Molecular Analysis of a Bifunctional Fatty Acid Conjugase/Desaturase from Tung. Implications for the Evolution of Plant Fatty Acid Diversity1

摘要

The seed oil derived from the tung (Aleurites fordii Hemsl.) tree contains approximately 80% α-eleostearic acid (18:3Δ9cis,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 Δ12 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 (18:1Δ9cis) into linoleic acid (18:2Δ9cis,12cis) and that FADX converted linoleic acid into α-eleostearic acid. Additional characterization revealed that FADX exhibited remarkable enzymatic plasticity, capable of generating a variety of alternative conjugated and Δ12-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 Δ12 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.