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

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.

파라과이 원산의 국화과 식물 Stevia rebaudiana Bertoni는 steviol (ent-13-hydroxy kaur-16-en-19-oic acid)을 기본 골격으로 하는 stevia 감미료를 건물 중량의 5-10%를 생합성하여 축적을 한다. 감미성분은 Gibberellin 생합성의 전구체로 알려진 ent-kaurenoic acid (ent-kaur-16-en-19-oic acid; ent-KA)의 7번과 13번의 어느 탄소에 hydroxylation을 시키느냐에 따라 gibberellin 대사와 steviol 대사로 나뉘어진다. ent-KA 13-hydroxylase를 stevia 엽록체의 stroma에 존재하는 것을 확인했다. Stroma 분획 단백질 $100\;{\mu}g$으로 효소 반응의 cofactor 요구성을 조사해 본 결과, 반응 혼합액에 NADPH를 첨가하지 않았을 때는 85%의 효소활성의 감소를 보였고, NADPH 대신 NADH를 첨가해도 34%의 감소를 보였다. anaerobic 조건에서는 2.1%의 활성을 뛰었다. 이 결과로 ent-KA 13-hydroxylase는 NADPH와 $O_2$ 요구성임을 밝혔다. FAD, FMN, riboflavin을 첨가함으로써 FAD는 1.5배, riboflavin은 1.7배의 효소활성 증가 효과가 나타났다. 효소의 기질 특이성을 조사한 결과, t-cinnamic acid, 4-hydroxyphenyl acetic acid, choline과 resorcinol에는 전혀 활성이 검출되지 않았으며, $[^(14)C]-methyl-KA$를 ent-KA 대신에 기질로 사용했을 때는 16.7% 의 활성이 검출되어 ent-KA 13-hydroxylase는 기질특이성이 높은것으로 확인되었다.