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

Abstract

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 germacryl mechanism. 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 P450 hydroxylase.