Primary structures of the precursor and mature forms of stearoyl-acyl carrier protein desaturase from safflower embryos and requirement of ferredoxin for enzyme activity.
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.
Thompson
G A
GA
Calgene, Inc., Davis, CA 95616.
Scherer
D E
DE
Foxall-Van Aken
S
S
Kenny
J W
JW
Young
H L
HL
Shintani
D K
DK
Kridl
J C
JC
Knauf
V C
VC
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005288:Ferredoxins / Q000378:metabolism*; D015723:Gene Library; D017343:Genes, Plant; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010944:Plants / Q000235:genetics; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012639:Seeds / Q000201:enzymology*
D005288:Ferredoxins; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D011994:Recombinant Proteins; D006899:Mixed Function Oxygenases; C018747:acyl-(acyl-carrier-protein)desaturase