| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2003: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2002: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Research Abstract |
1.Squalene cyclizing enzymes.
(1)Newly synthesized C_<35> polyprene was enzymatically converted to unnatural hexacyclic polyprenoid by squalene : hopene cyclase from Alicyclobacillus acidocaldarius. This is the first demonstration of the remarkable ability of the squalene cyclizing enzyme to perform construction of unnatural hexacyclic skeleton. Further, methylidene-extended squalene analogues were converted to unnatural pentacyclic novel polyprenoides by the enzyme. (2)Recombinant β-amyrin synthase from Pisum sativum converted 22,23-dihydro-2,3-oxidosqualene into bacchar-12-en-3β-ol ; the first demonstration of the enzymatic formation of the baccharene skeleton with a six-membered D-ring. It is remarkable that the formation of the anti-Markovnikov six-membered D-ring did not depend on he participation of the termianl π-electrons. Furthermore, the recombinant enzyme also converted 24,30-bisnor-2,3-oxidosqualene into 29,30-bisnor-β-amyrin.
2.Plant polyketide synthases (PKSs).
(1)Substrate
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specificities of plant polyketide synthases (PKSs) were investigated using analogs of malonyl-CoA and 4-coumaroyl-CoA, plant PKSs afforded an unnatural C_6-C_5 aromatic polyketide. In the chalcone synthase (CHS) reaction, it was shown that both the starter molecule and the extension unit of the poyketide chain elongation reaction could be simultaneously replaced with non-physiological substrates.
(2)One of the most characteristic features of benzalacetone synthase (BAS) is that it lacks active site Phe215 ; the residues ^<214>LF conserved in type III PKSs are uniquely replaced by IL. Our observation that BAS I214L/L215F mutant exhibited chalcone forming activity in pH dependent manner supported a hypothesis that the absence of Phe 215 in BAS accounts for the interruption of the polyketide chain elongation at the diketide stage. These results confirmed the critical role of Phe215 in the polyketide formation reactions and provided structural basis for understanding the structure-function relationship of the plant type III PKSs. Less
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