Publications

• Involvement of ferredoxin in desaturation of lipid-bound oleate in chloroplasts.

  Schmidt H., Heinz E.

  Intact spinach (Spinacia oleracea) chloroplasts, pulse-labeled with [(14)C]acetate, desaturate newly formed fatty acids as ester groups of monogalactosyl diacylglycerol in a subsequent chase in the dark. Rupture of pulse-labeled chloroplasts by addition of a detergent solution 3-([3-cholamidopropy ... >> More

  Intact spinach (Spinacia oleracea) chloroplasts, pulse-labeled with [(14)C]acetate, desaturate newly formed fatty acids as ester groups of monogalactosyl diacylglycerol in a subsequent chase in the dark. Rupture of pulse-labeled chloroplasts by addition of a detergent solution 3-([3-cholamidopropyl]dimethylammonio)-1-propane sulfonate preserves part of this desaturation activity. Direct addition of different free fatty acids together with appropriate cofactors to detergent-ruptured chloroplasts results in fatty acid labeling of monogalactosyl diacylglycerol. During subsequent incubation these lipid-linked fatty acids are desaturated, i.e. 18:1 to 18:2 and 18:3 and to a small extent also 16:0 to 16:3. The formation of 18:2 was also observed after incorporation of 18:1 into sulfolipid and phosphatidyl choline. Density gradient centrifugation separated a membrane fraction from detergent-ruptured chloroplasts which in the presence of appropriate cofactors incorporated 18:1 and 18:2 into the above-mentioned lipids. In the light, desaturation was dependent on added ferredoxin, whereas in the dark, in addition to ferredoxin NAD(P)H was also required. Preliminary evidence for the involvement of membrane-bound ferredoxin:NADP oxidoreductase (FNR) as a third component of desaturation in the dark was obtained by inhibitor studies including antibodies against FNR. Desaturation of lipid-bound 18:1 and 18:2 resembles stearoyl-ACP desaturation with respect to its requirement of reduced ferredoxin and oxygen. << Less

  Plant Physiol 94:214-220(1990) [PubMed] [EuropePMC]

• Purification and PCR-based cDNA cloning of a plastidial n-6 desaturase.

  Schmidt H., Dresselhaus T., Buck F., Heinz E.

  A plastidial membrane-bound n-6 desaturase from spinach (Spinacia oleracea) was purified from chloroplast envelope membranes by anion exchange, cation exchange and ferredoxin-affinity chromatography. The molecular mass of the protein was estimated by SDS-PAGE to be 40 kDa. The highest specific act ... >> More

  A plastidial membrane-bound n-6 desaturase from spinach (Spinacia oleracea) was purified from chloroplast envelope membranes by anion exchange, cation exchange and ferredoxin-affinity chromatography. The molecular mass of the protein was estimated by SDS-PAGE to be 40 kDa. The highest specific activity of the desaturase in the final preparation was 196 nmol/min per mg protein with free oleic acid as the substrate. The N-terminal amino acid sequence of the blotted protein was determined and used for the construction of a degenerated and inosine-containing oligonucleotide primer for PCR experiments with cDNA transcribed from leaf mRNA. A 3'-RACE experiment with this primer amplified a single band of 1500 bp that after sequencing showed an open reading frame of 382 amino acids corresponding to a protein of 43 kDa. The 5' end of the cDNA was amplified by a 5'-RACE experiment and isolated as a 500 bp fragment. Sequencing of this DNA revealed an additional 65 amino acids at the N-terminus of the native protein that are attributed to a plastidial leader peptide. With appropriate primers derived from these sequences a full-length clone was amplified by PCR and sequenced. Comparison of the plastidial oleate desaturase with the homologous enzyme from cyanobacteria showed about 50% amino acid homology. Comparison with other desaturases revealed three histidine boxes with the general sequence HXXXH that are highly conserved in all membrane-bound desaturases. These boxes might be involved in metal ion complexation required for reduction of oxygen. << Less

  Plant Mol. Biol. 26:631-642(1994) [PubMed] [EuropePMC]

• Desaturation of oleoyl groups in envelope membranes from spinach chloroplasts.

  Schmidt H., Heinz E.

  Envelope membranes isolated from chloroplasts of spinach (Spinacia oleracea) desaturate oleoyl groups in monogalactosyl diacylglycerol to linoleoyl groups. The desaturation requires NADPH in combination with ferredoxin and is not restricted to monogalactosyl diacylglycerol, since it is also observ ... >> More

  Envelope membranes isolated from chloroplasts of spinach (Spinacia oleracea) desaturate oleoyl groups in monogalactosyl diacylglycerol to linoleoyl groups. The desaturation requires NADPH in combination with ferredoxin and is not restricted to monogalactosyl diacylglycerol, since it is also observed in biosynthetic intermediates as, for example, in phosphatidic acid. This indicates a certain degree of unspecificity of the oleate desaturase in isolated envelope membranes. Lipid desaturation is another important function of chloroplast envelopes. << Less

  Proc Natl Acad Sci U S A 87:9477-9480(1990) [PubMed] [EuropePMC]

• Identification of a gene that complements an Arabidopsis mutant deficient in chloroplast omega 6 desaturase activity.

  Falcone D.L., Gibson S., Lemieux B., Somerville C.R.

  Membrane lipids of the fad6 (formerly fadC) mutant of Arabidopsis, which is deficient in chloroplast omega 6 desaturase activity, have increased levels of monounsaturated fatty acids and are deficient in trienoic fatty acids. A putative fad6 cDNA clone was isolated by probing a cDNA library with a ... >> More

  Membrane lipids of the fad6 (formerly fadC) mutant of Arabidopsis, which is deficient in chloroplast omega 6 desaturase activity, have increased levels of monounsaturated fatty acids and are deficient in trienoic fatty acids. A putative fad6 cDNA clone was isolated by probing a cDNA library with a degenerate oligonucleotide based on a conserved region within known omega 3 desaturase genes. Expression of the cDNA in transgenic plants of a fad6 mutant restored normal levels of all fatty acids. When used as a hybridization probe, the cDNA identified a restriction fragment-length polymorphism that co-segregated with the fad6 mutation. Thus, on the basis of a genetic complementation test and genetic map position, the fad6 gene is encoded by the cDNA. The cDNA encoded a 418-amino acid polypeptide of 47,727 D that displayed a high degree of sequence similarity to a delta 12 desaturase from the cyanobacterium Synechocystis. The fad6 gene exhibited less sequence homology to any known higher plant desaturase, including an endoplasmic reticulum-localized omega 6 desaturase corresponding to the Arabidopsis fad2 gene. << Less

  Plant Physiol. 106:1453-1459(1994) [PubMed] [EuropePMC]

• Cloning of a higher-plant plastid omega-6 fatty acid desaturase cDNA and its expression in a cyanobacterium.

  Hitz W.D., Carlson T.J., Booth J.R. Jr., Kinney A.J., Stecca K.L., Yadav N.S.

  Oligomers based on amino acids conserved between known plant omega-3 and cyanobacterium omega-6 fatty acid desaturases were used to screen an Arabidopsis cDNA library for related sequences. An identified clone encoding a novel desaturase-like polypeptide was used to isolate its homologs from Glyci ... >> More

  Oligomers based on amino acids conserved between known plant omega-3 and cyanobacterium omega-6 fatty acid desaturases were used to screen an Arabidopsis cDNA library for related sequences. An identified clone encoding a novel desaturase-like polypeptide was used to isolate its homologs from Glycine max and Brassica napus. The plant deduced amino acid sequences showed less than 27% similarity to known plant omega-6 and omega-3 desaturases but more than 48% similarity to cyanobacterial omega-6 desaturase, and they contained putative plastid transit sequences. Thus, we deduce that the plant cDNAs encode the plastid omega-6 desaturase. The identity was supported by expression of the B. napus cDNA in cyanobacterium. Synechococcus transformed with a chimeric gene that contains a prokaryotic promoter fused to the rapeseed cDNA encoding all but the first 73 amino acids partially converted its oleic acid fatty acid to linoleic acid, and the 16:1(9c) fatty acid was converted primarily to 16:2(9c, 12) in vivo. Thus, the plant omega-6 desaturase, which utilizes 16:1(7c) in plants, can utilize 16:1(9c) in the cyanobacterium. The plastid and cytosolic homologs of plant omega-6 desaturases are much more distantly related than those of omega-3 desaturases. << Less

  Plant Physiol. 105:635-641(1994) [PubMed] [EuropePMC]