Purification of the acyl-CoA elongase complex from developing rapeseed and characterization of the 3-ketoacyl-CoA synthase and the 3-hydroxyacyl-CoA dehydratase |
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Authors: | Frédéric Domergue Sylvette Chevalier Anne Créach C Cassagne René Lessiré |
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Affiliation: | 1. Institute für Allgemeine Botanik, Universit?t Hamburg, 22609, Hamburg, Germany 2. Laboratoire de Biogenèse Membranaire, Université Victor Segalen Bordeaux 2 (CNRS-UMR 5544), 146 rue Léo Saignat, 33076, Bordeaux Cédex, France 3. USTL UPRES-A CNRS 8013 “ELICO”, 59655 Villeneuve d'Ascq Cédex, France
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Abstract: | Oleoyl-CoA elongase catalyzes four successive reactions: condensation of malonyl-CoA to oleoyl-CoA, reduction, dehydration,
and another reduction. Evidence supporting this mechanism and the multienzymatic nature of the elongation complex are reported.
A particulate membrane fraction from rapeseed is able to elongate intermediates (R,S) 3-hydroxy-20∶0-CoA and (E) 2,3–20∶1-CoA to very long chain fatty acids in the presence of malonyl-CoA. Studies of the 3-ketoacyl-CoA synthase activities
showed that maximal activity could be measured by using 15 to 30 μM 18∶1-CoA and 30 μM malonyl-CoA, and that 18∶0-CoA and
18∶1-CoA were the best substrates. Comparison of the condensation and the overall elongation activities indicated that condensation
is the rate-limiting step of the elongation process. The 3-hydroxyacyl-CoA dehydratase activity was maximal in the presence
of 75 μM Triton X-100 and 25 μg of proteins. Finally, the acyl-CoA elongase complex was solubilized and purified. During the
purification process, the 3-hydroxyacyl-CoA dehydratase copurified with the elongase complex, strongly suggesting that this
enzyme belongs to the elongase complex. The apparent molecular mass of 700 kDa determined for the elongase complex, and the
fact that four different polypeptide bands were detected after sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis
of the purified fraction, further suggest that the acyl-CoA elongase is a multienzymatic complex. |
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