SYNTHESIS OF 1,3-DIHYDROXY-N-METHYLACRIDONE AND ITS CONVERSION TO RUTACRIDONE BY CELL-FREE-EXTRACTS OF RUTA-GRAVEOLENS CELL-CULTURES

Acridone synthase was isolated from cell suspension cultures of Ruta g raveolens which catalysed the formation of 1,3-dihydroxy-N-methylacrid one from N-methylanthraniloyl-CoA and malonyl-CoA. No cofactors were r equired for this enzyme reaction. Potassium phosphate buffer was super ior compared to Tris-HCl. Sodium ascorbate instead of mercaptoethanol as oxidation protectant showed an advantageous effect on acridone synt hase activity. The enzyme was strongly inhibited by 1,3-dihydroxy-N-me thylacridone and by the antibiotic cerulenin. Microsomal preparations from Ruta graveolens cell suspension cultures catalysed an NADPH- and oxygen-dependent condensation of 1,3-dihydroxy-N-methylacridone and is opentenyl pyrophosphate. The reaction product was identified as rutacr idone. Mg2+ or Mn2+ ions were necessary for optimal rutacridone syntha se activity. The enzyme was inhibited by a number of inhibitors of cyt ochrome P-450 enzymes. A prenylated acridone, viz. glycocitrine-II was identified as an essential intermediate. Under in vivo conditions gly cocitrine-II is incorporated into rutacridone, but a clear-cut convers ion of glycocitrine-II by microsomal preparations (cyclase) was not ob served. Microsomes converted rutacridone into furofoline-I. A number o f detergents was used for solubilization of membrane-bound proteins of Ruta microsomes. Highest specific glycocitrine-II synthase (prenyltra nsferase) activity was obtained after solubilization with dodecylmalto side.