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J. Biol. Chem., Vol. 278, Issue 37, 35115-35126, September 12, 2003

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Acyl Carriers Used as Substrates by the Desaturases and Elongases Involved in Very Long-chain Polyunsaturated Fatty Acids Biosynthesis Reconstituted in Yeast^*

Frédéric Domergue  , Amine Abbadi , Claudia Ott , Thorsten K. Zank  ¶, Ulrich Zähringer || and Ernst Heinz 

From the Institut für Allgemeine Botanik, Universität Hamburg, Ohnhorststrasse 18, 22609 Hamburg, Germany and the ^||Division of Immunochemistry, Research Center Borstel, Parkallee 22, 23845, Borstel, Germany

The health benefits attributed to very long-chain polyunsaturated fatty acids and the long term goal to produce them in transgenic oilseed crops have led to the cloning of all the genes coding for the desaturases and elongases involved in their biosynthesis. The encoded activities have been confirmed in vivo by heterologous expression, but very little is known about the actual acyl substrates involved in these pathways. Using a 6-elongase and front-end desaturases from different organisms, we have reconstituted in Saccharomyces cerevisiae the biosynthesis of arachidonic acid from exogenously supplied linoleic acid in order to identify these acyl carriers. Acyl-CoA measurements strongly suggest that the elongation step involved in polyunsaturated fatty acids biosynthesis is taking place within the acyl-CoA pool. In contrast, detailed analyses of lipids revealed that the two desaturation steps (5 and 6) occur predominantly at the sn-2 position of phosphatidylcholine when using 5- and 6-desaturases from lower plants, fungi, worms, and algae. The specificity of these 6-desaturases for the fatty acid acylated at this particular position as well as a limiting re-equilibration with the acyl-CoA pool result in the accumulation of -linolenic acid at the sn-2 position of phosphatidylcholine and prevent efficient arachidonic acid biosynthesis in yeast. We confirm by using a similar experimental approach that, in contrast, the human 6-desaturase uses linoleoyl-CoA as substrate, which results in high efficiency of the subsequent elongation step. In addition, we report that 12-desaturases have no specificity toward the lipid polar headgroup or the sn-position.

Received for publication, June 6, 2003 , and in revised form, June 27, 2003.

^* This research was supported by a Marie Curie Fellowship of the European Community Program Human Potential under the contract number HPMF-CT-1999-00148 (to F. D.), by BASF Plant Science GmbH, BPS-A30 (Ludwigshafen, Germany), and by the BMBF project NAPUS 2000 (part FK 0312252 F). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Present address: BASF Plant Science GmbH, BPS-A30, Ludwigshafen, Germany.

To whom correspondence should be addressed: Institut für Allgemeine Botanik, Universität Hamburg, Ohnhorststrasse 18, 22609 Hamburg, Germany. Tel.: 49-40-42816-373; Fax: 49-40-42816-254; E-mail: fredDo{at}botanik.uni-hamburg.de.

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