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KMID : 0903519970400060501
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Journal of the Korean Society of Agricultural Chemistry and Biotechnology
1997 Volume.40 No. 6 p.501 ~ p.507
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Characterization of ent-Kaurenoic Acid 13-Hydroxylase in Steviol Biosynthesis of Stevia rebaudiana Bertoni
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Abstract
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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 strotna fraction of either Spinacia deracea 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.
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