ArticlePDF Available

Identification and Analysis of a Gene from Calendula officinalis Encoding a Fatty Acid Conjugase


Abstract and Figures

Two homologous cDNAs, CoFad2 and CoFac2, were isolated from a Calendula officinalis developing seed by a polymerase chain reaction-based cloning strategy. Both sequences share similarity to FAD2 desaturases and FAD2-related enzymes. In C. officinalis plants CoFad2 was expressed in all tissues tested, whereas CoFac2 expression was specific to developing seeds. Expression of CoFad2 cDNA in yeast (Saccharomyces cerevisiae) indicated it encodes a Delta12 desaturase that introduces a double bond at the 12 position of 16:1(9Z) and 18:1(9Z). Expression of CoFac2 in yeast revealed that the encoded enzyme acts as a fatty acid conjugase converting 18:2(9Z, 12Z) to calendic acid 18:3(8E, 10E, 12Z). The enzyme also has weak activity on the mono-unsaturates 16:1(9Z) and 18:1(9Z) producing compounds with the properties of 8,10 conjugated dienes.
Content may be subject to copyright.
Identification and Analysis of a Gene from
Calendula officinalis Encoding a Fatty
Acid Conjugase
Xiao Qiu
*, Darwin W. Reed, Haiping Hong
, Samuel L. MacKenzie, and Patrick S. Covello
Research and Development, Bioriginal Food and Science Corporation, 102 Melville Street, Saskatoon,
Saskatchewan, Canada S7J 0R1 (X.Q., H.H.); and National Research Council of Canada, Plant Biotechnology
Institute, 110 Gymnasium Place, Saskatoon, Saskatchewan, Canada S7N 0W9 (D.W.R., S.L.M., P.S.C.)
Two homologous cDNAs, CoFad2 and CoFac2, were isolated from a Calendula officinalis developing seed by a polymerase
chain reaction-based cloning strategy. Both sequences share similarity to FAD2 desaturases and FAD2-related enzymes. In
C. officinalis plants CoFad2 was expressed in all tissues tested, whereas CoFac2 expression was specific to developing seeds.
Expression of CoFad2 cDNA in yeast (Saccharomyces cerevisiae) indicated it encodes a 12 desaturase that introduces a double
bond at the 12 position of 16:1(9Z) and 18:1(9Z). Expression of CoFac2 in yeast revealed that the encoded enzyme acts as a
fatty acid conjugase converting 18:2(9Z,12Z) to calendic acid 18:3(8E,10E,12Z). The enzyme also has weak activity on the
mono-unsaturates 16:1(9Z) and 18:1(9Z) producing compounds with the properties of 8,10 conjugated dienes.
Hundreds of different fatty acids, many of which
have potential for industrial and pharmaceutical use,
have been identified in nature (Smith, 1970). How-
ever, these fatty acids are mostly produced in the
wild plant species or microorganisms that are not
readily cultivated and cultured commercially. Con-
ventional oilseed crops have high yields and oil con-
tents, but they produce a limited set of fatty acids,
which usually contain less than three double bonds
in their acyl chains.
Generally speaking, in polyunsaturated fatty acids
(PUFA), double bonds tend to be methylene-
interrupted and in the cis configuration. However,
fatty acids containing conjugated double bonds with
various stereochemical configurations do occur in
bacteria, algae, and plants. In the marine algae Boss-
iella orbigniana and Ptilota filicina, a substantial pro-
portion of the PUFA contain conjugated double
bonds (Burgess et al., 1991; Wise et al., 1994). In
plants, various conjugated linolenic acid isomers ac-
cumulate in seeds. Examples includes
acid [18:3(9Z,11E,13E)] in Momordica charantia (Liu
et al., 1997), punicic acid [18:3(9Z,11E,13Z)] in Pu-
nica granatum and Cayaponia africana, and jarcaric acid
[18:3(8Z,10E,12Z)] in Jacaranda mimosifolia (Chisholm
and Hopkins, 1967b; Hopkins and Chisholm, 1968).
Calendula officinalis is an annual flowering plant that
can accumulate more than 40% of calendic acid
[18:3(8E,10E,12Z)] of the seed lipid fatty acids
(Chisholm and Hopkins, 1967a). Although oils con-
taining conjugated linolenic acids have potential
value as drying oils, only
-eleostearic acid-
containing oil from tung (Aleurites fordii) seeds is
currently of commercial significance.
As compared with conjugated polyunsaturated ac-
ids, conjugated linoleic acids (CLAs) appear less
commonly in nature. A few reports have docu-
mented the occurrence of this fatty acid in the foods
derived from ruminant animals (Fritsche and
Fritsche, 1998) and a number of anaerobic bacteria
such as rumen bacterium Butyrivibrio fibrisolvens (Ke-
pler et al., 1966; Kepler and Tove, 1967). It is believed
that CLAs are originally generated by rumen bacteria
and then absorbed by the animal host (Pariza, 1997).
The diversity of fatty acids in nature is largely due
to various combinations of the numbers and loca-
tions of double and triple bonds and other functional
groups (hydroxyl and epoxy). These are produced by
a family of structurally related enzymes (with three
conservative His-rich motifs), including desaturases
and their diverged forms such as hydroxylases, ep-
oxygenases, acetylenases, and the so-called fatty acid
conjugases (Lee et al., 1998; Shanklin and Cahoon,
1998; Cahoon et al., 1999). For microsomal enzymes
in this category it is believed that they use fatty acids
esterified to complex lipid as the substrate and accept
electrons from an electron transport chain consisting
of NAD(P)H, cytochrome b
reductase, and cyto-
chrome b
Based on the information that microsomal desatu-
rases and related enzymes have similar primary
structure, we undertook a PCR approach to clone
genes that are involved in the biosynthesis of conju-
gated fatty acids in C. officinalis. Two unique cDNAs
(CoFad2 and CoFac2) were identified. Expression of
the two cDNAs in yeast (Saccharomyces cerevisiae) re-
Present address: National Research Council of Canada, Plant
Biotechnology Institute, 110 Gymnasium Place, Saskatoon, SK,
Canada S7N 0W9.
* Corresponding author; e-mail; fax 306–975–
Plant Physiology, February 2001, Vol. 125, pp. 847–855, © 2001 American Society of Plant Physiologists 847
vealed that CoFAD2 is a 12 desaturase and CoFAC2
is a fatty acid conjugase that could convert the 9
double bond of linoleic acid and, to a lesser extent, of
palmitoleic and oleic acids, into two conjugated dou-
ble bonds at 8 and 10 position. To our knowledge
this is the first example of identification of an enzyme
that can produce CLAs.
C. officinalis is an annual flowering plant that has
recently drawn scientific attention due to health
claims of the essential oil in the flowers and the
industrial potential of calendic acid in the seed oil.
Calendic is the major fatty acid in the seeds, account-
ing for more than 40% of the total fatty acids. We are
interested in the molecular basis for the biosynthesis
of this special fatty acid.
Identification of a cDNA Coding for a Putative Fatty
Acid Conjugase
To identify genes encoding conjugated double
bond-forming enzymes in C. officinalis, a PCR-based
cloning strategy was adopted. Sequencing of PCR
products revealed three types of inserts related to
desaturases. One had high sequence similarity to
desaturases (FAD3). The other two shared amino
acid sequence similarity to various 12 desaturases
(FAD2) and related enzymes, such as an acetylenase
from Crepis alpina (Lee et al., 1998).
To isolate full-length cDNA clones the two types of
Fad2-like inserts were used as probes to screen a
cDNA library from developing seeds, which resulted
in identification of several cDNA clones in each
group. Sequencing identified two unique full-length
of cDNAs, CoFad2 and CoFac2. CoFad2 is 1,411 bp and
codes for 383 amino acids with an M
of 44,000.
CoFac2 is 1,301 bp in length and codes for 374 amino
acids with a molecular mass of 43.6 kD. Sequence
comparison revealed 46% amino acid identity be-
tween the two deduced proteins. The identity occurs
all along the polypeptides with the highest among
three conservative His-rich areas (Fig. 1).
Sequence comparisons indicate that CoFAD2
shares 73% to 89% amino acid identity with the 12
desaturases from various plants (Okuley et al., 1994;
Lee et al., 1998; GenBank accession nos. AF188264
and AAC 31698). Whereas CoFAC2 shares approxi-
mately equal sequence identity (50%) both to FAD2
desaturases and to other FAD2-like fatty acid-
modifying enzymes including FAD2 from C. officina-
lis (CoFAD2, this paper), Indian mustard (GenBank
accession Q39287 ), and borage (GenBank accession
no. AAC31698), the 12 acetylenase of C. alpina (Lee
et al., 1998), the bifunctional enzyme (oleate 12-
hydroxylase:12-desaturase) of Lesquerella fendleri
(Broun et al., 1998), the 12,13-epoxygenase of Crepis
palaestina (Lee et al., 1998), and fatty acid conjugases
from C. officinalis (Fritsche et al., 1999), Impatiens
balsamina, and Momordica charantia (Cahoon et al.,
Phylogenetic analysis indicates that CoFAC2 dis-
tinguishes itself as one of the most deeply branching
within the plant FAD2-like sequences (Fig. 2). Boot-
strap analysis does indicate that this branching pat-
tern is not particularly reliable and it is possible that
CoFAC2 could cluster with other fatty acid conju-
gases, an epoxygenase, and an acetylenase. On the
other hand, CoFAD2 is clearly grouped within a
main branch of FAD2-like enzymes, which includes
the FAD2s per se, as well as the L. fendleri bifunc-
tional enzyme (Broun et al., 1998) and Ricinus com-
munis hydroxylase (van de Loo et al., 1995). These
results suggest the possible functions of CoFAD2 and
CoFAC2 as those of the extraplastidial 12 fatty acid
desaturase and a fatty acid modifier likely to be
involved in calendic acid biosynthesis, respectively.
Northern-Blot Analysis of CoFac2 and CoFad2
Northern-blot analysis indicated that the CoFac2
was exclusively expressed in the developing seeds of
C. officinalis (Fig. 3). It was not expressed in vegeta-
tive tissues such as leaves and in reproductive tissues
such as flower buds. In contrast, CoFad2 was ex-
pressed in all tissues tested such as leaves, flower
buds, and developing seeds, but preferentially in
flower buds and developing seeds. Expression pat-
terns of the two genes were consistent with the pat-
tern of calendic acid accumulation, which occurs
only in seeds. In C. officinalis calendic acid accumu-
lated only in seeds, whereas linoleic acid, the product
of the 12 desaturase (CoFAD2), was present in all
Figure 1. Comparison of CoFad2 and CoFac2 protein sequences of
. Vertical bars indicate identical amino acids.
Qiu et al.
848 Plant Physiol. Vol. 125, 2001
three tissues examined, but the flower buds and de-
veloping seeds contain a higher amount of this fatty
Expression of CoFac2 and CoFad2 in Yeast
To investigate the function of CoFac2 the full-length
cDNA was expressed in the yeast strain AMY-2
which the stearoyl-coenzyme A desaturase gene, ole1,
is disrupted. The strain is unable to grow in minimal
media without supplementation of mono-unsaturated
fatty acids and allows for experimental control of the
fatty composition of the yeast. In our experiments the
strain was grown in minimal medium supplemented
with 17:1(10Z), a non-substrate of CoFAC2, which
enabled us to study the substrate specificity of the
enzyme toward various substrates, especially mono-
unsaturates. A number of possible substrates includ-
ing 16:0, 16:1(9Z), 17:1(10Z), 18:0, 18:1(9Z), 18:1(9E),
18:1(11Z), 18:1(11E), 18:1(12Z), 18:1(15Z), 18:2(9Z,
12Z), 18:3(9Z,12Z,15Z), 20:0, 20:2(11Z,14Z), and
22:1(13Z) were tested. As indicated in Figures 4 and
5 and Table I, only 18:2(9Z,12Z) and, to a lesser
extent, 16:1(9Z) and 18:1(9Z) were converted to con-
jugated fatty acids by the enzyme. For cultures sup-
plemented separately with the three substrates, when
gas chromatograms of fatty acid methyl esters
(FAMEs) derived from strains expressing CoFac2
were compared with those for vector controls, extra
peaks were detected as shown in Figure 4. These peaks
were selectively ablated when a Diels-Alder reaction
with 4-methyl-1,2,4-triazoline-3,5-dione (MTAD) was
performed prior to gas chromatography (GC) analy-
sis (data not shown). The sets of m/z peaks indicated
in Figure 5 are highly diagnostic for the original
double bond positions of the conjugated fatty acid
analyte. Mass spectral (MS) analysis of the MTAD
derivatives indicates that the products of 16:1, 18:1,
and 18:2 conversion are 16:2(8, 10) and 18:2(8, 10)
(Fig. 5) and 18:3(8, 10, 12) (data not shown). Assign-
ment of the product of 18:1(9) conversion is also
supported by the agreement of its GC peak retention
Figure 2. Phylogenetic analysis of FAD2-like enzymes. The dendro-
gram represents the result of a neighbor-joining analysis of amino
acid sequence distances with bootstrap values in percent shown at
nodes. The tree was arbitrarily rooted with
sequence. Sequences used for the analysis were obtained from ac-
cession numbers: AAB61352.1, Sydesb,
-3 desaturase of
PCC7002; CAB64256.1, CoFac1, (8, 11)-linoleoyl desaturase
C. officinalis
; P46313, AtFad2, Arabidopsis 12 desaturase;
CAA76158.1, CaFad2, 12 fatty acid acetylenase from
C. alpina
AAF05915.1, IbFac, 12 oleic acid desaturase-like protein from
; CAA76157.1, CpFad2, 12 fatty acid desaturase from
Crepis palaestina
; AAC32755.1, LfFdh, bifunctional oleate 12-hy-
droxylase:desaturase from
L. fendleri
; T14269, HaFad2, 12 oleate
desaturase from common sunflower; T09839, RcFah, oleate 12-
hydroxylase from castor bean; CAA76156.1, CpEpo, 12 fatty acid
epoxygenase from
C. palaestina
; AAF05916.1, McFac, 12 oleic
acid desaturase-like protein from
Momordica charantia
; and
AAF08684.1, MaFad2, 12 fatty acid desaturase from
Figure 3. Northern-blot analysis of
. A, Autora-
diogram of northern blot hybridized with
probes. B, Ethidium bromide gel indicating RNA loading. F, Flower
buds; L, leaves; S, developing seeds (see “Materials and Methods”).
Calendula Fatty Acid Conjugase
Plant Physiol. Vol. 125, 2001 849
time with one of a mixture of standard CLA isomers
(data not shown). The mass spectrum for the analyte
identified as 18:3(8, 10, 12) is consistent with two
compounds for which the Diels-Alder reaction has
occurred at the 8 and 10 positions or the 10 and 12
positions of an 18:3 isomer. This compound has the
same GC retention time as the major FAME derived
from C. officinalis seeds and is in all likelihood 18:
3(8E,10E,12Z). In control experiments with the
/pYES2 strain no peaks corresponding to
conjugated fatty acids were detected (Fig. 4 and GC/
MS, data not shown). The peaks in Figure 4 marked
with asterisks were also ablated by reaction with
MTAD. Their retention times and GC/MS analysis of
the FAMES suggests that they are (conjugated) iso-
mers of the major conjugated products. The amounts
of conjugated fatty acids that accumulate in yeast
cultures are shown in Table I. Qualitatively similar
results were obtained in experiments with the un-
supplemented and fatty acid-supplemented INVSc2/
pCoFac2 strain (data not shown). Within the limita-
tions of these in vivo experiments it would appear
that the conjugase has a much higher activity on 18:2
than on the monoenes.
As for the function of CoFad2, the expression of it in
the wild-type yeast strain INVSc2 indicated that the
encoded enzyme is a 12 desaturase that introduces
a double bond at position 12 of 1:1(9Z) and 18:1(9Z)
(data not shown).
In this report we described the identification and
characterization of two homologous cDNAs, CoFac2
and CoFad2, from C. officinalis. Both products have
sequence similarity to the FAD2 desaturases and re-
lated enzymes from plants. CoFAD2 has higher
amino acid identity to the FAD2 desaturases (approx-
imately 80%), whereas CoFAC2 has approximately
equal sequence identity (approximately 50%) to both
FAD2 desaturases and FAD2-related enzymes, in-
cluding the 12 acetylenase of C. alpina (Lee et al.,
1998), a bifunctional enzyme (oleate 12-hydroxylase:
12-desaturase) of L. fendleri (Broun et al., 1998), an
epoxygenases from C. palaestina (Lee et al., 1998), fatty
acid conjugases from C. officinalis (Fritsche et al., 1999),
I. balsamina, and M. charantia (Cahoon et al., 1999).
Expression of CoFad2 cDNA in yeast indicated it en-
codes a 12 desaturase, whereas expression of CoFac2
in yeast revealed that the encoded enzyme produced
conjugated linoleic and linolenic acids from 18:1(9Z)
and 18:2(9Z,12Z) substrates, respectively.
The name “conjugase” was previously coined to
refer to enzymes that are responsible for introducing
conjugated double bonds into acyl chains. Two con-
jugases from I. balsamina and M. charantia were found
to be able to convert the 12 double bond of linoleic
acid into two conjugated double bonds at the 11 and
13 positions, resulting in the production of conju-
gated linolenic acid [18:3(9Z,11E,13E)]. Expression
of CoFac2 in yeast showed that this “conjugase” could
convert 9 double bonds of 16:1(9Z), 18:1(9Z), and
18:2(9Z,12Z) into two conjugated double bonds at
the 8 and 10 positions to produce their corresponding
conjugated fatty acids.
Two major routes to the biosynthesis of conjugated
fatty acids have been elucidated. Isomerization of a
common fatty acid into its conjugated counterpart
without the introduction of additional double bonds
was first described during biohydrogenation of lino-
leic acid by anaerobic rumen bacteria (Polan et al.,
1964). In marine algae a recently identified enzyme
can isomerize several PUFA into their corresponding
conjugated polyenoic acids (Wise et al., 1994, 1997).
The process is strictly an isomerization; there is no
oxidized intermediate or net desaturation involved.
In plants the mechanism underlying biosynthesis
of conjugated linolenic acids was studied by radiola-
beling (Crombie and Holloway, 1985; Liu et al., 1997).
Kinetics of the time course of metabolism of the
radiolabeled precursors indicated linoleic acid ester-
ified to phosphatidylcholine is an intermediate pre-
cursor of conjugated linolenic acid, implying that
Figure 4. GC analysis of FAMEs from yeast strain AMY-2
formed with pCoFac2 (a) or control plasmid pYES2 (b) supplemented
with 17:1(10) and separately with 16:1(9), (1), 18:1(9), (2), or 18:2(9,
12), (3) (see “Materials and Methods”). indicates 18:1 and 20:1
impurities in the 16:1 and 18:1 substrates, respectively. An asterisk
indicates peaks corresponding to isomers of the major conjugated
products (see “Results”).
Qiu et al.
850 Plant Physiol. Vol. 125, 2001
there is desaturation involved. Substrate specificity
studies of CoFAD2, along with that of conjugases
from I. balsamina and M. charantia (Cahoon et al.,
1999), favor the hypothesis that conjugated fatty ac-
ids in plants are produced by a process similar to
desaturation, which can result in introduction of one
additional double bond in the existing fatty acid
substrate. Crombie and Holloway (1985) previously
observed that during conversion of linoleic acid to
calendic acid in C. officinalis developing seeds, there
is no loss of labeled hydrogens at C-9, C-10, C-12, and
C-13, but there is a loss of a hydrogen from C-8 and
C-11. Thus, Fritsche et al. (1999) speculated that the
C. officinalis fatty acid conjugase could abstract hy-
drogens at carbon 8 and 11 positions, resulting in two
conjugated double bonds in the 8 and 10. Two genes
have now been cloned from C. officinalis whose prod-
ucts appear to catalyze the production of calendic
acid. However, it is still not clear whether both
cloned enzymes actually act via an “8,11 desatura-
tion” mechanism.
It was unexpected that 18:1 (9) acts as a substrate,
albeit a weak one, for CoFAC2 giving rise to conju-
gated linoleic acid [18:2(8, 10)] in yeast. CLA is a
newly recognized nutraceutical compound that has
recently drawn the attention of the pharmaceutical
and nutraceutical industries because of its various
physiological effects in animals and humans (Hau-
mann, 1996; Ip, 1997; Pariza, 1997). Dietary CLA (two
major isomers: 9Z,11E and 10E,12Z) was shown to
reduce the development of atherosclerosis in rabbits
(Lee et al., 1994) and to inhibit development of vari-
ous cancers in model animals (Pariza et al., 1999).
Feeding CLAs at low concentration (0.5% of diet) to
rodents can enhance immune function (Miller et al.,
1994). In addition, CLAs were recently found to de-
crease fat composition and increase lean body masses
and to improve feed efficiency in chickens and pigs
(Park et al., 1997). With the realization of the benefits
of CLAs, market demand for the product is growing.
There is, unfortunately, no rich natural source for
CLAs. Although some animal foods such as dairy
products and meat derived from ruminants contain
CLAs, the proportion is low. Linoleic acid can be
converted to CLA by chemical methods (Berdeaux et
al., 1998; Chen et al., 1999). However, CLA derived
from the chemical process is a mixture of several
isomers. The two major isomers (9Z,11E and 10E,
12Z) in about equal proportions account for about
80% of the product. The rest are other CLA isomers.
CLA produced by CoFAC2 in yeast is an unusual
isomer with two conjugated double bonds at the 8
and 10 positions. The stereochemistry of the product
remains to be determined. It is likely that it is 8E and
10E, since calendic acid [18:3(8E,10E,12Z)] is also a
product of the enzyme in yeast.
The finding that CoFAC2 can use oleic substrate to
synthesize the CLA has opened up a question regard-
ing the potential uses: does this CLA isomer have any
physiological effects on human and animal as com-
mon CLA does? To answer the question, preparation
of large amounts of the isomer is the first essential step
since feeding experiments and clinical trials would
consume a large amount of the fatty acid. If the effi-
ciency of conversion of 18:1 to CLA could be im-
proved, it may be possible to produce the 8,10 isomer
commercially in genetically modified organisms.
Plant Materials
Calendula officinalis was grown in a growth chamber at
22°C with a 16-h photoperiod at a photon flux density of
150 to 200
. The developing seeds at 15 to 30 d
after flowering were collected. The embryos were dissected
from seeds and used for RNA isolation.
Construction and Screening of cDNA Library
The total RNA was isolated from developing embryos
according to Qiu and Erickson (1994). The cDNA library
was constructed from the total RNA. The first strand cDNA
was synthesized by superscript II reverse transcriptase
from Gibco-BRL (Gaithersburg, MD). The second strand
cDNA was synthesized by DNA polymerase I from Strat-
agene (La Jolla, CA). After size fractionation, cDNA inserts
larger than 1 kb were ligated into
Uni-Zap XR vector
(Stratagene). The recombinant
DNAs were then packaged
with Gigapack III Gold packaging extract (Stratagene) and
plated on NZ amine-yeast extract plates. The resulting
library represented more than 8 10
independent clones.
Screening of the cDNA library was performed according to
standard methods (Sambrook et al., 1989).
Table I.
Conversion of exogenous fatty acids by the yeast strain AMY-2
See “Materials and Methods” for culture conditions. Values are the means and SDs (in parentheses)
of three experiments. For control experiments using the AMY-2
/pYES2 strain, no significant peaks were
detected at the retention time of the desaturation product.
Substrate Supplied Substrate Accumulation Product Product Accumulation
% total fatty acids % total fatty acids
16:1(9) 45 (12) 16:2 (8, 10) 0.29 (0.04)
18:1(9) 42 (10) 18:2 (8, 10) 0.09 (0.02)
18:2(9, 12) 18 (3) 18:3 (8
) 0.56 (0.3)
Calendula Fatty Acid Conjugase
Plant Physiol. Vol. 125, 2001 851
Reverse Transcriptase-PCR
For reverse-transcriptase experiments the single strand
cDNA was synthesized by superscript II reverse transcrip-
tase (Gibco-BRL) from total RNA and was then used as the
template for PCR reaction. Two degenerate primers (the
TC/GA and the reverse primer: CATXGTXG/CA/TG/
AAAXAG/AG/ATGG/ATG) were designed to target the
conserved His-rich domains of desaturases. The PCR ampli-
fication consisted of 35 cycles with 1 min at 94°C, 1.5 min at
55°C, and 2 min at 72°C followed by an extension step at
72°C for 10 min. The amplified products from 400 to 600 bp
Figure 5. GCMS EI spectra of the MTAD derivatives of novel fatty acids in AMY-2
/pCoFac2 cultures supplemented with
), (A) and 18:1(9
), (B). The structures assigned to the derivatives are shown with asterisks indicating the original
position of the double bonds in the fatty acid. The pairs of peaks with
values 236 and 308 in A and 264 and 308 in B
are diagnostic for the loss of R
and R
fragments, respectively, for 16:2(8, 10) and 18:2(8, 10) derivatives.
Qiu et al.
852 Plant Physiol. Vol. 125, 2001
were isolated from agarose gel and purified by a kit (Qiaex
II gel purification, Qiagen, Valencia, CA), and subsequently
cloned into the TA cloning vector pCR 2.1 (Invitrogen, Carls-
bad, CA). The cloned inserts were then sequenced by PRISM
DyeDeoxy Terminator Cycle Sequencing System (Perkin
Elmer/Applied Biosystems, Foster City, CA).
Phylogenetic Analysis
For phylogenetic analysis, predicted amino acid se-
quences were aligned using CLUSTALW (version 1.60;
Thompson et al., 1998) with the default parameters, includ-
ing gap open and extension penalties of 10 and 0.05, re-
spectively, for pairwise and multiple alignments. The BLO-
SUM 30 protein weight matrix was used for pairwise
alignments and the BLOSUM series for multiple align-
ments. CLUSTALW was used to determine dendrograms
representing a neighbor-joining analysis of sequence dis-
tances. Bootstrap analysis was performed with 1,000 itera-
tions and visualized with the TreeView program (Page,
Northern-Blot Analysis
For northern-blot analysis, 7
g of total RNAs isolated
from flower buds, leaves, and developing seeds of C. offi-
cinalis as described above were fractionated in a
formaldehyde-agarose gel. After electrophoresis RNAs
were transferred to Hybond membrane (Amersham Phar-
macia, Uppsala) using 10 SSC transferring solution and
were then fixed to the membrane by UV crosslinking.
Filter-bound RNAs were then hybridized with the radiola-
beled cDNA probes at 68°C for1hinQuickhyb (Strat-
agene). After hybridization the blots were washed once at
room temperature for one-half an hour with a solution of
2 SSC and 1% (w/v) SDS, and once at 65°C for one-half
an hour with a solution of 0.1 SSC and 0.1% (w/v) SDS.
Expression of CoFad2 and CoFac2 in Yeast
(Saccharomyces cerevisiae)
The open reading frames of CoFad2 and CoFac2 were
amplified by PCR using the Precision Plus enzyme (Strat-
agene) and cloned into a TA cloning vector (pCR 2.1,
Invitrogen). Having confirmed that the PCR products were
identical to the original cDNAs by sequencing, the frag-
ments were then released by a BamHI-EcoRI double diges-
tion and inserted into the yeast expression vector pYES2
(Invitrogen) under the control of the inducible promoter
Yeast strains InvSc2 (Invitrogen) and AMY-2
[the ge-
notype: MAT
, CYTb5, ole1(BstEII)::LEU2, trp1-1, can1-
100, ura3-1, ade2-1, HIS3; Mitchell and Martin, 1995] were
transformed with the expression constructs using the lith-
ium acetate method and transformants were selected on
minimal medium plates lacking uracil (Gietz et al., 1992;
Covello and Reed, 1996).
Transformants were first grown in minimal medium
lacking uracil and containing Glc (CM-ura, Ausubel et al.,
1995) at 28°C. After overnight culture the cells were spun
down, washed, and resuspended in distilled water. Mini-
mal medium with 2% (w/v) Gal replacing Glc, and with or
without 0.3 mm substrate fatty acids in the presence of 0.1%
(w/v) Tergitol was inoculated with the yeast transformant
cell suspension and incubated at 20°C for 3 d followed by
15°C for 3 d. For the AMY2
strain media were supple-
mented with 0.3 mm 17:1(10Z) and 0.1% (w/v) Tergitol.
Fatty Acid Analysis
Yeast cultures were pelleted by centrifugation (4,000g,10
min.) and pellets were washed with 10 mL of 1% (w/v)
Tergitol solution and 2 10 mL of water. The yeast pellet
was dried under vacuum at ambient temperature. To the
dried pellet in a glass culture tube was added 1 mL of
methanol and the pellet was dispersed using a high speed
homogenizer. To this mixture was added 2 mL of 0.5 m
sodium methoxide in methanol. The tube was flushed with
nitrogen, sealed, and heated to 50°C for 1 h. The cooled
mixture was extracted with 2 2 mL of hexane. The pooled
hexane was washed with 2 mL of water and was concen-
trated under N
for GC or GC/MS analysis.
FAME analysis was carried out using a gas chromato-
graph (6890, Hewlett-Packard, Palo Alto, CA) equipped
with a DB-23 fused silica column (30 m 0.25 mm i.d.,
m film thickness; J&W Scientific, Fulsom, CA) with a
temperature program of 180°C for 1 min, 4°C/min to
240°C, hold for 15 min.
For conjugated polyene analysis, FAME were derivat-
ized with MTAD (Dobson, 1998). One hundred microliters
of a dilute solution of MTAD (1 mg/mL, slight pink
color) in CHCl
at 0°C was added to dry FAME from yeast
cells with agitation for 5 to 10 s. A dilute solution of
1,3-hexadiene (excess) was then added to neutralize reac-
tants (removal of color). The tube was dried under nitrogen
and the residue was re-dissolved in CHCl
GC/MS analysis was performed in standard EI mode
using a Fisons VG TRIO 2000 mass spectrometer (VG An-
alytical, Manchester, UK) controlled by Masslynx version
2.0 software, coupled to a GC 8000 Series gas chromato-
graph. For FAME analysis, a DB-23 column was used with
the temperature program described above. For MTAD deriv-
ative analysis, a DB-5 column (60 m 0.32 mm i.d., 0.25-
film thickness, J&W Scientific) that was temperature-
programmed at 50°C for 1 min, increased at 20°C/min to
160°C, then 5°C/min to 350°C and held for 15 min.
In some experiments, C. officinalis oil extracted from
seeds or a mixture of CLAs (Sigma, St. Louis) was used as
the standard.
The authors wish to thank Dr. Ron Wilen for providing
C. officinalis seeds, Dr. Charles Martin for providing yeast
mutant strain, and Stephen Ambrose for GC/MS
Received June 6, 2000; returned for revision July 20, 2000;
accepted October 12, 2000.
Calendula Fatty Acid Conjugase
Plant Physiol. Vol. 125, 2001 853
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman
JG, Smith JA, Struhl K, Albright LM, Coen DM, Varki
A (1995) Current Protocols in Molecular Biology. John
Wiley & Sons, New York
Berdeaux O, Voinot L, Angioni E, Juaneda P, Sebedio JL
(1998) A simple method of preparation of methyl trans-
10, cis-12 and cis-9, trans-11-octadecadienoates from
methyl linoleate. JAOCS 75(12): 1749–1755
Broun P, Boddupalli S, Somerville C (1998) A bifunctional
oleate 12-hydroxylase: desaturase from Lesquerella
fendleri. Plant J 13(2): 201–210
Burgess JR, de la Rosa RI, Jacobs RS, Butler A (1991) A
new eicosapentaenoic acid formed from arachidonic acid
in the coralline red algae Bossiella orbigniana. Lipids 26:
Cahoon EB, Carlson TJ, Ripp KG, Schweiger BJ, Cook
GA, Hall SE, Kinney AJ (1999) Biosynthetic origin of
conjugated double bonds: production of fatty acid com-
ponents of high-value drying oils in transgenic soybean
embryos. Proc Natl Acad Sci USA 96: 12935–12940
Chen CA, Lu W, Sih CJ (1999) Synthesis of 9Z,11E-
octadecadienoic and 10E,12Z-octadecadienoic acids, the
major components of conjugated linoleic acid. Lipids
34(8): 879–884
Chisholm MJ, Hopkins CY (1967a) Calendic acid in seed
oils of the genus Calendula. Can J Biochem 45: 251–255
Chisholm MJ, Hopkins CY (1967b) Conjugated fatty acids
in some Cucurbitaceae seed oils. Can J Biochem 45:
Covello PS, Reed DW (1996) Functional expression of the
extraplastidial Arabidopsis thaliana oleate desaturase gene
(FAD2)inSaccharomyces cerevisiae. Plant Physiol 111:
Crombie L, Holloway JH (1985) The biosynthesis of calen-
dic acid, octadeca-(8E, 10E, 12E)-trienoic acid, by devel-
oping marigold seeds: origin of (E,E,Z) and (Z,E,Z) con-
jugated triene acids in higher plants. J Chem Soc Perkin
Trans 1: 2425–2434
Dobson G (1998) Identification of conjugated fatty acids by
gas chromatography-mass spectrometry of 4-methyl-
1,2,4-triazoline-3,5-dione adducts. JAOCS 75(2): 137–142
Fritsche K, Hornung E, Peitzsch N, Renz A, Feussner I
(1999) Isolation and characterization of a calendic acid
producing (8, 11)-linoleoyl desaturase. FEBS Lett 462:
Fritsche S, Fritsche J (1998) Occurrence of conjugated li-
noleic acid isomers in beef. JAOCS 75: 1449–1451
Gietz D, St. Jean A, Woods RA, Schiestl RH (1992) Im-
proved method for high efficiency transformation of in-
tact yeast cells. Nucleic Acids Res 20: 1425
Haumann BF (1996) Conjugated linoleic acid offers re-
search promise. Inform 7: 152–159
Hopkins CY, Chisholm MJ (1968) A survey of the conju-
gated fatty acids of seed oils. JAOCS 45: 176–182
Ip C (1997) Review of the effects of trans fatty acids, oleic
acid, n-3 polyunsaturated fatty acids, and conjugated
linoleic acid on mammary carcinogenesis in animals.
Am J Clin Nutr 66: S1523–S1529
Kepler CR, Hirons KP, McNeill JJ, Tove SB (1966) Inter-
mediates and products of biohydrogenation of linoleic
acid by Butyrivibrio fibrsolvens. J Biol Chem 241(6):
Kepler CR, Tove SB (1967) Biohydrogenation of unsatur-
ated fatty acids, III purification and properties of a li-
noleate 12-cis, 11 trans-isomerase from Butyrivibrio
fibrsolvens. J Biol Chem 212(24): 5686–5692
Lee KN, Kritchevsky D, Pariza MW (1994) Conjugated
linoleic acids and atherosclerosis in rabbits. Atheroscle-
rosis 108: 19–25
Lee M, Lenman M, Banas A, Bafor M, Singh S, Schweizer
M, Nilsson R, Liljenberg C, Dahlqvist A, Gummeson P,
Sjoedahl S, Green A, Stymne S (1998) Identification of
non-heme diiron proteins that catalyze triple bond and
epoxy group formation. Science 280(5365): 915–918
Liu L, Hammond EG, Nikolau BJ (1997) In vivo studies of
the biosynthesis of
-eleostearic acid in the seed of Mo-
mordica charantia L. Plant Physiol 113: 1343–1349
Miller CC, Park Y, Pariza MW, Cook ME (1994) Feeding
conjugated linoleic acid to animals partially overcomes-
catabolic responses due to endotoxin injection. Biochem
Biophys Res Commun 198: 1107–1112
Mitchell AG, Martin CE (1995) A novel cytochrome b5-like
domain is linked to the carboxyl terminus of the Saccha-
romyces cerevisiae -9 fatty acid. J Biol Chem 270:
Okuley J, Lightner J, Feldman KA, Yadav NS, Lark E,
Browse J (1994) Arabidopsis FAD2 gene encodes the en-
zyme that is essential for polyunsaturated lipid synthe-
sis. Plant Cell 6: 147–158
Page RDM (1996) TREEVIEW: an application to display
phylogenetic trees on personal computers. Computer
Appl Biosci 12: 357–358
Park Y, Albright KJ, Liu W, Storkson JM, Cook ME,
Pariza MW (1997) Effect of conjugated linoleic acid on
body composition in mice. Lipids 32: 853–858
Pariza MW (1997) Conjugated linoleic acid, a newly recog-
nized nutrient. Chem Industry 16: 464–466
Pariza MW, Park Y, Cook ME (1999) Conjugated linoleic
acid and the control of cancer and obesity. Toxicol Sci 52:
Polan CE, McNeill JJ, Tove SB (1964) Biohydrogenation of
unsaturated fatty acids by rumen bacteria. J Biol Chem
88(4): 1056–1064
Qiu X, Erickson L (1994) A simple and effective method for
isolating RNA from alfalfa pollen. Plant Mol Biol Re-
porter 12: 209–214
Sambrook J, Fritsch EF, Maniatis T (1989): Molecular
Cloning: A Laboratory Manual. Cold Spring Harbor Lab-
oratory Press, Cold Spring Harbor, NY
Shanklin J, Cahoon EB (1998) Desaturation and related
modifications of fatty acids. Annu Rev Plant Physiol
Plant Mol Biol 49: 611–641
Smith CR (1970) Occurrence of unusual fatty acids in
plants. In RT Holman, ed, Progress in the Chemistry of
Fats and Other Lipids. Vol XI, part 1. Pergamon Press,
Oxford, pp 137–177
Thompson JD, Higgins DG, Gibson TJ (1998) CLUSTAL
Qiu et al.
854 Plant Physiol. Vol. 125, 2001
W: improving the sensitivity of progressive multiple se-
quence alignment through sequence weighting, position-
specific gap penalties and weight matrix choice. Nucleic
Acids Res 22: 4673–4680
van de Loo FJ, Broun P, Turner S, Somerville C (1995) An
oleate 12-hydroxylase from Ricinus communis L. is a fatty
acyl desaturase homolog. Proc Natl Acad Sci USA 92(15):
Wise ML, Hamberg M, Gerwick WH (1994) Biosynthesis
of conjugated triene-containing fatty acids by a novel
isomerase from the red marine alga Ptilota filicina. Bio-
chemistry 33: 15223–15232
Wise ML, Hamberg M, Gerwick WH (1997) Characteriza-
tion of the substrate binding site of polyenoic fatty acids
isomerase, a novel enzyme from the marine alga Ptilota
filicina. Biochemistry 36: 2985–2992
Calendula Fatty Acid Conjugase
Plant Physiol. Vol. 125, 2001 855
Full-text available
Marigold (Calendula), an important asteraceous genus, has a history of many centuries of therapeutic use in traditional and officinal medicines all over the world. The scientific study of Calendula metabolites was initiated at the end of the 18th century and has been successfully performed for more than a century. The result is an investigation of five species (i.e., C. officinalis, C. arvensis, C. suffruticosa, C. stellata, and C. tripterocarpa) and the discovery of 656 metabolites (i.e., mono-, sesqui-, di-, and triterpenes, phenols, coumarins, hydroxycinnamates, flavonoids, fatty acids, carbohydrates , etc.), which are discussed in this review. The identified compounds were analyzed by various separation techniques as gas chromatography and liquid chromatography which are summarized here. Thus, the genus Calendula is still a high-demand plant-based medicine and a valuable bioactive agent, and research on it will continue for a long time.
Calendula officinalis, commonly known as “marigold,” is a plant with yellow-orange flowers belonging to the Asteraceae family. The plant, which has been used since ancient times, is widely used in both traditional and homeopathic medicine for the treatment of various diseases. This chapter firstly summarized the description, distribution, and chemical composition of the plant. In vivo, in vitro, and clinical studies on the plant have been shown in detail. In addition, toxicity and mutagenicity studies have been reported.KeywordsCalendula officinalis L.Chemical compositionTraditional useBiological activities
Full-text available
Fatty acid desaturase catalyzes the desaturation reactions by inserting double bonds into the fatty acyl chain, producing unsaturated fatty acids, which play a vital part in the synthesis of polyunsaturated fatty acids. Though soluble fatty acid desaturases have been described extensively in advanced organisms, there are very limited studies of membrane fatty acid desaturases due to their difficulties in producing a sufficient amount of recombinant desaturases. However, the advancement of technology has shown substantial progress towards the development of elucidating crystal structures of membrane fatty acid desaturase, thus, allowing modification of structure to be manipulated. Understanding the structure, mechanism, and biosynthesis of fatty acid desaturase lay a foundation for the potential production of various strategies associated with alteration and modifications of polyunsaturated fatty acids. This manuscript presents the current state of knowledge and understanding about the structure, mechanisms, and biosynthesis of fatty acid desaturase. In addition, the role of unsaturated fatty acid desaturases in health and diseases is also encompassed. This will be useful in understanding the molecular basis and structural protein of fatty acid desaturase that are significant for the advancement of therapeutic strategies associated with the improvement of health status. Key points • Current state of knowledge and understanding about the biosynthesis, mechanisms, and structure of fatty acid desaturase. • The role of unsaturated fatty acid desaturase. • The molecular basis and structural protein elucidated the crystal structure of fatty acid desaturase.
Full-text available
Dietary bioactive lipids, one of the three primary nutrients, is not only essential for growth and provides nutrients and energy for life's activities but can also help to guard against disease, such as Alzheimer's and cardiovascular diseases, which further strengthen the immune system and maintain many body functions. Many microorganisms, such as yeast, algae, and marine fungi, have been widely developed for dietary bioactive lipids production. These biosynthetic processes were not limited by the climate and ground, which are also responsible for superiority of shorter periods and high conversion rate. However, the production process was also exposed to the challenges of low stability, concentration, and productivity, which was derived from the limited knowledge about the critical enzyme in the metabolic pathway. Fortunately, the development of enzymatic research methods provides powerful tools to understand the catalytic process, including site-specific mutagenesis, protein dynamic simulation, and metabolic engineering technology. Thus, we review the characteristics of critical desaturase and elongase involved in the fatty acids' synthesis metabolic pathway, which aims to not only provide extensive data for enzyme rational design and modification but also provides a more profound and comprehensive understanding of the dietary bioactive lipids' synthetic process.
Full-text available
Main conclusion Nitro fatty acids (NO2-FA)have relevant physiological roles as signaling molecules in biotic and abiotic stress, growth, and development, but the mechanism of action remains controversial. The two main mechanisms involving nitric oxide release and thiol modification are discussed. Abstract Fatty acids (FAs) are major components of membranes and contribute to cellular energetic demands. Besides, FAs are precursors of signaling molecules, including oxylipins and other oxidized fatty acids derived from the activity of lipoxygenases. In addition, non-canonical modified fatty acids, such as nitro-fatty acids (NO2-FAs), are formed in animals and plants. The synthesis NO2-FAs involves a nitration reaction between unsaturated fatty acids and reactive nitrogen species (RNS). This review will focus on recent findings showing that, in plants, NO2-FAs such as nitro-linolenic acid (NO2-Ln) and nitro-oleic acid (NO2-OA) have relevant physiological roles as signaling molecules in biotic and abiotic stress, growth, and development. Moreover, since there is controversy on mechanisms of action of NO2-FAs as signaling molecules, we will provide evidence showing why this aspect needs further evaluation.
Full-text available
Environmental insults such as extremes of temperature, extremes of water status, and deteriorating soil conditions pose major threats to agriculture and food security. Employing contemporary tools and techniques from all branches of science, attempts are being made worldwide to understand how plants respond to abiotic stresses with the aim to manipulate plant performance that is better suited to withstand these stresses. This book searches for possible answers to several basic questions related to plant responses towards abiotic stresses. Synthesizing developments in plant stress biology, the book offers strategies that can be used in breeding, including genomic, molecular, physiological, and biotechnological approaches that have the potential to develop resilient plants and improve crop productivity worldwide.
Nature, the supreme artist, and scientists have designed almost an infinite range of plant molecular bioactive molecules for drugs operative for the remedy of innumerable human disorders in the biosynthetic laboratory of plants. From 250,000 to 300,000 plant species which exist on Earth, only about 5000 were investigated for chemical compounds with pharmacological and biological activities. More than 25% of pharmaceutical molecules are plant based through sequestration of the novel bioactive compounds. The 200,000 known secondary metabolites with widespread chemical structures are manly categorized into phenolics, terpenoids or terpenes, and steroids and alkaloids. Secondary metabolites perform significant functions in plants including role as signaling molecules, chemical defense mechanism and adaptation, pollination and seed dispersal, protection from predators, herbivores, pathogens, and allelopathic agents. Medicinal and aromatic plants (MAPs) are bestowed with both the aromatic and medicinal properties and contain bioactive secondary metabolites with a broad range of pharmacological and therapeutic potentials such as antioxidant, antitumor, anticancer, antiviral, antimicrobial, anti-inflammatory, antiatherosclerotic, antidepressant, antidiabetic, hepatoprotective, antithrombotic, vasoprotective, and immunoprotective effects, cardiovascular improver, memory enhancer, anti-AIDS, anti-Alzheimer’s, anti-Parkinson’s disease, and anti-cognitive impairment.
Full-text available
This chapter highlights on the applications of marigold plant extracts as an antibacterial and antimicrobial best dyer for textiles. Tagetes erecta usually known as Marigold is a vital wellspring of carotenoids and lutein, developed as a nursery plant. Marigold blossoms are yellow to orange red in colour. Now a days, lutein is transforming into an unquestionably common powerful fixing, used as a part of the medicines, food industry and textile coatings. This has increased more noticeable vitality of marigold and its exceptional concealing properties. Regardless of the way that marigold blooms; its extract has been used as a measure of veterinary supports. The examination was directed to contemplate the usage of a concentrate of marigold as a trademark shading, which is antibacterial and antimicrobial. The marigold extract ability was focused on colouring of the cotton fabrics. Investigations of the dye ability, wash fastness, light fastness, antibacterial tests and antimicrobial tests can be endeavoured. Studies have exhibited that surface concealing was not impacted by washing and drying in the shadow/sunlight. These surprises reveal that the concentrate of marigold extract can be used for cotton fabrics.
L’acide ricinoléique est un acide gras hydroxylé inhabituel d’origine végétale qui possède de nombreuses applications. Sa demande ne cesse de croître et la culture de la plante tropicale qui produit cette huile plafonne. La surexpression d’une hydroxylase de champignon CpFAH12 dans une souche ingénierée de la levure oléagineuse Yarrowia lipolytica a permis de produire de façon hétérologue cette molécule d’intérêt mais en quantité insuffisante pour en faire une voie de production alternative. L’objectif de ces travaux de thèse était d’augmenter la production en acide ricinoléique de la souche précédemment utilisée.Dans une première partie de ma thèse, une nouvelle méthode de production de type « fermentation extractive » a été développée pour réduire la variabilité de la production d’acide ricinoléique. La présence d’une phase organique a eu un effet positif sur l’état physiologique des cellules et sur la variabilité des concentrations en acides gras mesurées. Une souche permettant une intégration ciblée des cassettes d’expression a également été construite et a permis d’améliorer la reproductibilité entre les expériences.Deux stratégies ont alors été suivies pour l’amélioration de la production d’acide ricinoléique. La première porte sur la compréhension des déterminants de la spécificité désaturation/hydroxylation de l’hydroxylase bifonctionnelle du champignon Claviceps purpurea, CpFAH12. La construction de chimères et de mutants par comparaison avec un homologue ne présentant qu’une activité de désaturation et la création d’un modèle tridimensionnel de l’hydroxylase ont permis d’identifier un acide aminé clef pour la spécificité enzymatique, mais aucune amélioration du taux d’hydroxylation n’a été obtenue.La seconde stratégie est la modification du métabolisme lipidique de Y. lipolytica. La réaction d’hydroxylation a pour substrat un acide oléique estérifié sur une phosphatidylcholine. Deux stratégies visant à augmenter le pool de substrat et à favoriser la libération du produit ont été testées. Toutes deux montrent des résultats intéressants qui restent à confirmer. La dernière semble toutefois la plus prometteuse puisque la surexpression de Yl.TGL5 semble avoir permis une amélioration de près de 50% de la production d’acide ricinoléique.Enfin, une dernière voie a été testée, l’expression d’une hydroxylase monofonctionnelle comme l’hydroxylase du ricin, RcFAH12. Mais son expression, ainsi que celle d’autres enzymes de plantes (conjugases et époxydases), n’a pas permis d’obtenir une activité significative. Nous avons donc, par des approches de chimères, tenté de comprendre quelles étaient les raisons de la non-fonctionnalité de ces enzymes de plantes chez la levure. Il a été possible de montrer que le problème n’était pas lié à l’intégration dans la membrane. D’autres hypothèses restent à creuser pour déterminer quel est le facteur limitant, comme les interactions avec les enzymes de la chaîne membranaire de transport d’électron.
Full-text available
Claviceps purpurea bifunctional Δ12‐hydroxylase/desaturase, CpFAH12, and monofunctional desaturase CpFAD2, share 86% of sequence identity. In order to identify the underlying determinants of the hydroxylation/desaturation specificity, chimeras of these two enzymes were tested for their fatty acid production in an engineered Yarrowia lipolytica strain. It reveals that transmembrane helices are not involved in the hydroxylation/desaturation specificity whereas all cytosolic domains have an impact on it. Especially, replacing the CpFAH12 cytosolic part near the second histidine‐box by the corresponding CpFAD2 part annihilates all hydroxylation activity. Further mutagenesis experiments within this domain identified isoleucine 198 as the crucial element for the hydroxylation activity of CpFAH12. Monofunctional variants performing only desaturation were obtained when this position was exchanged by the threonine of CpFAD2. Saturation mutagenesis at this position showed modulation in the hydroxylation/desaturation specificity in the different variants. The WT enzyme was demonstrated as the most efficient for ricinoleic acid production and some variants showed a better desaturation activity. A model based on the recently discovered membrane desaturase structures indicate that these changes in specificity are more likely due to modifications in the di‐iron center geometry rather than changes in the substrate binding mode. This article is protected by copyright. All rights reserved.
Full-text available
Incubation of Butyrivibrio fibrisolvens with different geometrical isomers of linoleic acid indicated the specificity of the biohydrogenation system for linoleic acid or for a conjugated dienoic acid. cis-9-Octadecenoic acid, trans-9-octadecenoic acid, and trans-11-octadecenoic acid were not hydrogenated by this bacterium. The intermediates and products of biohydrogenation of linoleic acid were identified as a Δ9,11-cis-trans (or trans-cis, or both)-octadecadienoic acid and a mixture of trans-9-octadecenoic acid and trans-11-octadecenoic acid. When different positional isomers of cis-trans-conjugated octadecadienoic acid were incubated, various trans-octadecenoic acids were produced, reflecting the double bond positions of the conjugated diene substrates. The first reaction in the biohydrogenation of linoleic acid by B. fibrisolvens is the isomerization of linoleic acid to the cis-trans (or trans-cis, or both)-conjugated octadecadienoic acid. This intermediate is then hydrogenated to form a mixture of the two trans-monoenoic acids.
Full-text available
Recent spectroscopic evidence implicating a binuclear iron site at the reaction center of fatty acyl desaturases suggested to us that certain fatty acyl hydroxylases may share significant amino acid sequence similarity with desaturases. To test this theory, we prepared a cDNA library from developing endosperm of the castor-oil plant (Ricinus communis L.) and obtained partial nucleotide sequences for 468 anonymous clones that were not expressed at high levels in leaves, a tissue deficient in 12-hydroxyoleic acid. This resulted in the identification of several cDNA clones encoding a polypeptide of 387 amino acids with a predicted molecular weight of 44,407 and with approximately 67% sequence homology to microsomal oleate desaturase from Arabidopsis. Expression of a full-length clone under control of the cauliflower mosaic virus 35S promoter in transgenic tobacco resulted in the accumulation of low levels of 12-hydroxyoleic acid in seeds, indicating that the clone encodes the castor oleate hydroxylase. These results suggest that fatty acyl desaturases and hydroxylases share similar reaction mechanisms and provide an example of enzyme evolution.
Full-text available
The polyunsaturated fatty acids linoleate and alpha-linolenate are important membrane components and are the essential fatty acids of human nutrition. The major enzyme responsible for the synthesis of these compounds is the plant oleate desaturase of the endoplasmic reticulum, and its activity is controlled in Arabidopsis by the fatty acid desaturation 2 (fad2) locus. A fad2 allele was identified in a population of Arabidopsis in which mutations had been created by T-DNA insertions. Genomic DNA flanking the T-DNA was cloned by plasmid rescue and used to isolate cDNA and genomic clones of FAD2. A cDNA containing the entire FAD2 coding sequence was expressed in fad2 mutant plants and shown to complement the mutant fatty acid phenotype. The deduced amino acid sequence from the cDNA showed homology to other plant desaturases, and this confirmed that FAD2 is the structural gene for the desaturase. Gel blot analyses of FAD2 mRNA levels showed that the gene is expressed throughout the plant and suggest that transcript levels are in excess of the amount needed to account for oleate desaturation. Sequence analysis identified histidine-rich motifs that could contribute to an iron binding site in the cytoplasmic domain of the protein. Such a position would facilitate interaction between the desaturase and cytochrome b5, which is the direct source of electrons for the desaturation reaction, but would limit interaction of the active site with the fatty acyl substrate.
Acetylenic bonds are present in more than 600 naturally occurring compounds. Plant enzymes that catalyze the formation of the Delta12 acetylenic bond in 9-octadecen-12-ynoic acid and the Delta12 epoxy group in 12,13-epoxy-9-octadecenoic acid were characterized, and two genes, similar in sequence, were cloned. When these complementary DNAs were expressed in Arabidopsis thaliana, the content of acetylenic or epoxidated fatty acids in the seeds increased from 0 to 25 or 15 percent, respectively. Both enzymes have characteristics similar to the membrane proteins containing non-heme iron that have histidine-rich motifs.
Incubation of Butyrivibrio fibrisolvens with linoleic acid produced a conjugated dienoic acid which was identified as cis-9,trans-11-octadecadienoic acid. When linolenic acid (Δ9,12,15) was used as substrate, it also was isomerized to a conjugated acid which was tentatively identified as cis-9,trans-11,cis-15-octadecatrienoic acid. Subsequent hydrogenation of this conjugated trienoic acid produced a nonconjugated cis,trans-dienoic acid but no monoenoic acid. Incubation of whole bacteria with linoleic acid in D2O produced cis-9,trans-11-octadecadienoic acid which contained a single deuterium atom at C-13. A spectrophotometric assay was developed for the presence of linoleate Δ12-cis,Δ11-trans-isomerase, an enzyme localized in the cell envelope. The enzyme did not require the addition of nucleotide cofactors or the presence of a hydrogen atmosphere. The Km for linoleic acid was 1.2 x 10-5 m, and for linolenic acid it was 2.3 x 10-5 m. The average equilibrium constant for the isomerization of linoleic acid was 61 when measured from the forward and reverse directions.
Fatty acids with conjugated unsaturation occur in many seed oils. Thirty of these acids are reviewed with emphasis on their detection, isolation, and structure determination. Their distribution among plant families is shown, and a botanical source of each acid is given. Some reactions, derivatives, and methods of determining configuration are described. Current theories of their biosynthesis in the seed, involving oxygenated precursors, are summarized.
Seed oils of 17 definite and putative species of Calendula were examined. The major fatty acid of C. arvensis oil was identified as trans-8, trans-10, cis-12-octadecatrienoic acid (calendic acid). Spectrometric evidence demonstrated that all 17 species contained the same acid, in amounts varying from 38.9 to 58.4% of the oil.Seed was collected at intervals during maturation from growing plants of C. officinalis. Analysis of the seed and the oil showed that the conjugated trienoic acid was present at an early stage of development and increased steadily in amount to maturity. Accumulation of hydroxydienoic or conjugated dienoic acid in quantity was not observed at any stage although small amounts may have been present. It is concluded that calendic acid is formed in the seed by a rapid continuous process.
Punicic acid was identified as a component of the seed oils of Cyclanthera explodens (26%) and Cayaponia africana (38%). α-Eleostearic acid was identified in Momordica dioica (55%) and its occurrence in Telfairia occidentalis (7 and 12%) was confirmed. Evidence of conjugated acids was noted in Fevillea peruviana and Bryonia alba. The oils of four other species of Cucurbitaceae had no conjugated acids. The distribution pattern of fatty acids in this family is discussed.
The biosynthesis of conjugated triene-containing fatty acids by the red alga Ptilota filicina is catalyzed by a novel enzyme, polyenoic fatty acid isomerase. The enzyme has been highly purified and is described here for the first time. Matrix-assisted laser-induced desorption mass spectrometry was used to determine that the major protein in the purified enzyme is composed of similar or identical subunits of M(r) 58,119 Da. The native enzyme emerges with an apparent M(r) of 174,000 Da from a gel permeation chromatography column. While this enzyme catalyzes the formation of conjugated trienes from a variety of polyunsaturated fatty acid precursors [arachidonate ((5Z,8Z,11Z,14Z)- eicosatetraenoate) is converted to (5Z,7E,9E,14Z)-eicosatetraenoate; gamma-linolenate ((6Z,9Z,12Z)-octadecatrienoate) is converted to 6Z,8E,-10E-octadecatrienoate], this occurs most rapidly with eicosapentaenoate [(5Z,7E,9E,14Z,17Z)- eicosapentaenoate], which is likely the native substrate. Through a series of experiments utilizing gamma-linolenates stereospecifically labeled with deuterium, we have determined that the enzyme intramolecularly transfers the bis-allylic pro-S hydrogen from the C11 position to the C13 position. Furthermore, the bis-allylic pro-R hydrogen at C8 in gamma-linolenate is lost to the solvent. Using arachidonate as substrate, we demonstrated that the C11 olefinic position becomes protonated by a solvent-derived proton. There appears to be no requirement for molecular oxygen, and the transformation is catalyzed by this single enzyme.