Enzymes in artemisinin biosynthesis : cloning and catalytic characteristics ; part I hydrogen migration in cyclization of farnesyl diphosphate by amorpha-4, 11-diene synthase ; part II molecular cloning of cytochrome P450 gene from artemisia annua
Artemisinin 생합성관여 효소 : 유전자 클로닝 및 촉매 특성연구
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- 서울대학교 대학원
- 개똥쑥 (A annua); A annua; Artemisinin 생합성; X-mechanism; Bisaboyl mechanism; Germacryl mechanism; Artemisinin biosynthesis; Cytochrome P450.
- Thesis (master`s)--서울대학교 대학원 :농생명공학부,2003.
- Artemisia annua, an indigeneous plant to Korea, contains an antimalarial
sesquiterpene, artemisinin. Amorpha-4,11-diene synthase (ADS) is first commited
step and a key enzyme in artemisinin biosynthesis of Artemisia annua. The
purpose of this study is to understand the mechanism of cyclization of FDP to
amorpha-4,11-diene. Three possible mechanisms proposed by Akhila et al. [Xmechanism],
Merck et al. (or Chang et al.¡Çs a) [bisaboyl mechanism] and Chang
et al. b [germacryl mechanism]. In each mechanism, a hydrogen at C1 was
transferred to different positions. So, deuterium labeling at C1 would discern
three kinds of pathways by determining the position of deuterium.
The deuterium labeled substrates were supplied to partially purified ADS, and
the resulting products were analyzed by mass spectrometry and NMR to determined
the fate of deuterium.
The migration pattern of the label was concluded that amorpha-4,11-diene
synthase catalyzed the cyclization reaction by bisaboyl mechanism or germacryl
mechanism. Also, when we exchange the medium with D2O, we observed that one
deuterium enter the substrate. It was not clear where the deuterium was shifted
and proposed data did not explain this result.
According these result, we propose new mechanism based on bisaboyl mechanism and
The conversion of amorpha-4,11-diene to artemisinic acid is still hypothetical.
Biochemical evidence from previous studies in A. annua suggested that the
biosynthetic pathway of artemisinin mediated by cytochrome P450 monooxygenase.
To investigate the pathway, the precursor, amorpha-4,11-diene and A. annua
extract were prepared. The structure was confirmed through GC/MS analysis. The
cDNA from A. annua using heme-binding domain was isolated seven distinct P450
families (Annua1, Annua2, Annua3, Annua4, Annua5 Annua11, Annua31).
Approximately 80 % of the sequenced DNAs contained signature sequences typical
of P450 enzymes as revealed by BlastX database. The full sequence of Annua31 was
isolated and expressed in Pichia pastoris system. To ascribe functional identity
of full-length cDNA for Annua31, microsomal fraction was prepared and reacted
with A. annua extract. After GC/MS analysis 16 molecular mass was increased,
which suggested that he cloned full-length cDNA might be on of the cytochrome
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