The Molecular Cloning of Artemisinic Aldehyde Δ11(13) Reductase and Its Role in Glandular Trichome-dependent Biosynthesis of Artemisinin in Artemisia annua

Yansheng Zhang,Keat H Teoh,Darwin W Reed,Lies Maes,Alain Goossens,Douglas J.H Olson,Andrew R.S Ross,Patrick S Covello

doi:10.1074/jbc.m803090200

Yansheng Zhang, Keat H Teoh + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m803090200

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Abstract

At some point during biosynthesis of the antimalarial artemisinin in glandular trichomes of Artemisia annua, the Delta11(13) double bond originating in amorpha-4,11-diene is reduced. This is thought to occur in artemisinic aldehyde, but other intermediates have been suggested. In an effort to understand double bond reduction in artemisinin biosynthesis, extracts of A. annua flower buds were investigated and found to contain artemisinic aldehyde Delta11(13) double bond reductase activity. Through a combination of partial protein purification, mass spectrometry, and expressed sequence tag analysis, a cDNA clone corresponding to the enzyme was isolated. The corresponding gene Dbr2, encoding a member of the enoate reductase family with similarity to plant 12-oxophytodienoate reductases, was found to be highly expressed in glandular trichomes. Recombinant Dbr2 was subsequently characterized and shown to be relatively specific for artemisinic aldehyde and to have some activity on small alpha,beta-unsaturated carbonyl compounds. Expression in yeast of Dbr2 and genes encoding four other enzymes in the artemisinin pathway resulted in the accumulation of dihydroartemsinic acid. The relevance of Dbr2 to trichome-specific artemisinin biosynthesis is discussed.

Highlights

In a continuation of our investigation of the enzymes involved in artemisinin biosynthesis, we report here on the combined use of enzyme purification, mass spectrometry, and expressed sequence tag (EST) analysis leading to the molecular cloning and characterization of a sesquiterpenoid double bond reductase from A. annua
Artemisinic Aldehyde ⌬11(13) Reductase Activity in A. annua Extracts—The nature of the enzyme involved in the sesquiterpenoid ⌬11(13) double bond reduction was initially investigated in extracts of A. annua 2/39 tissues
The molecular cloning of Dbr2 is an example of the power of the combined use of protein purification, mass spectrometry, and expressed sequence tag analysis

Summary

EXPERIMENTAL PROCEDURES

Plant Materials—A. annua seeds were obtained from Pedro Melillo de Magalhaes (State University of Campinas, Brazil; line 2/39). GC/MS analysis of reductase assays using arteannuin B and artemisitene was accomplished using a short DB-5 column (10-m ϫ 0.25-mm inner diameter; Agilent) with all heated zones set to Ͻ150 °C to prevent thermal decomposition of any heat labile compounds (including artemisinin) and an oven temperature program of 120 –150 °C at 2 °C minϪ1 Under these conditions no degradation of a standard of artemisinin was detected. The open reading frames of Dbr and Opr were introduced separately into pER420 using Gateway௡ LR ClonaseTM recombination reactions (Invitrogen) with pENTR/D-AaDBR2 and pENTR/DAtOPR3 to give the plasmids pER420-AaDBR2 and pER420AtOPR3, respectively Both expression vectors were transferred to the Agrobacterium tumefaciens strain C58C1[pMP90] for transformation of A. thaliana cotyledons using a protocol developed by J.-D. Yeast Engineering—The preparation of the plasmids pESCHIS-FPS-ADS, pESC-LEU-CYP-CPR, and pYESDEST52AaDBR2 and their use in transformed yeast are described in the supplemental data

RESULTS

Artemisinic aldehyde

DISCUSSION