| Co-Investigator(Kenkyū-buntansha) |
ABE Ikuro Univ.of Shizuoka, School of Pharmaceutical Sciences, Department of Pharmacognosy, Lecturer, 薬学部, 講師 (40305496)
UMEHARA Kaoru Univ.of Shizuoka, School of Pharm.Sci., Dept of Pharmacognosy, Research Associate, 薬学部, 助教 (40185070)
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| Budget Amount *help |
¥15,800,000 (Direct Cost: ¥15,800,000)
Fiscal Year 2006: ¥6,100,000 (Direct Cost: ¥6,100,000)
Fiscal Year 2005: ¥9,700,000 (Direct Cost: ¥9,700,000)
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| Research Abstract |
(1)The chalcone synthase (CHS) superfamily of type III polyketide synthases (PKSs) produces a variety of plant secondary metabolites with remarkable structural diversity and biological activities (e.g., chalcones, stilbenes, benzophenones, acrydones, phloroglucinols, resorcinols, pyrones, and chromones). An octaketide-producing novel plant-specific type III PKS from aloe (Aloe arborescens) shares 50-60% amino acid sequence identity with other plant CHS-superfamily enzymes. A recombinant enzyme expressed in Escherichia coli catalyzed seven successive decarboxylative condensations of malonyl-CoA to yield aromatic octaketides SEK4 and SEK4b, the longest polyketides known to be synthesized by the structurally simple type III PKS. Surprisingly, site-directed mutagenesis revealed that a single residue Gly207 (corresponding to the CHS's active site Thr197) determines the polyketide chain length and product specificity. Small-to-large substitutions (G207A,G207T,G207M,G207L,G207F, and G207W) re
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sulted in loss of the octaketide-forming activity and concomitant formation of shorter chain length polyketides (from triketide to heptaketide) including a pentaketide chromone, 2,7-dihydroxy-5-methylchromone, and a hexaketide pyrone, 6-(2,4-dihydroxy-6-methylphenyl)-4-hydroxy-2-pyrone, depending on the size of the side chain. Notably, the functional diversity of the type III PKS was shown to evolve from simple steric modulation of the chemically inert single residue lining the active-site cavity accompanied by conservation of the Cys-His-Asn catalytic triad. This provided novel strategies for the engineered biosynthesis of pharmaceutically important plant polyketides. Pentaketide chromone synthase (PCS) from Aloe arborescens is a novel plant-specific type III polyketide synthase (PKS) that produces 5,7-dihydroxy-2-methylchromone from five molecules of malonyl-CoA. On the basis of the crystal structures of wild-type and M207G mutant PCS, the F80A/Y82A/M207G triple mutant was constructed and shown to produce an unnatural novel nonaketide naphthopyrone by sequential condensations of nine molecules of malonyl-CoA. This is the first demonstration of the formation of a nonaketide by the structurally simple type III PKS. A homology model predicted that the active-site cavity volume of the triple mutant is increased to 4 times that of the wild-type PCS.
(2)Two indole-containing substrate analogs in which a C20 isoprene unit is connected to indole ; 3-(geranylgeranyl)indole and 3-(farnesyl-dimethyIallyl)indole, were synthesized and tested for enzymatic cyclization by squalene : hopene cyclase from Alicyclobacillus acidocaldarius. Interestingly, 3-(Geranylgeranyl)indole was not a substrate for the bacterial squalene cyclase, while 3-(farnesyl-dimethylallyl)indole was efficiently converted to a 2:1 mixture of an unnatural novel tetracycic and pentacyclic product. Less
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