First published online November 7, 2002; 10.1104/pp.102.010835
Plant Physiol, December 2002, Vol. 130, pp. 2027-2038
Molecular Analysis of a Bifunctional Fatty Acid
Conjugase/Desaturase from Tung. Implications for the Evolution of Plant
Fatty Acid Diversity1
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.)
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.
1
This work was supported by the U.S. Department
of Agriculture-Agricultural Research Service (Current Research
Information System project no. 6435-41000-049-00D), by the Natural
Sciences and Engineering Research Council of Canada (grant no. 217291), and by the Ontario Premier's Research in Excellence Award (to R.T.M.).
*
Corresponding author; e-mail jdyer{at}nola.srrc.usda.gov; fax
504-286-4419.
© 2002 American Society of Plant Biologists
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