Biosynthesis of Docosahexaenoic Acid in Euglena gracilis: Biochemical and Molecular Evidence for the Involvement of a 4-Fatty
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- 作者：Astrid Meyer ; Petra Cirpus ; Claudia Ott ; Rainer Schlecker ; Ulrich Zä ; hringer ; and Ernst Heinz
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：August 19, 2003
- 年：2003
- 卷：42
- 期：32
- 页码：9779 - 9788
- 全文大小：213K
- 年卷期：v.42,no.32(August 19, 2003)
- ISSN：1520-4995
文摘
Docosahexaenoic acid (DHA) can be synthesized via alternative routes from which only the
3/6-pathways involve the action of a 
4-fatty acid desaturase. We examined the suitability of Euglenagracilis, Thraustochytrium sp., Schizochytrium sp., and Crypthecodinium cohnii to serve as sources forcloning a cDNA encoding a 4-fatty acid desaturase. For this purpose we carried out in vivo labelingstudies with radiolabeled C22 polyunsaturated fatty acid substrates. Schizochytrium sp. was unable toconvert exogenously supplied [2-14C]-docosapentaenoic acid (DPA, 22:57,10,13,16,19) to DHA, while E.gracilis and Thraustochytrium sp. carried out this desaturation very efficiently. Hydrogenation and
-oxidation of the labeled DHA isolated from these two organisms showed that it was the result of direct4-desaturation and not of substrate breakdown and resynthesis. To clone the desaturase gene, a cDNAlibrary of E. gracilis was subjected to mass sequencing. A full-length clone with highest homology to the4-desaturase of Thraustochytrium sp. was isolated, and its function was verified by heterologous expressionin yeast. The desaturase efficiently converted DPA to DHA. Analysis of the substrate specificitydemonstrated that the enzyme activity was not limited to C22 fatty acids, since it also efficiently desaturatedC16 fatty acids. The enzyme showed strict 4-regioselectivity and required the presence of a 7-doublebond in the substrate. Positional analysis of phosphatidylcholine revealed that the proportion of the 4-desaturated products was up to 20 times higher in the sn-2 position than in the sn-1 position.
3/6-pathways involve the action of a 4-fatty acid desaturase. We examined the suitability of Euglenagracilis, Thraustochytrium sp., Schizochytrium sp., and Crypthecodinium cohnii to serve as sources forcloning a cDNA encoding a 4-fatty acid desaturase. For this purpose we carried out in vivo labelingstudies with radiolabeled C22 polyunsaturated fatty acid substrates. Schizochytrium sp. was unable toconvert exogenously supplied [2-14C]-docosapentaenoic acid (DPA, 22:57,10,13,16,19) to DHA, while E.gracilis and Thraustochytrium sp. carried out this desaturation very efficiently. Hydrogenation and-oxidation of the labeled DHA isolated from these two organisms showed that it was the result of direct4-desaturation and not of substrate breakdown and resynthesis. To clone the desaturase gene, a cDNAlibrary of E. gracilis was subjected to mass sequencing. A full-length clone with highest homology to the4-desaturase of Thraustochytrium sp. was isolated, and its function was verified by heterologous expressionin yeast. The desaturase efficiently converted DPA to DHA. Analysis of the substrate specificitydemonstrated that the enzyme activity was not limited to C22 fatty acids, since it also efficiently desaturatedC16 fatty acids. The enzyme showed strict 4-regioselectivity and required the presence of a 7-doublebond in the substrate. Positional analysis of phosphatidylcholine revealed that the proportion of the 4-desaturated products was up to 20 times higher in the sn-2 position than in the sn-1 position.