Δ6 Hexadecenoic acid is synthesized by the activity of a soluble Δ6 palmitoyl-acyl carrier protein desaturase in Thunbergia alata endosperm

Edgar B Cahoon, Ann M. Cranmer, John Shanklin, John B. Ohlrogge

Research output: Contribution to journalArticle

55 Citations (Scopus)

Abstract

Δ6 Hexadecenoic acid (16:1Δ6) composes more than 80% of the seed oil of Thunbergia alata. Studies were conducted to determine the biosynthetic origin of the double bond of this unusual fatty acid. Assays of fractions of developing T. alata seed endosperm with [1-14C]palmitoyl (16:0)-acyl carrier protein (ACP) revealed the presence of a soluble Δ6 desaturase activity. This activity was greatest when 16:0-ACP was provided as a substrate, whereas no desaturation of the coenzyme A ester of this fatty acid was detected. In addition, Δ616:0-ACP desaturase activity in T. alata endosperm extracts was dependent on the presence of ferredoxin and molecular oxygen and was stimulated by catalase. To further characterize this enzyme, a cDNA encoding a diverged acyl-ACP desaturase was isolated from a T. alata endosperm cDNA library using polymerase chain reaction with degenerate oligonucleotides corresponding to conserved amino acid sequences in Δ9stearoyl (18:0)- and Δ416:0-ACP desaturases. The primary structure of the mature peptide encoded by this cDNA shares 66% identity with the mature castor Δ918:0-ACP desaturase and 57% identity with the mature coriander Δ416:0-ACP desaturase. Extracts of Escherichia coli that express the T. alata cDNA catalyzed the Δ6 desaturation of 16:0-ACP. These results demonstrate that 16:1Δ6 in T. alata endosperm is formed by the activity of a soluble Δ616:0-ACP desaturase that is structurally related to the Δ918:0- and Δ416:0-ACP desaturases. Implications of this work to an understanding of active site structures of acyl-ACP desaturases are discussed.

Original languageEnglish (US)
Pages (from-to)27519-27526
Number of pages8
JournalJournal of Biological Chemistry
Volume269
Issue number44
StatePublished - Nov 4 1994

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Acanthaceae
Acyl Carrier Protein
Endosperm
acyl-(acyl-carrier-protein)desaturase
Complementary DNA
Seeds
Fatty Acids
Coriandrum
hexadecenoic acid
Ferredoxins
Oilseeds
Molecular oxygen
Polymerase chain reaction
Coenzyme A
Gene Library
Oligonucleotides
Catalase
Escherichia coli
Seed
Amino Acid Sequence

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Δ6 Hexadecenoic acid is synthesized by the activity of a soluble Δ6 palmitoyl-acyl carrier protein desaturase in Thunbergia alata endosperm. / Cahoon, Edgar B; Cranmer, Ann M.; Shanklin, John; Ohlrogge, John B.

In: Journal of Biological Chemistry, Vol. 269, No. 44, 04.11.1994, p. 27519-27526.

Research output: Contribution to journalArticle

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abstract = "Δ6 Hexadecenoic acid (16:1Δ6) composes more than 80{\%} of the seed oil of Thunbergia alata. Studies were conducted to determine the biosynthetic origin of the double bond of this unusual fatty acid. Assays of fractions of developing T. alata seed endosperm with [1-14C]palmitoyl (16:0)-acyl carrier protein (ACP) revealed the presence of a soluble Δ6 desaturase activity. This activity was greatest when 16:0-ACP was provided as a substrate, whereas no desaturation of the coenzyme A ester of this fatty acid was detected. In addition, Δ616:0-ACP desaturase activity in T. alata endosperm extracts was dependent on the presence of ferredoxin and molecular oxygen and was stimulated by catalase. To further characterize this enzyme, a cDNA encoding a diverged acyl-ACP desaturase was isolated from a T. alata endosperm cDNA library using polymerase chain reaction with degenerate oligonucleotides corresponding to conserved amino acid sequences in Δ9stearoyl (18:0)- and Δ416:0-ACP desaturases. The primary structure of the mature peptide encoded by this cDNA shares 66{\%} identity with the mature castor Δ918:0-ACP desaturase and 57{\%} identity with the mature coriander Δ416:0-ACP desaturase. Extracts of Escherichia coli that express the T. alata cDNA catalyzed the Δ6 desaturation of 16:0-ACP. These results demonstrate that 16:1Δ6 in T. alata endosperm is formed by the activity of a soluble Δ616:0-ACP desaturase that is structurally related to the Δ918:0- and Δ416:0-ACP desaturases. Implications of this work to an understanding of active site structures of acyl-ACP desaturases are discussed.",
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N2 - Δ6 Hexadecenoic acid (16:1Δ6) composes more than 80% of the seed oil of Thunbergia alata. Studies were conducted to determine the biosynthetic origin of the double bond of this unusual fatty acid. Assays of fractions of developing T. alata seed endosperm with [1-14C]palmitoyl (16:0)-acyl carrier protein (ACP) revealed the presence of a soluble Δ6 desaturase activity. This activity was greatest when 16:0-ACP was provided as a substrate, whereas no desaturation of the coenzyme A ester of this fatty acid was detected. In addition, Δ616:0-ACP desaturase activity in T. alata endosperm extracts was dependent on the presence of ferredoxin and molecular oxygen and was stimulated by catalase. To further characterize this enzyme, a cDNA encoding a diverged acyl-ACP desaturase was isolated from a T. alata endosperm cDNA library using polymerase chain reaction with degenerate oligonucleotides corresponding to conserved amino acid sequences in Δ9stearoyl (18:0)- and Δ416:0-ACP desaturases. The primary structure of the mature peptide encoded by this cDNA shares 66% identity with the mature castor Δ918:0-ACP desaturase and 57% identity with the mature coriander Δ416:0-ACP desaturase. Extracts of Escherichia coli that express the T. alata cDNA catalyzed the Δ6 desaturation of 16:0-ACP. These results demonstrate that 16:1Δ6 in T. alata endosperm is formed by the activity of a soluble Δ616:0-ACP desaturase that is structurally related to the Δ918:0- and Δ416:0-ACP desaturases. Implications of this work to an understanding of active site structures of acyl-ACP desaturases are discussed.

AB - Δ6 Hexadecenoic acid (16:1Δ6) composes more than 80% of the seed oil of Thunbergia alata. Studies were conducted to determine the biosynthetic origin of the double bond of this unusual fatty acid. Assays of fractions of developing T. alata seed endosperm with [1-14C]palmitoyl (16:0)-acyl carrier protein (ACP) revealed the presence of a soluble Δ6 desaturase activity. This activity was greatest when 16:0-ACP was provided as a substrate, whereas no desaturation of the coenzyme A ester of this fatty acid was detected. In addition, Δ616:0-ACP desaturase activity in T. alata endosperm extracts was dependent on the presence of ferredoxin and molecular oxygen and was stimulated by catalase. To further characterize this enzyme, a cDNA encoding a diverged acyl-ACP desaturase was isolated from a T. alata endosperm cDNA library using polymerase chain reaction with degenerate oligonucleotides corresponding to conserved amino acid sequences in Δ9stearoyl (18:0)- and Δ416:0-ACP desaturases. The primary structure of the mature peptide encoded by this cDNA shares 66% identity with the mature castor Δ918:0-ACP desaturase and 57% identity with the mature coriander Δ416:0-ACP desaturase. Extracts of Escherichia coli that express the T. alata cDNA catalyzed the Δ6 desaturation of 16:0-ACP. These results demonstrate that 16:1Δ6 in T. alata endosperm is formed by the activity of a soluble Δ616:0-ACP desaturase that is structurally related to the Δ918:0- and Δ416:0-ACP desaturases. Implications of this work to an understanding of active site structures of acyl-ACP desaturases are discussed.

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