Plant Molecular Biology 33: 1105–1110, 1997.
1997 Kluwer Academic Publishers. Printed in Belgium.
Characterization of a structurally and functionally diverged acyl-acyl
carrier protein desaturase from milkweed seed
Edgar B. Cahoon
, Sean J. Coughlan
and John Shanklin
Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA (
author for correspondence);
Department of Biotechnology Research, Pioneer Hi-Bred International, Inc., Johnston, IA 50131, USA
Received 17 October 1996; accepted in revised form 15 January 1997
Key words: fatty acid desaturase, Asclepias syriaca
A cDNA for a structurally variant acyl-acylcarrier protein (ACP) desaturase was isolated from milkweed (Asclepias
syriaca) seed, a tissue enriched in palmitoleic (16:1
and cis-vaccenic (18:1
) acids. Extracts of Escherichia
coli that express the milkweed cDNA catalyzed
desaturation of acyl-ACP substrates, and the recombinant
enzyme exhibited seven- to ten-fold greater speciﬁcity for palmitoyl (16:0)-ACP and 30-fold greater speciﬁcity for
myristoyl (14:0)-ACPthan did known
-stearoyl(18:0)-ACPdesaturases. Like other variant acyl-ACPdesaturases
reported to date, the milkweed enzyme contains fewer amino acids near its N-terminus compared to previously
-18:0-ACP desaturases. Based on the activity of an N-terminal deletion mutant of a
desaturase, this structural feature likely does not account for differences in substrate speciﬁcities.
Acyl-acyl carrier protein(ACP) desaturasesaresoluble
enzymes that catalyze the insertion of a double bond
into saturated fatty acids bound to ACP in plants and
photoauxotrophic Euglena [17, 18]. The most widely-
occurring member of this family is the
(18:0)-ACP desaturase [10, 17], which is found in
nearly all plant tissues. In addition to this enzyme,
three variant acyl-ACP desaturases have been reported
to date: the
-palmitoyl (16:0)-ACP desaturase of
coriander (Coriandrum sativum) seed [3, 4], the
16:0-ACPdesaturaseof Thunbergiaalata seed , and
-myristoyl (14:0)-ACP desaturase of geranium
(Pelargonium xhortorum) trichomes . In spite of
their different functional properties, these enzymes
70% amino acid sequence similarity. The
most notable difference between the primary struc-
Fatty acid nomenclature:
indicates that the double bond is
located (or inserted) at the x carbon atom relative to the carboxyl
terminus of a fatty acid. X:Y indicates that a fatty acid contains X
number of carbon atoms and Y number of double bonds (e.g. 18:1).
The nucleotide sequence reported will appear in the GenBank,
EMBL and DDBJ Nucleotide Sequence Databases under the acces-
sion number U60277.
tures of the
-18:0-ACP desaturase and the three
diverged acyl-ACP desaturases are variations in num-
bers of amino acids in a region near their N-termini.
For example, the
-16:0-ACP desaturase contains 15
fewer amino acids in this region relative to known
18:0-ACP desaturases .
Weare currently attemptingto understand thestruc-
tural basis for the different chain-length and double
bond positional speciﬁcities of acyl-ACP desaturases.
One approach that we are using is site-directed muta-
genesis based on amino acid sequence alignments of
these enzymes. The goal of these studies is to con-
vert the activity of one type of acyl-ACP desaturase to
that of another by the replacement of speciﬁc residues.
In addition, with the recent availability of a three-
dimensional structure for the
, it is now possible to derive comparative models
of the substrate binding pockets of different types of
To enhance the usefulness of amino acid sequence
alignments and active site modeling, we are continu-
ing to pursue the isolation of cDNAs for acyl-ACP
desaturases with previously uncharacterized activities.
GR: 201001892, Pips nr. 133452 BIO2KAP
pl375us.tex; 2/04/1997; 7:48; v.7; p.1