Characterization of ent-Kaurenoic Acid 13-Hydroxylase in Steviol Biosynthesis of Stevia rebaudiana Bertoni

Stevia rebaudiana Bertoni의 Steviol 생합성 효소 ent-Kaurenoic Acid 13-Hydroxylase의 특성

  • Shibata, Hitoshi (Department of Agricultural chemistry, Gyeongsang National University) ;
  • Kim, Keun-Ki (Department of Agriculture chemistry, Gyeongsang National University)
  • ;
  • 김근기 (경상대학교 농과대학 농화학과)
  • Published : 1997.12.31

Abstract

Chloroplasts isolated from Stevia rebaudiana Bertoni leaves contained an enzyme activity which catalyzed hydroxylation of ent-kaurenoic acid (ent-kaur-16-en-19-oic acid; ent-KA) to steviol (ent-13-hydroxy kaur-16-en-19-oic acid), the diterpenoid carboxylic alcohol which is the aglycone of sweet stevioside-related glycosides. $[^(14)C]-methylated$ ent-KA was used to localize ent-KA hydroxylase. $[^(14)C]-methyl-KA$ was most actively was transformed into methyl-steviol in chloroplast. The enzymatic activity was found in stroma fraction but not in thylakoid membrane in Stevia rebaudiana Bertoni. However, ent-KA 13-hydroxylase activity was not detected in stroma fraction of either Spinacia oleracea and Solidago altissima. The reaction products using $[^(14)C]-methyl-KA$ were purified and identified on TLC autoradiogram. The hydroxylation of ent-KA from stromal protein to form steviol required NADPH and oxygen. FAD and riboflavin stimulated the enzyme activity 1.5-and 1.7-fold, respectively. It also turned out that the activity of this enzyme using methyl-KA as a substrate was 16.7% that of ent-KA. The purified ent-KA 13-hydroxylase did not act on t-cinnamic acid, 4-hydroxyphenyl acetic acid, choline and resorcinol, known as monooxygenase and hydroxylase substrates.