RESUMO
Stearoyl-acyl carrier protein (ACP) desaturase (EC 1.14.99.6) catalyzes the principal conversion of saturated fatty acids to unsaturated fatty acids in the synthesis of vegetable oils. Stearoyl-ACP desaturase was purified from developing embryos of safflower seed, and extensive amino acid sequence was determined. The amino acid sequence was used in conjunction with polymerase chain reactions to clone a full-length cDNA. The primary structure of the protein, as deduced from the nucleotide sequence of the cDNA, includes a 33-amino-acid transit peptide not found in the purified enzyme. Expression in Escherichia coli of a gene encoding the mature form of stearoyl-ACP desaturase did not result in an altered fatty acid composition. However, active enzyme was detected when assayed in vitro with added spinach ferredoxin. The lack of significant activity in vitro without added ferredoxin and the lack of observed change in fatty acid composition indicate that ferredoxin is a required cofactor for the enzyme and that E. coli ferredoxin functions poorly, if at all, as an electron donor for the plant enzyme.
Assuntos
Ferredoxinas/metabolismo , Oxigenases de Função Mista/genética , Sementes/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Escherichia coli/genética , Biblioteca Gênica , Genes de Plantas , Oxigenases de Função Mista/isolamento & purificação , Oxigenases de Função Mista/metabolismo , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Plantas/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Mapeamento por RestriçãoRESUMO
A 715 base pair cDNA clone coding for an acyl carrier protein (ACP) in spinach leaves has been isolated and characterized. The amino acid sequence indicated by the cDNA sequence closely matches the amino acid sequence of the ACP-I isoform. The presence of polyadenylation and DNA sequence coding for a precursor protein with a putative transit peptide, and the absence of hybridization between the cloned DNA and isolated spinach plastid DNA collectively show that the ACP-I gene is nuclear-encoded. The ACP-I cloned DNA did not cross-hybridize with mRNA from spinach tissues in which ACP-II has been found. Cross-hybridization with mRNA from tissues of Brassica campestris was either weak or undetectable. The cloning of an ACP-I gene represents an initial step in the molecular dissection of fatty acid synthetase in plants.
