Plant Physiol. (1998) 117: 593-598
A Determinant of Substrate Specificity Predicted from the
Acyl-Acyl Carrier Protein Desaturase of Developing
Cat's Claw
Seed1
Edgar B. Cahoon2, 3,
Salehuzzaman Shah2, 4,
John Shanklin, and
John Browse*
Biology Department, Brookhaven National Laboratory, Upton, New York
11976 (E.B.C., J.S.); and Institute of Biological Chemistry,
Washington State University, P.O. Box 646340, Pullman, Washington
99164-6340 (S.S., J.B.)
 |
ABSTRACT |
Cat's
claw (Doxantha unguis-cati L.) vine accumulates nearly
80% palmitoleic acid (16:1
9) plus cis-vaccenic acid
(18:111) in its seed oil. To characterize the biosynthetic origin of
these unusual fatty acids, cDNAs for acyl-acyl carrier protein
(acyl-ACP) desaturases were isolated from developing cat's claw seeds.
The predominant acyl-ACP desaturase cDNA identified encoded a
polypeptide that is closely related to the stearoyl (9-18:0)-ACP
desaturase from castor (Ricinis communis
L.) and other species. Upon expression in
Escherichia coli, the cat's claw polypeptide functioned
as a 9 acyl-ACP desaturase but displayed a distinct substrate
specificity for palmitate (16:0)-ACP rather than stearate (18:0)-ACP.
Comparison of the predicted amino acid sequence of the cat's claw
enzyme with that of the castor 9-18:0-ACP desaturase suggested that a single amino acid substitution (L118W) might account in large part
for the differences in substrate specificity between the two
desaturases. Consistent with this prediction, conversion of leucine-118
to tryptophan in the mature castor 9-18:0-ACP desaturase resulted
in an 80-fold increase in the relative specificity of this enzyme for
16:0-ACP. The alteration in substrate specificity observed in the L118W
mutant is in agreement with a crystallographic model of the proposed
substrate-binding pocket of the castor 9-18:0-ACP desaturase.
| |
INTRODUCTION |
The seed oils of higher plants contain many different fatty acids
that determine the value of a particular oil for human nutrition or as
a source of industrial chemicals (Murphy, 1994
; Ohlrogge, 1994). For
this reason, there is considerable interest in bringing about useful
changes in the fatty acid composition of oilseed crops. One of the most
promising areas of research in this regard relates to protein
engineering of acyl-ACP desaturases (Cahoon et al., 1997b). The
acyl-ACP desaturases are a family of closely related, soluble enzymes
that catalyze insertion of the first double bond in a saturated fatty
acyl chain (Cahoon et al., 1997a). The most widely occurring member of
the family is the 9-18:0-ACP desaturase, which is responsible for
18:1 synthesis in plants (Nagai and Bloch, 1968; Shanklin and
Somerville, 1991; Thompson et al., 1991). In addition, several other
acyl-ACP desaturases have been described: the 4-16:0-ACP desaturase
of coriander seed (Cahoon et al., 1992), the 6-16:0-ACP desaturase
from blacked-eyed Susan (Thunbergia alata) seed (Cahoon et
al., 1994a), and the 9-14:0-ACP desaturase of geranium trichomes
(Schultz et al., 1996). The characterization of these variant enzymes
and the availability of a crystal structure for the castor
Ricinus communis L. 9-18:0-ACP desaturase (Lindqvist et
al., 1996) raises the possibility that protein engineering can be
used to manipulate the double-bond position and substrate specificities
of these enzymes. Unusual monounsaturated fatty acids produced by
redesigned desaturases may be useful for generating seed oils with
altered physical properties and new commercial applications.
Based on previous studies (Cahoon et al., 1992,
1994a; Cahoon and Ohlrogge, 1994b; Schultz et al., 1996), seeds and
other plant tissues that accumulate large amounts of unusual
monounsaturated fatty acids represent potential sources of variant
forms of acyl-ACP desaturases. The seed oils of several plant species
are rich sources of 16:19 and its elongation product, 18:111
(Chisholm and Hopkins, 1965). These unusual fatty acids can account for
25 to 80% of the seed oils of such species. A likely route of
synthesis of 16:19 and 18:111 would initially involve the 9
desaturation of 16:0-ACP by an acyl-ACP desaturase with enhanced
specificity for this substrate relative to known 9-18:0-ACP
desaturases. In this regard, a diverged acyl-ACP desaturase was
recently identified in milkweed seed (Cahoon et al., 1997a), a tissue
that accumulates 10% 16:19 and 15% 18:111. Although this enzyme
displayed elevated activity with 16:0-ACP compared with known
9-18:0-ACP desaturases, it was most active in vitro with 18:0-ACP.
A similar substrate-specificity profile was also observed when acyl-ACP
desaturase activity was assayed in crude homogenates of developing
milkweed seed.
Because 16:19 is found in the seed oils of very divergent species,
it is likely that 9-16:0-ACP desaturases have evolved separately
several times. This suggests the possibility that distinct molecular
changes were involved in producing different 9-16:0-ACP desaturases
from the ancestral 9-18:0-ACP enzyme. As an alternative source
of a 9-16:0-ACP desaturase, we have examined acyl-ACP desaturase
cDNAs in developing seed of cat's claw (Doxantha
unguis-cati L.). This species is unrelated to milkweed and
accumulates nearly 80% 16:19 plus 18:111 in its seed oil
(Chisholm and Hopkins, 1965). In this report we describe the cloning of
a cat's claw cDNA that encodes a 16:0-specific 9-ACP desaturase. By
comparing the amino acid sequence of this enzyme with that of the
castor 9-18:0-ACP desaturase, we were able to identify a single
amino acid substitution located at the base of the substrate cleft, which, when introduced into the castor enzyme, dramatically increased its relative specificity for 16:0- versus 18:0-ACP.
| |
MATERIALS AND METHODS |
Immature fruits of cat's claw (Doxantha unguis-cati
L.) were harvested from plants on the campus of Louisiana
State University (Baton Rouge). Developing seeds were extracted from
fruit pods, frozen in liquid N2, and stored at
80°C until they were used for RNA isolation.
RNA Preparation and Northern Blotting
Approximately 1 g of plant tissue was ground in liquid
N2 in a precooled mortar and pestle. The fine
powder was transferred to 5 mL of a 1:1 mixture of phenol and
extraction buffer (0.1 M Tris-HCl, pH 8.0, 10 mM EDTA, and 1% SDS) at 80°C. This suspension was mixed
for 1 min. Chloroform:isoamyl alcohol (24:1, v/v) was then added and
the suspension was vortexed again. The sample was briefly centrifuged
to separate the phases, and the aqueous phase was recovered and mixed
with an equal volume of 4 M LiCl. The RNA was then allowed
to precipitate overnight at 4°C. The RNA pellet obtained was
dissolved in water after centrifugation and precipitated again with 0.1 volume of 3 M sodium acetate, pH 5.2, and 2 volumes of
ethanol. The pellet was washed with 70% alcohol, dried, dissolved in
water, and stored at 80°C.
Total RNA was run in a formaldehyde-formamide-denaturing agarose gel
and transferred to a nylon membrane with 20× SSC (Sambrook et al.,
1989). For hybridization, DNA was labeled with
[32P]dCTP to a specific activity of 3 × 108 dpm/mg DNA using random hexanucleotide
primers. Hybridization and washing of the blot were done at 65°C as
described previously (Shah et al., 1997).
Construction and Screening of a cDNA Library
mRNA was isolated from total RNA by adding biotinylated oligo(dT)
to the RNA sample and mixing thoroughly. The mRNA-oligo(dT) hybrid
molecules were captured with streptavidin-coated paramagnetic particles
in a magnetic separation strand (PolyATract mRNA isolation system,
Promega). Double-stranded cDNA was synthesized from the mRNA using a
Superscript cDNA synthesis kit obtained from GIBCO-BRL. Size-fractionated cDNA was ligated to the EcoRI arms of
Lambda-Ziplox (GIBCO-BRL) and packaged using Gigapack III
gold-packaging extract (Stratagene).
The cDNA library was screened by hybridization with DNA probes labeled
with [32P]dCTP. Blots were hybridized and
washed at 57°C. Plasmids carrying positive cDNA inserts were released
from the Lambda-Ziplox DNA by cre-loxP-mediated
recombination in Escherichia coli strain DH10BZIP according
to the manufacturer's protocol. Both strands of cDNA were sequenced by
fluorescent dideoxy-termination using a DNA sequencer (model 373A,
Applied Biosystems). Sequence information was analyzed with Genetics
Computer Group (Madison, WI) programs using default settings for
parameters unless otherwise indicated.
Desaturase Expression in E. coli
One of the positive cDNA clones isolated from the library pDU1 was
amplified by PCR. The 5
primer corresponded to amino acids 30 to 38 of
the polypeptide encoded by pDU1 and contained a flanking SphI site. The 3 primer was designed according to the
sequence immediately downstream of the stop codon, and a terminal
HindIII site was included. The PCR product was cloned in the
SphI-HindIII site of the E. coli expression vector pQE-32 (Qiagen, Chatsworth, CA), which
carries six His amino acid residues as a tag at the N terminus.
Ligation in the correct reading frame with the His tag was confirmed by
sequencing. The recombinant plasmid was introduced and expressed in
E. coli strain DH10B, which also carries the repressor
plasmid pREP4.
Six liters of E. coli DH10B cells harboring recombinant
plasmids carrying cat's claw cDNA c566 was grown to an
A600 = 0.4 at 37°C in BTNa medium (10 g/L
bacto-tryptone and 5 g/L NaCl), then induced by addition of
isopropylthio-
-galactoside (0.4 mM), and grown for an
additional 4 h at 30°C. Bacterial cells were harvested by
centrifugation and resuspended in 75 mL of a buffer consisting of 6.7 mM Mes/6.7 mM Hepes/6.7 mM Mops (pH
7.0) and 1 mM PMSF. The cells were lysed using a French
pressure cell at 100 mPa and the extract was centrifuged at
100,000g. The resulting soluble protein was loaded onto
a 1.7-mL Poros 20 HS cation-exchange column (PerSeptive Biosystems,
Inc., Framingham, MA) interfaced with a Biocad Sprint HPLC system
(PerSeptive Biosystems). Protein was loaded in cell lysis buffer
lacking PMSF and eluted over a gradient of 20 column-volumes from 0 to
600 mM NaCl in the Mes/Hepes/Mops buffer described above.
The cat's claw acyl-ACP desaturase was recovered at approximately 90%
purity, as judged by SDS-PAGE using Coomassie blue staining. The
enriched enzyme was exchanged into a buffer consisting of 40 mM Tris (pH 7.5), 50 mM NaCl, and 10% glycerol
by gel filtration using a PD-10 column (Pharmacia). The protein was
stored in aliquots at 70°C until use in enzyme assays.
In Vitro Assay
Activities of acyl-ACP desaturases were measured as described
elsewhere (Cahoon et al., 1994a) with minor modifications.
Anabaena nidulans vegetative Fd (22 µg/150 µL
assay) and maize root Fd:NADP+ oxidoreductase
(400 milliunits/assay) were used in place of spinach Fd and Fd
reductase. Both cofactors were purified from E. coli expressing the corresponding cDNA (Ritchie et al., 1994; Cheng et al.,
1995). No attempt was made to optimize assay conditions for the cat's
claw desaturase. However, the conditions used are likely to be suitable
because the cat's claw and castor (Ricinus communis L.)
enzymes both operate in the stroma of seed plastids.
[14C]Acyl-ACPs were synthesized according to
the method of Rock and Garwin (1979) using spinach ACP-I purified from
recombinant sources. [1-14C]Myristic, palmitic,
and stearic acids (American Radiolabeled Chemicals, Inc., St. Louis,
MO) were used for the synthesis of acyl-ACPs, and the specific
activity of each was 55 mCi/mmol.
Assay products were separated from the unreacted substrate by
argentation TLC as previously described (Cahoon and Ohlrogge, 1994b).
Radiation on TLC plates was detected using a phosphor imager (Molecular
Dynamics, Sunnyvale, CA), and the distribution of radiolabel between
the product and the unreacted substrate was determined using ImageQuant
software (Molecular Dynamics). Double-bond positions were determined by
the mobility of methyl esters of desaturation products on argentation
TLC relative to authentic standards.
Preparation of Mutant L118W of the Castor 9-18:0-ACP
Desaturase
Mutation L118W was introduced into the castor 9-18:0-ACP
desaturase by overlap-extension PCR (Ho et al., 1989). Two separate PCR
reactions were conducted using the coding sequence of the mature
wild-type castor 9-18:0-ACP desaturase in the vector
pET9d (Novagen, Madison, WI) as the template. One reaction was
performed using the primer combination T7 primer and
5-CCGAACTCCATCCCAGGTATTCAGCA-3 (the mutation is
underlined). The second reaction was conducted with the primer
combination 5-TGCTGAATACCTGGGATGGAGTTCGG-3 and 5-GCAAAAGCCAAAACGGTACCATCAGGATCA-3 (primer 1). Following agarose-gel purification, the products of the two reactions were
combined and amplified in a third PCR reaction using the T7 primer and primer 1 (without added template). The product of this reaction was gel
purified, digested with XbaI and BamHI, and
inserted in place of the corresponding portion of the mature wild-type
castor 9-18:0-ACP desaturase cDNA in pET9d. The presence of the
desired mutation was confirmed by DNA sequencing of the final
construct. The activity of the L118W mutant with radiolabeled acyl-ACP
substrates was determined following expression of this polypeptide in
E. coli and subsequent purification by cation-exchange
chromatography using the methods described above for analysis of the
recombinant cat's claw acyl-ACP desaturase.
The methods used to map residues lining the substrate-binding pocket
have been described (Lindqvist et al., 1996; Cahoon et al., 1997b). The
projection of eight residues of the castor enzyme followed that used by
Cahoon et al. (1997b). The substituted Trp in the L118W mutant was
drawn with the bulky side group extending into the substrate-binding
pocket.
| |
RESULTS AND DISCUSSION |
Isolation and Sequence Analysis of an Acyl-ACP Desaturase from
Cat's Claw Seed
The seed oil of cat's claw contains more than 75% 16:19 and
18:111 fatty acids (Chisholm and Hopkins, 1965). To assess the possible role of an acyl-ACP desaturase in the biosynthesis of these
unusual fatty acids, cDNAs for acyl-ACP desaturases from immature
cat's claw seeds were characterized. Initially, a
32P-labeled probe derived from a cDNA of the castor
9-18:0-ACP desaturase was used to screen approximately 30,000 plaques from a cDNA library derived from developing cat's claw seeds.
Twenty randomly selected, positive clones were converted to plasmids and subjected to restriction-enzyme mapping. All 20 clones shared a
common set of restriction fragments, indicating that they represented cDNAs from a single gene (data not shown). The inserts from 8 of these
plasmids were approximately the same size and longer than the remaining
12. One of these, designated pDU1, was chosen for further analysis.
The insert in pDU1 was fully sequenced in both directions, and this
sequence has been deposited in the GenBank database under accession no.
AF051134. Sequence analysis revealed an ORF of 1188 bp flanked by 19 bp
of 5-untranslated sequence and 300 bp of 3-untranslated sequence plus
a poly(A+) tail. The sequence of nucleotides flanking the
proposed translation initiation site, AAAATGGC, differs from the
eukaryotic consensus sequence ACAATGGC (Joshi, 1987) by only one
nucleotide and is identical to the corresponding sequences in cDNAs
encoding the 16:0-ACP desaturases of black-eyed Susan and coriander
(Cahoon and Ohlrogge, 1994b; Cahoon et al., 1994a). No in-frame stop
codon is present upstream of the ATG, but alignment of this ATG with the start codons in other acyl-ACP desaturases support its
identification as the correct translation start.
The translated ORF of pDU1 corresponded to a polypeptide of 396 amino
acids. Based on similarities with other acyl-ACP desaturases, the first
33 amino acids likely represent a plastid transit peptide. The
calculated molecular mass of the mature protein was 41.7 kD. When
compared with sequences available in the GenBank database, the
predicted protein shares considerable sequence similarity with all of
the acyl-ACP desaturases. The desaturases from coriander, geranium,
milkweed, and black-eyed Susan all show deletions (from 6 to 22 residues) relative to the castor sequence near the amino terminus of
the mature protein (Cahoon et al., 1997a). By contrast, the cat's claw
protein had no deletions and was in fact entirely colinear with
the castor 9-18:0-ACP desaturase (Fig.
1). The cat's claw and castor sequences
were 85% identical (90% similar with conservative amino acid
substitutions). This remarkably high degree of homology compares with
62 to 65% identity (72-74% similarity) for pairwise comparisons
between the cat's claw or castor sequence and the enzyme from
milkweed.
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| Figure 1.
Comparison of the deduced amino acid sequences of
the cat's claw, castor, and milkweed acyl-ACP desaturases. Identical
and similar residues are shown on backgrounds of black and gray,
respectively. The cleavage site for the plastid transit peptide of the
castor enzyme is indicated by the arrowhead. Eight residues that lie near the bottom of the substrate-binding pocket in the crystal structure of the castor enzyme are indicated by asterisks. The GenBank
accession numbers for the sequences are: cat's claw, AF051134; castor,
M59857; and milkweed, U60277.
|
|
Characterization of the Seed-Specific 9-16:0-ACP
Desaturase
A gel blot containing total RNA from seed and leaf tissue of
cat's claw was hybridized with a probe derived from the insert of
pDU1. A transcript hybridizing to the probe was extremely abundant in
seed tissue but very little transcript was present in the leaf sample
(Fig. 2). We cannot exclude the
possibility that the gene corresponding to the pDU1 cDNA is expressed
at a low level in leaves, but, given the high amino acid sequence
similarity within the acyl-ACP desaturase family, it is also possible
that the pDU1 probe hybridized to some extent to a related transcript
that in leaves would be expected to encode a chloroplast 9-18:0-ACP
desaturase. The results shown in Figure 2, and the fact that the 20 clones analyzed from the cat's claw seed library represented the same gene, demonstrate that there is a single, dominant acyl-ACP desaturase in these seeds. The fact that fatty acids derived from the
9-18:0-ACP desaturase (18:19 plus 18:2) represent only 8% of
the fatty acids in cat's claw oil (Chisholm and Hopkins, 1965)
suggests that pDU1 might encode a relatively specific 9-16:0-ACP
desaturase.
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| Figure 2.
Gel-blot analysis of acyl-ACP transcript levels in
developing seeds (S) and leaves (L) of cat's claw. Equal amounts (10 µg) of total RNA from seeds and leaves were run in each lane and
probed with the insert from pDU1. Ethidium bromide staining of the
major rRNA bands was used to confirm equal loading of total RNA.
|
|
To investigate the substrate specificity of the cat's claw acyl-ACP
desaturase, the ORF from pDU1 was expressed in E. coli DH10B
cells using the vector pQE32. The recombinant protein produced in these
cells contained six additional His residues at its N terminus that were
derived from the vector. The desaturase was partially purified from
bacterial lysates by cation-exchange chromatography and assayed for
activity with acyl-ACPs of different chain lengths. The enzyme
functioned primarily as a 9-16:0-ACP desaturase (Fig. 3), with approximately 4-fold lower
relative activities when 14:0-ACP or 18:0-ACP were used as the
substrates. In contrast to these results, the milkweed acyl-ACP
desaturase showed a 12-fold higher activity with 18:0-ACP compared with
that detected with 16:0-ACP (Cahoon et al., 1997b). It is interesting
that two independent attempts to express the cat's claw cDNA in pET
vectors (Novagen) lacking the His tag resulted in the accumulation of
little if any recombinant protein, as determined by Coomassie blue
staining of SDS-PAGE gels of crude bacterial extracts.
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| Figure 3.
Substrate specificities of the recombinant cat's
claw acyl-ACP desaturase assayed with 14:0-, 16:0-, and 18:0-ACPs.
Partially purified enzyme from E. coli lysate was
assayed as described in ``Materials and Methods''. The reaction rate
with 16:0-ACP was 0.47 nmol min 1 mg1
protein.
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|
A Mutant Form of the Castor Enzyme Favors 9 Desaturation of
16:0-ACP
The amino acid sequences of all of the acyl-ACP desaturases are
highly homologous and are colinear over most of their length. This
suggests that the three-dimensional structure determined for the castor
9-18:0-ACP desaturase (Lindqvist et al., 1996) provides an
excellent model for predicting the structure of the other enzymes.
These predictions and site-specific mutagenesis studies have been used
to identify eight residues located near the bottom of the
substrate-binding pocket that help to determine the chain-length
specificity of the enzymes (Cahoon et al., 1997b). The first of these
residues, M114 of the mature castor protein, corresponds to M167 of the
cat's claw sequence and M133 of the milkweed sequence shown in Figure
1, and the sequences are colinear throughout the remaining seven
residues. Three of the eight residues are altered in the milkweed
enzyme relative to the castor sequence (L115I, T117R, and P179T) but
only one change occurs in the cat's claw protein L118W. A model of
16:0 and 18:0 bound to the active site is shown in Figure
4 along with stick-model representations of the eight previously identified residues. Clearly, the more bulky
side chain of Trp at residue 118 can be predicted to reduce the depth
of the substrate pocket and to favor the binding of 16:0-ACP over the
longer substrate, 18:0-ACP.
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| Figure 4.
A model of 18:0 (gray) and 16:0 (black) bound to
the active site of acyl-ACP desaturases. The structure of seven common
residues lining the substrate pocket of the castor and cat's claw
enzymes and L118 of the castor enzyme are shown in gray. The W118
residue of the cat's claw enzyme and the L118W castor mutant is shown in black. The position of the catalytic di-iron center is indicated and
the arrow shows the position of double-bond insertion. Amino acid
numbering is given with respect to the sequence of the mature castor
9-18:0-ACP desaturase.
|
|
To test this hypothesis, we used site-specific mutagenesis to engineer
the L118W variant of the 9-18:0-ACP castor desaturase. When this
enzyme was assayed with acyl-ACP substrates of different chain lengths,
its activity with 16:0-ACP was 115% relative to its activity with
18:0-ACP (Fig. 5). By contrast, the
wild-type castor desaturase was more than 70 times more active with
18:0-ACP than with 16:0-ACP. Thus, the substitution of a single residue in the archetypal 9-18:0-ACP desaturase accounts for approximately one-half of the difference in relative substrate specificity between the castor and cat's claw enzymes (Fig. 3). Like the previously reported L118F/P179I mutant (Cahoon et al., 1997a), the L118W mutant of
the castor 9-18:0-ACP desaturase is most active with 16:0-ACP.
However, the L118F/P179I mutant displays a greater relative specificity
for 16:0- versus 18:0-ACP than does the L118W mutant. The ratio of
specific activity with 16:0-ACP:18:0-ACP is 2.4 for the L118F/P179I
mutant compared with 1.1 for the L118W mutant. This difference
demonstrates that, although W118 is a major determinant of substrate
specificity, other residues likely contribute to creating the substrate
profile displayed by the cat's claw acyl-ACP desaturase, which is more
similar to that of the L118F/P179I mutant.
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| Figure 5.
Substrate specificities of recombinant wild-type
castor 9-18:0-ACP desaturase (left) and the mutant L118W (right)
assayed with 14:0-, 16:0-, and 18:0-ACPs. Partially purified enzymes
from E. coli were assayed as described in ``Materials and Methods''. The maximum reaction rate (100%) was 823 nmol
min1 mg1 protein for the wild-type enzyme
and 28.8 nmol min1 mg1 protein for the
L118W mutant.
|
|
Identification of the underlying structural bases for the substrate
specificity in studies of variant acyl-ACP desaturases further
validates the crystallographic model of desaturase specificity (Lindqvist et al., 1996; Cahoon et al., 1997a, 1997b). Cat's claw and
milkweed acyl-ACP desaturases have different amino acid substitutions that occlude the base of the substrate-binding cavity, resulting in
similar reductions in chain-length specificity. This highlights the
plasticity of protein structure-function relationships in this class of
enzymes and supports the idea that acyl-ACP desaturases represent an
excellent target for protein engineering. Such engineered enzymes have
the potential to form the basis for a new generation of crop plants
containing unusual fatty acids.
| |
FOOTNOTES |
1
This work was supported by the U.S. Department
of Agriculture-National Research Initiative Competitive Grants Program
(grant no. 97-35301-4426 to J.B.), the Office of Basic Energy Sciences of the U.S. Department of Energy (E.C., J.S.), and the Agricultural Research Center, Washington State University.
2
These authors contributed equally to the work
and are considered joint first authors.
3
Present address: DuPont Agricultural Products,
Experimental Station, Building 402, Wilmington, DE 19880.
4
Present address: Alberta Research Council, P.O.
Box 4000, Vegreville, Alberta T9C 1T4, Canada.
*
Corresponding author; e-mail jab{at}wsu.edu; fax 1-509-335-7643.
Received December 9, 1997;
accepted March 12, 1998.
| |
ABBREVIATIONS |
Abbreviations:
ACP, acyl carrier protein.
ORF, open reading
frameX:Y a fatty acyl group containing X carbon atoms and Y
cis double bonds.
Z, site of a double bond or of
double-bond insertion as the Z carbons from the carboxyl end of the
fatty acid chain .
14:0, myristate.
16:0, palmitate.
16:19, palmitoleate.
18:0, stearate.
18:1, oleate.
18:111, cis-vaccenate.
| |
ACKNOWLEDGMENTS |
We wish to thank P. Vijayan for collecting developing cat's
claw seeds and other tissues and for shipping them to us. We are grateful to Jay Shockey for advice and help with computer
analysis of the sequences. We also thank Armin Dorner for technical
assistance.
| |
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Abstract
Introduction
