Plant Physiology Preview
Published on November 7, 2002; 10.1104/pp.102.010835
Received July 8, 2002
Returned for revision August 30, 2002
Accepted September 2, 2002
Molecular Analysis of a Bifunctional Fatty Acid Conjugase/Desaturase from Tung. Implications for the Evolution of Plant Fatty Acid Diversity
John M. Dyer *, Dorselyn C. Chapital , Jui-Chang W. Kuan , Robert T. Mullen , Charlotta Turner , Thomas A. McKeon , and Armand B. Pepperman
United States Department of Agriculture-Agricultural Research Service Southern Regional Research Center, 1100 Robert E. Lee Boulevard, New Orleans, Louisiana 70124 (J.M.D., D.C.C., J.-C.W.K., A.B.P.); Department of Botany, University of Guelph, Guelph, Ontario, Canada N1G 2W1 (R.T.M.); and United States Department of Agriculture-Agricultural Research Service Western Regional Research Center, 800 Buchanan Street, Albany, California 94710 (C.T., T.A.M.)
* Corresponding author; email: jdyer{at}nola.srrc.usda.gov.
The seed oil derived from the tung (Aleurites fordii Hemsl.) tree contains approximately 80%  -eleostearic acid (18:3 9cis,11trans,13trans), an unusual conjugated fatty acid that imparts industrially important drying qualities to tung oil. Here, we describe the cloning and functional analysis of two closely related 12 oleate desaturase-like enzymes that constitute consecutive steps in the biosynthetic pathway of eleostearic acid. Polymerase chain reaction screening of a tung seed cDNA library using degenerate oligonucleotide primers resulted in identification of two desaturases, FAD2 and FADX, that shared 73% amino acid identity. Both enzymes were localized to the endoplasmic reticulum of tobacco (Nicotiana tabacum cv Bright-Yellow 2) cells, and reverse transcriptase-polymerase chain reaction revealed that FADX was expressed exclusively within developing tung seeds. Expression of the cDNAs encoding these enzymes in yeast (Saccharomyces cerevisiae) revealed that FAD2 converted oleic acid (18:19cis) into linoleic acid (18:29cis,12cis) and that FADX converted linoleic acid into -eleostearic acid. Additional characterization revealed that FADX exhibited remarkable enzymatic plasticity, capable of generating a variety of alternative conjugated and 12-desaturated fatty acid products in yeast cells cultured in the presence of exogenously supplied fatty acid substrates. Unlike other desaturases reported to date, the double bond introduced by FADX during fatty acid desaturation was in the trans, rather than cis, configuration. Phylogenetic analysis revealed that tung FADX is grouped with 12 fatty acid desaturases and hydroxylases rather than conjugases, which is consistent with its desaturase activity. Comparison of FADX and other lipid-modifying enzymes (desaturase, hydroxylase, epoxygenase, acetylenase, and conjugase) revealed several amino acid positions near the active site that may be important determinants of enzymatic activity.
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