Cloning and characterization of unusual fatty acid desaturases from Anemone leveillei: identification of an acyl-coenzyme A C20 Delta5-desaturase responsible for the synthesis of sciadonic acid.
The seed oil of Anemone leveillei contains significant amounts of sciadonic acid (20:3Delta(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 C(20) Delta(5cis)-desaturase from developing seeds of A. leveillei were functionally characterized in transgenic Arabidopsis (Arabidopsis thaliana) plants. The open reading frames of both Delta(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 C(18) Delta(9)-elongase in transgenic Arabidopsis plants resulted in the production of SA and juniperonic fatty acid (20:4Delta(5,11,14,17)). Thus, AL10 acted only on C(20) polyunsaturated fatty acids in a manner analogous to "front-end" desaturases. However, neither AL10 nor AL21 contain the cytochrome b(5) domain normally present in this class of enzymes. Acyl-CoA profiling of transgenic Arabidopsis plants and developing A. leveillei seeds revealed significant accumulation of Delta(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 Delta(5)-desaturated fatty acids were present in both sn-2 and sn-1 + sn-3 positions, although the majority of 16:1Delta(5), 18:1Delta(5), and SA was present at the sn-2 position. Our data provide biochemical evidence for the A. leveillei Delta(5)-desaturases using acyl-CoA substrates.
Sayanova
Olga
O
Rothamsted Research, Harpenden, Herts, United Kingdom. olga.sayanova@bbsrc.ac.uk
Haslam
Richard
R
Venegas Caleron
Monica
M
Napier
Johnathan A
JA
D000595:Amino Acid Sequence; D031883:Anemone / Q000196:embryology / Q000201:enzymology* / Q000235:genetics; D017360:Arabidopsis / Q000235:genetics; D001095:Arachidonic Acids / Q000096:biosynthesis*; D003001:Cloning, Molecular; D000090943:Delta-5 Fatty Acid Desaturase; D044943:Fatty Acid Desaturases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism; D050356:Lipid Metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000378:metabolism; D012639:Seeds / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014170:Transformation, Genetic
D001095:Arachidonic Acids; D000090943:Delta-5 Fatty Acid Desaturase; D005227:Fatty Acids; D010940:Plant Proteins; C045533:eicosa-5,11,14-trienoic acid; D044943:Fatty Acid Desaturases