45 オルセリン酸合成酵素とその反応の立体化学(口頭発表の部)

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A wide variety of natural products are formed by the so-called polyketide pathway. The difficulties in achieving the cell-free system of polyketides syntheses have retarded their enzymological investigation. To date, only two polyketide synthases have been purified, 6-methylsalicylic acid synthase and chalcone synthase. Orsellinic acid synthase was chosen as our target enzyme because orsellinic acid (1) is the simplest aromatic tetraketide. We found the orsellinic acid synthase activity in the cell-free extract of Penicillium cyclopium which produces penicillic acid. The enzymatic formation of orsellinic acid (1) was confirmed by carrier dilution experiment. The enzyme was unstable and partially stabilized by the addition of glycerol, DTT, and EDTA to the buffer. By preparative centrifugation (210,000xg for 1hr), the enzyme was pelleted and solubilized by Tween 80. The solubilized enzyme was partially purified by DEAE-cellulose column chromatography. Questions about the stereochemistry of orsellinic acid synthase reaction was approached by,using chiral malonate (2). (2R,3S)-[2,3-^<13>C_2,3-^2H_1]Malate and (2R,3R)-[2,3-^<13>C_2,2,3-^2H_2]malate, which are configurationally stable precursors of S- and R-malonate, respectively, were synthesized. The chiral malonates were prepared by KmnO_4 oxidation and their chiralities were confirmed by CoA transferase and fatty acid synthase system. Then, chiral malonates were reacted with CoA transferase and orsellinic acid synthase system. Mass spectroscopic analyses of the orsellinic acids formed from chiral malonates suggested that elimination of methylene hydrogen in the enolization (or aromatization) occurs in non-stereospecific.
天然有機化合物討論会の論文
1988-09-26

45 オルセリン酸合成酵素とその反応の立体化学(口頭発表の部)

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