Molecular cloning of an aldehyde dehydrogenase implicated in artemisinin biosynthesis in Artemisia annua
Molecular cloning of an aldehyde dehydrogenase implicated in artemisinin biosynthesis in Artemisia annua
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DOI：
10.1139/b09-032
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发表时间：
2009-06-01
期刊：
影响因子：
1.1
通讯作者：
Covello, Patrick S.
中科院分区：
文献类型：
--
作者：
Teoh, Keat H.;Polichuk, Devin R.;Covello, Patrick S.
来自青蒿的抗疟化合物青蒿素的供应有限，引起了人们对了解其生物合成和提高其产量的兴趣。最近的生化和分子遗传学数据表明二氢青蒿醛是相应酸的前体，然后转化为青蒿素。因此，了解参与二氢青蒿醛氧化的酶很重要。鉴于其对青蒿醛的活性，我们研究了细胞色素 P450 CYP71AV1 氧化二氢青蒿醛的能力。然而，没有检测到净活动。为了寻找可以催化氧化的替代酶，我们对青蒿的表达序列标签 (EST) 集合进行了相关 cDNA 的研究。这导致分离出编码乙醛脱氢酶同源物的全长 cDNA，名为 Aldh1，它在毛状体中高度表达。 cDNA 在大肠杆菌中的表达和纯化重组酶的表征表明，该基因产物催化假定的青蒿素前体、青蒿醛和二氢青蒿醛以及有限范围的其他醛的 NAD(P) 依赖性氧化。观察到的 Aldh1 酶活性和相应基因的表达模式表明在青蒿腺分泌毛中青蒿素生物合成中的作用。
Limitations in the supply of the antimalarial compound artemisinin from Artemisia annua L. have led to an interest in understanding its biosynthesis and enhancing its production. Recent biochemical and molecular genetic data have implicated dihydroartemisinic aldehyde as a precursor to the corresponding acid, which is then converted to artemisinin. Thus, it is important to understand the enzyme or enzymes involved in dihydroartemisinic aldehyde oxidation. Given its activity on artemisinic aldehyde, the cytochrome P450 CYP71AV1 was investigated for its ability to oxidize dihydroartemisinic aldehyde. However, no net activity was detected. In a search for alternative enzymes that could catalyze the oxidation, an expressed sequence tag (EST) collection from A. annua was investigated for relevant cDNAs. This led to the isolation of a full-length cDNA encoding an aldehyde dehydrogenase homologue, named Aldh1, which is highly expressed in trichomes. Expression of the cDNA in E. coli and characterization of the purified recombinant enzyme revealed that the gene product catalyses the NAD(P)-dependent oxidation of the putative artemisinin precursors, artemisinic and dihydroartemsinic aldehydes, and a limited range of other aldehydes. The observed enzyme activity of Aldh1 and the expression pattern of the corresponding gene suggest a role in artemisinin biosynthesis in the glandular secretory trichomes of A. annua.