Acyl carrier proteins from sunflower (Helianthus annuus L.) seeds and their influence on FatA and FatB acyl-ACP thioesterase activities.
The kinetics of acyl-ACP thioesterases from sunflower importantly changed when endogenous ACPs were used. Sunflower FatB was much more specific towards saturated acyl-ACPs when assayed with them. Acyl carrier proteins (ACPs) are small (~9 kDa), soluble, acidic proteins involved in fatty acid synthesis in plants and bacteria. ACPs bind to fatty acids through a thioester bond, generating the acyl-ACP lipoproteins that are substrates for fatty acid synthase (FAS) complexes, and that are required for fatty acid chain elongation, acting as important intermediates in de novo fatty acid synthesis in plants. Plants, usually express several ACP isoforms with distinct functionalities. We report here the cloning of three ACPs from developing sunflower seeds: HaACP1, HaACP2, and HaACP3. These proteins were plastidial ACPs expressed strongly in seeds, and as such they are probably involved in the synthesis of sunflower oil. The recombinant sunflower ACPs were expressed in bacteria but they were lethal to the prokaryote host. Thus, they were finally produced using the GST gene fusion system, which allowed the apo-enzyme to be produced and later activated to the holo form. Radiolabelled acyl-ACPs from the newly cloned holo-ACP forms were also synthesized and used to characterize the activity of recombinant sunflower FatA and FatB thioesterases, important enzymes in plant fatty acids synthesis. The activity of these enzymes changed significantly when the endogenous ACPs were used. Thus, FatA importantly increased its activity levels, whereas FatB displayed a different specificity profile, with much high activity levels towards saturated acyl-CoA derivatives. All these data pointed to an important influence of the ACP moieties on the activity of enzymes involved in lipid synthesis.
Kansas State University, 141 Chalmers Hall, Manhattan, KS, 66506, USA.
Jose A
JA
Aznar-Moreno
Instituto de la Grasa (CSIC), Edificio 46, Campus Universitario Pablo de Olavide, Carretera de Utrera Km 1, 41013, Seville, Spain.
Mónica
M
Venegas-Calerón
Instituto de la Grasa (CSIC), Edificio 46, Campus Universitario Pablo de Olavide, Carretera de Utrera Km 1, 41013, Seville, Spain.
Enrique
E
Martínez-Force
Instituto de la Grasa (CSIC), Edificio 46, Campus Universitario Pablo de Olavide, Carretera de Utrera Km 1, 41013, Seville, Spain.
Rafael
R
Garcés
Instituto de la Grasa (CSIC), Edificio 46, Campus Universitario Pablo de Olavide, Carretera de Utrera Km 1, 41013, Seville, Spain. jjsalas@cica.es.
Joaquín J
JJ
Salas
D003001:Cloning, Molecular; D005227:Fatty Acids / Q000096:biosynthesis*; D006368:Helianthus / Q000235:genetics / Q000378:metabolism*; D050356:Lipid Metabolism; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D000072417:Protein Domains; D012639:Seeds / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000378:metabolism*
D005227:Fatty Acids; D010940:Plant Proteins; D013869:Thiolester Hydrolases; C018748:oleoyl-(acyl-carrier-protein) hydrolase
Acyl carrier protein; Acyl-ACP thioesterase; Fatty acid synthesis; Sunflower, Helianthus annuus