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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Cloning and Characterization of Unusual Fatty Acid Desaturases from Anemone leveillei: Identification of an Acyl-Coenzyme A C₂₀ ⁵-Desaturase Responsible for the Synthesis of Sciadonic Acid¹

Rothamsted Research, Harpenden, Herts AL5 2JQ, United Kingdom

The seed oil of Anemone leveillei contains significant amounts of sciadonic acid (20:3^5,11,14; SA), an unusual non-methylene-interrupted fatty acid with pharmaceutical potential similar to arachidonic acid. Two candidate cDNAs (AL10 and AL21) for the C₂₀ ^5cis-desaturase from developing seeds of A. leveillei were functionally characterized in transgenic Arabidopsis (Arabidopsis thaliana) plants. The open reading frames of both ⁵-desaturases showed some similarity to presumptive acyl-coenzyme A (CoA) desaturases found in animals and plants. When expressed in transgenic Arabidopsis, AL21 showed a broad range of substrate specificity, utilizing both saturated (16:0 and 18:0) and unsaturated (18:2, n-6 and 18:3, n-3) substrates. In contrast, AL10 did not show any activity in wild-type Arabidopsis. Coexpression of AL10 or AL21 with a C₁₈ ⁹-elongase in transgenic Arabidopsis plants resulted in the production of SA and juniperonic fatty acid (20:4^5,11,14,17). Thus, AL10 acted only on C₂₀ polyunsaturated fatty acids in a manner analogous to "front-end" desaturases. However, neither AL10 nor AL21 contain the cytochrome b₅ domain normally present in this class of enzymes. Acyl-CoA profiling of transgenic Arabidopsis plants and developing A. leveillei seeds revealed significant accumulation of ⁵-unsaturated fatty acids as acyl-CoAs compared to the accumulation of these fatty acids in total lipids. Positional analysis of triacylglycerols of A. leveillei seeds showed that ⁵-desaturated fatty acids were present in both sn-2 and sn-1 + sn-3 positions, although the majority of 16:1⁵, 18:1⁵, and SA was present at the sn-2 position. Our data provide biochemical evidence for the A. leveillei ⁵-desaturases using acyl-CoA substrates.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Olga Sayanova (olga.sayanova{at}bbsrc.ac.uk).

www.plantphysiol.org/cgi/doi/10.1104/pp.107.098202

^* Corresponding author; e-mail olga.sayanova{at}bbsrc.ac.uk; fax 44–1582–763010.

Received February 16, 2007; accepted March 15, 2007; published March 23, 2007.

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