Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1991 Mar 15;88(6):2578–2582. doi: 10.1073/pnas.88.6.2578

Primary structures of the precursor and mature forms of stearoyl-acyl carrier protein desaturase from safflower embryos and requirement of ferredoxin for enzyme activity.

G A Thompson 1, D E Scherer 1, S Foxall-Van Aken 1, J W Kenny 1, H L Young 1, D K Shintani 1, J C Kridl 1, V C Knauf 1
PMCID: PMC51276  PMID: 2006194

Abstract

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.

Full text

PDF
2578

Images in this article

2581Image
on p.2581

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alexander D. C. An efficient vector-primer cDNA cloning system. Methods Enzymol. 1987;154:41–64. doi: 10.1016/0076-6879(87)54069-1. [DOI] [PubMed] [Google Scholar]
  2. Boylan M. T., Quail P. H. Oat Phytochrome Is Biologically Active in Transgenic Tomatoes. Plant Cell. 1989 Aug;1(8):765–773. doi: 10.1105/tpc.1.8.765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Girgis S. I., Alevizaki M., Denny P., Ferrier G. J., Legon S. Generation of DNA probes for peptides with highly degenerate codons using mixed primer PCR. Nucleic Acids Res. 1988 Nov 11;16(21):10371–10371. doi: 10.1093/nar/16.21.10371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Goldberg R. B., Hoschek G., Tam S. H., Ditta G. S., Breidenbach R. W. Abundance, diversity, and regulation of mRNA sequence sets in soybean embryogenesis. Dev Biol. 1981 Apr 30;83(2):201–217. doi: 10.1016/0012-1606(81)90467-x. [DOI] [PubMed] [Google Scholar]
  5. Jackson A. O., Larkins B. A. Influence of Ionic Strength, pH, and Chelation of Divalent Metals on Isolation of Polyribosomes from Tobacco Leaves. Plant Physiol. 1976 Jan;57(1):5–10. doi: 10.1104/pp.57.1.5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Kay S. A., Nagatani A., Keith B., Deak M., Furuya M., Chua N. H. Rice Phytochrome Is Biologically Active in Transgenic Tobacco. Plant Cell. 1989 Aug;1(8):775–782. doi: 10.1105/tpc.1.8.775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Klein K., Steinberg R., Fiethen B., Overath P. Fatty acid degradation in Escherichia coli. An inducible system for the uptake of fatty acids and further characterization of old mutants. Eur J Biochem. 1971 Apr;19(3):442–450. doi: 10.1111/j.1432-1033.1971.tb01334.x. [DOI] [PubMed] [Google Scholar]
  8. Knoell H. E., Knappe J. Escherichia coli ferredoxin, an iron-sulfur protein of the adrenodoxin type. Eur J Biochem. 1974 Dec 16;50(1):245–252. doi: 10.1111/j.1432-1033.1974.tb03893.x. [DOI] [PubMed] [Google Scholar]
  9. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  10. Lütcke H. A., Chow K. C., Mickel F. S., Moss K. A., Kern H. F., Scheele G. A. Selection of AUG initiation codons differs in plants and animals. EMBO J. 1987 Jan;6(1):43–48. doi: 10.1002/j.1460-2075.1987.tb04716.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. McKeon T. A., Stumpf P. K. Purification and characterization of the stearoyl-acyl carrier protein desaturase and the acyl-acyl carrier protein thioesterase from maturing seeds of safflower. J Biol Chem. 1982 Oct 25;257(20):12141–12147. [PubMed] [Google Scholar]
  12. Nagai J., Bloch K. Enzymatic desaturation of stearyl acyl carrier protein. J Biol Chem. 1968 Sep 10;243(17):4626–4633. [PubMed] [Google Scholar]
  13. Post-Beittenmiller M. A., Schmid K. M., Ohlrogge J. B. Expression of holo and apo forms of spinach acyl carrier protein-I in leaves of transgenic tobacco plants. Plant Cell. 1989 Sep;1(9):889–899. doi: 10.1105/tpc.1.9.889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Rock C. O., Cronan J. E., Jr Acyl carrier protein from Escherichia coli. Methods Enzymol. 1981;71(Pt 100):341–351. doi: 10.1016/0076-6879(81)71043-7. [DOI] [PubMed] [Google Scholar]
  15. Rock C. O., Garwin J. L., Cronan J. E., Jr Preparative enzymatic synthesis of acyl-acyl carrier protein. Methods Enzymol. 1981;72:397–403. doi: 10.1016/s0076-6879(81)72029-9. [DOI] [PubMed] [Google Scholar]
  16. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Sheehy R. E., Kramer M., Hiatt W. R. Reduction of polygalacturonase activity in tomato fruit by antisense RNA. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8805–8809. doi: 10.1073/pnas.85.23.8805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Sparace S. A., Mudd J. B. Phosphatidylglycerol synthesis in spinach chloroplasts: characterization of the newly synthesized molecule. Plant Physiol. 1982 Nov;70(5):1260–1264. doi: 10.1104/pp.70.5.1260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  20. Talamo B. R., Bloch K. A new assay for fatty acid desaturation. Anal Biochem. 1969 May;29(2):300–304. doi: 10.1016/0003-2697(69)90313-3. [DOI] [PubMed] [Google Scholar]
  21. Thiede M. A., Ozols J., Strittmatter P. Construction and sequence of cDNA for rat liver stearyl coenzyme A desaturase. J Biol Chem. 1986 Oct 5;261(28):13230–13235. [PubMed] [Google Scholar]
  22. Wada M., Fukunaga N., Sasaki S. Mechanism of biosynthesis of unsaturated fatty acids in Pseudomonas sp. strain E-3, a psychrotrophic bacterium. J Bacteriol. 1989 Aug;171(8):4267–4271. doi: 10.1128/jb.171.8.4267-4271.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES