtaining the biosynthetic gene cluster composed of four ORF (Fig. 9). Several results will be presented, which were obtained by constructing over-expression system in E. coli.
天然有機化合物討論会の論文
2001-09-01

46(P-3) 微生物青紫色色素ビオラセインの生合成研究 : 生合成メカニズム及び仮想中間体の構造(ポスター発表の部)

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