Purification of the acyl-CoA elongase complex from developing rapeseed and characterization of the 3-ketoacyl-CoA synthase and the 3-hydroxyacyl-CoA dehydratase.
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 microM 18:1-CoA and 30 microM 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 microM Triton X-100 and 25 microg 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.
Domergue
F
F
Institute for Allgemeine Botanik, Universität Hamburg, Germany.
Chevalier
S
S
Créach
A
A
Cassagne
C
C
Lessire
R
R
D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000737:chemistry* / Q000378:metabolism; D051080:Acyl-Carrier Protein S-Malonyltransferase; D000217:Acyltransferases / Q000737:chemistry* / Q000302:isolation & purification*; D001937:Brassica / Q000201:enzymology*; D002847:Chromatography, Agarose; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006836:Hydro-Lyases / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008316:Malonyl Coenzyme A / Q000494:pharmacology; D017830:Octoxynol / Q000494:pharmacology; D012685:Sepharose / Q000737:chemistry; D013997:Time Factors
D003902:Detergents; D008316:Malonyl Coenzyme A; D017830:Octoxynol; D012685:Sepharose; D000217:Acyltransferases; D051080:Acyl-Carrier Protein S-Malonyltransferase; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; C065185:D-3-hydroxyacyl CoA dehydratase; D006836:Hydro-Lyases