From the Department of Plant Biology, Michigan State University, East Lansing, Michigan 48824
Received for publication, December 6, 2002, and in revised form, January 30, 2003
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ABSTRACT |
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Cyclopropane synthase from Sterculia
foetida developing seeds catalyzes the addition of a methylene
group from S-adenosylmethionine to the cis
double bond of oleic acid (Bao, X., Katz, S., Pollard, M., and
Ohlrogge, J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7172-7177). To understand this enzyme better, differential expression in leaf and seed tissues, protein properties, and substrate preferences of plant cyclopropane synthase were investigated. Immunoblot analysis with antibodies raised to recombinant S. foetida
cyclopropane synthase (SfCPA-FAS) revealed that
SfCPA-FAS is expressed in S. foetida
seeds, but not in leaves, and is a membrane protein localized to
microsomal fractions. Transformed tobacco cells expressing SfCPA-FAS were labeled in vivo with
L-[methyl-14C]methionine and
assayed in vitro with
S-adenosyl-L-[methyl-14C]methionine.
These kinetic experiments demonstrated that dihydrosterculate was
synthesized from oleic acid esterified at the sn-1 position of phosphatidylcholine (PC). Furthermore, analysis of acyl chains at
sn-1 and sn-2 positions that accumulated in PC
from S. foetida developing seeds and from tobacco cells
expressing SfCPA-FAS also demonstrated that greater than
90% of dihydrosterculate was esterified to the sn-1
position. Thus, we conclude that SfCPA-FAS is a microsomal localized membrane protein that catalyzes the addition of methylene groups derived from S-adenosyl-L-methionine
across the double bond of oleic acid esterified to the sn-1
position of PC. A survey of plant and bacterial genomes for sequences
related to SfCPA-FAS indicated that a peptide domain with a
putative flavin-binding site is either fused to the methyltransferase
domain of the plant protein or is often found encoded by a gene
adjacent to a bacterial cyclopropane synthase gene.
Cyclopropane fatty acids
(CPA-FAs)1 and cyclopropene
fatty acids (CPE-FAs) contain three-member carbocyclic rings derived by methyl group addition across a double bond. In the plant kingdom, sterculic and malvalic acid, containing 19 and 18 carbons,
respectively, are the most commonly found CPE-FAs in seed oils, whereas
dihydrosterculic acid is the major carbocyclic fatty acid in the seed
oil of Litchi chinensis and Euphoria longana
(2-4). Long-chain CPA- and CPE-FAs are also found in root, leaf, stem,
and callus tissue in plants of the Malvaceae family (5, 6). Although
the biological role of these fatty acids in plants is uncertain,
CPE-FAs might function as anti-fungal agents (7). In tropical areas,
CPE-FAs-containing seeds are still often consumed by humans (8, 9).
Among the CPE-FAs-containing oilseeds, cottonseeds are the most
prevalent, ranking third in world oilseed crushing (10). Cotton oil
contains ~1% CPE-FAs, which is the cause of many physiological
disorders in animals fed cottonseed meal, such as pink white in
eggs, depression of egg production, delay of sexual maturity in hens,
and higher levels of hard fats in cows (11, 12). These disorders all reduce product quality or quantity and therefore can be a source of
economic loss. The underlying cause of these disorders is due to the
fact that CPE-FAs are strong inhibitors of fatty-acid desaturases in
animals (13, 14). Therefore, vegetable oils containing CPE-FAs,
particularly cottonseed oil, are generally treated with high
temperature or hydrogenation before consumption to remove these CPE-FAs
(15). Because of the added processing costs and the creation of
trans fatty acids during hydrogenation, a more suitable
approach to eliminate CPE-FAs from oilseeds is through genetic
engineering. This approach might also be useful to increase carbocyclic
fatty acid production for specialty oil applications. Although CPE-FAs
are anti-nutritional factors in animal diets, the high chemical
reactivity of the cyclopropene ring can potentially be utilized for
many oleochemical applications (8). Therefore, methods of both
decreasing CPE-FA occurrence in edible oils and increasing their
production in industrial oils are desirable. The isolation of a plant
cyclopropane synthase cDNA provides an initial step toward
achieving these goals (1).
CPA-FAs are also constituents of bacterial and parasitic protozoa
lipids (16, 17) but are never accompanied by CPE-FAs. Formation of the
cyclopropane ring has been studied extensively in bacteria, and the
enzymatic reaction has been defined as the transfer of a methylene
group from S-adenosyl-L-methionine to the double
bond of an unsaturated acyl chain (18). The position requirement for
the cis double bonds is rather loose for the
Escherichia coli CPA-FA synthase (19, 20) but more stringent
for cyclopropane synthases from Mycobacterium (21).
E. coli and other bacterial cyclopropane synthases
prefer the monoenoic fatty acids esterified to the sn-2
position of phospholipids, most prominently phosphatidylethanolamine (PE), although the Clostridium butyricum enzyme is selective
for the acyl chain located on the sn-1 position of PE (22).
Recent analysis of the crystal structures of three cyclopropane
synthases from Mycobacterium suggests that acyl-ACP
might be a substrate for these enzymes (21).
Interestingly, alignment of the SfCPA-FAS with bacterial
homologs indicates similarity only to the C-terminal half of the plant
protein. The N-terminal half (amino acids 1-438) is unique to plant
cyclopropane synthase in that known bacterial cyclopropane synthases
lack this portion. The cloning of cyclopropane synthase cDNA from
developing seeds of S. foetida (1) has provided an opportunity to examine the SfCPA-FAS with regard to membrane
association, subcellular localization, substrate, and positional
specificity. We demonstrate that S. foetida cyclopropane
synthase catalyzes the addition of a methylene group, derived from
S-adenosyl-L-methionine, across the double bond
position of oleic acid esterified to PC. In contrast to the E. coli homolog and most other fatty acid modification enzymes of
plants, SfCPA-FAS acts on the sn-1 rather than
sn-2 position of phospholipid.
Plant Material and Chemicals Production of Antibodies to SfCPA-FAS--
SfCPA-FAS
is 864 amino acid long, and only the C-terminal half shares significant
similarity with known cyclopropane synthases from E. coli
and Mycobacteria. To be able to detect homologs of SfCPA-FAS in other species, the C-terminal half of
SfCPA-FAS (470-864 aa) was used as antigen. The
corresponding fragment was obtained through PCR with upstream primer
JO873 (CCGGAATTCTGTTCTCTTAAAACAGCTCTGAAAG TGC) and downstream primer
JO885 (CCCTCTAGAGCTCGGATAAATGAAAACTATTTCAATTATCCG). The resulting PCR
fragment was subcloned into the protein expression vector pET28a(+)
(Novagen, Madison, WI) at EcoRI and SacI sites. The expressed peptide contains 36 amino acids from the vector including
the histidine tag at the N terminus. Protein expression and inclusion
body purification were conducted according to the manufacturer's
instructions. The purified inclusion bodies were resolved by SDS-PAGE
and stained with Coomassie Brilliant Blue, and the partial
SfCPA-FAS protein band was excised and stored at Total Protein Extraction, Characterization of Membrane
Association, and Immunoblot Analysis--
Plant tissue (1 g) was
homogenized with a mortar and pestle in extraction buffer (5 ml)
containing 20 mM Tris-HCl, pH 7.6, 0.2 M NaCl,
10 mM Subcellular Localization of SfCPA-FAS in Transgenic Tobacco
Suspension Cells--
Transgenic tobacco suspension cells expressing
SfCPA-FAS (0.5 g) were harvested and homogenized with a
Potter homogenizer in 50 ml of homogenization buffer (30 mM
TES-KOH, pH 7.6, 0.35 M mannitol, 1 mM EDTA, 2 mM dithiothreitol, 14 mM
L-cysteine, 0.6% (w/v) polyvinylpolypyrrolidone) and
filtered through two layers of Miracloth into a polycarbonate
centrifuge tube on ice. All subsequent steps were performed at 4 °C.
Organelles were enriched by rate-zonal sedimentation using a procedure
adapted from previous protocols (24, 25). Plastids were sedimented by
centrifuging the homogenate at 5,000 × g for 2 min.
The supernatant was decanted into a fresh tube and centrifuged at
20,000 × g for 5 min to sediment intact mitochondria.
The 20,000 × g supernatant was centrifuged at
100,000 × g for 60 min to collect low density membrane
particles. The final 100,000 × g supernatant was
concentrated on an ultrafiltration membrane (5,000 molecular weight
cut-off). Sedimented organelles were gently resuspended in ice-cold
resuspension buffer (20 mM TES-KOH, pH 7.6, 0.3 M mannitol, 1 mM EDTA, 1 mM
dithiothreitol), and protein was quantified. Purity of organelle
fractions was determined by immunoblot analyses using antibodies
against plastid and mitochondrion marker proteins. Protein (100 µg)
from each fraction was resolved by SDS-PAGE under standard conditions
and subsequently transferred to nitrocellulose membranes for antibody probing. Anti-biotin polyclonal antibodies (Kirkegaard & Perry Laboratories, Gaithersburg, MD) detected the biotin carboxyl carrier protein subunit of the plastid heteromeric acetyl-CoA carboxylase (23)
and were therefore used to determine plastid enrichment. Monoclonal
antibodies to the mitochondrial pyruvate dehydrogenase In Vivo Radiolabeling of Transgenic Tobacco Cells--
The
tobacco cell line expressing SfCPA-FAS was maintained in
liquid medium and subcultured as described previously (1). After 3 days
of subculture, 20 µCi of
L-[methyl-14C]methionine was added
to 100 ml of the cell culture followed by continuous growth under the
same conditions. At 5-min intervals, 15 ml of cell culture was removed,
and cells were harvested by brief centrifugation. Lipids were extracted
from cell pellets according to Bligh and Dyer (27). Lipids were
transmethylated by vortexing at room temperature for 3 min with 0.5%
sodium methoxide in methanol:heptane (1:1, v/v). Lipid classes were
separated with K6 TLC using two development systems, the first to 12 cm
in chloroform:methanol:acetic acid:water (85:15:5:2), drying the plate
completely and then followed by second development to the top with
hexane:diethyl ether:acetic acid (80:20:1). Radioactivity was
quantified using an Instant-Imager. PC from each time point was
recovered from the TLC plate for positional analysis. To determine the
distribution of dihydrosterculic acid (DHSA) on PC, purified PC was
subjected to phospholipase A2 treatment as described by
Christie (28). Free fatty acids and lyso-PC were separated by TLC using
two different mobile phases. The first development was in full in
hexane:diethyl ether:acetic acid (60:40:2). Free fatty acids were
located by spraying with 0.2% (w/v) 2',7'-dichlorofluorescein in
ethanol and viewing under UV light. After completely drying the plate,
it was developed in chloroform:methanol:acetic acid:water (50:30:8:4)
to immediately below the free fatty acid bands. The distribution of
radioactivity between free fatty acids and lyso-PC was quantified using
an Instant-Imager. The radioactive lyso-PC of each time point was
recovered, and FAMEs were prepare as described before; the FAMEs were
separated with C18 reverse-phase TLC developed in
acetonitrile:methanol:water (75:25:0.5) and quantified with an
Instant-Imager. In a separate experiment, after the PC from tobacco
cells expressing SfCPA-FAS and S. foetida
developing seeds were treated with phospholipase A2, FAMEs
were prepared from both free fatty acids (from sn-2 of PC)
and lyso-PC (from sn-1 of PC), and followed by gas
chromatography-mass spectrometry analysis using a Hewlett-Packard 5890 gas chromatograph MSD 5972 mass analyzer.
In Vitro Assay of Cyclopropane Fatty-acid Synthase--
The
30,000 × g membrane-enriched pellets from tobacco
cells expressing SfCPA-FAS and control containing only empty
vector pE1776 were used for CPA-FA synthase assays. The
membrane-enriched pellet was resuspended in 0.1 M sodium
Tricine-NaOH, pH 7.0, 15% (v/v) glycerol, and 1 mM
2-mercaptoethanol. After protein concentrations were determined,
defatted bovine serum albumin was added to a final concentration of 1%
(w/v). CPA-FA synthase assays were performed at 30 °C, 0.05 mM
S-adenosyl[methyl-14C]methionine
substrate and with or without 0.05 mM oleoyl-CoA in a total
volume of 0.2 ml. The assay was terminated by either lipid extraction
or the addition of 0.5 ml of 10% (w/v) KOH and 1.0 ml of ethanol and
allowed to stand overnight to saponify lipids completely. The
subsequent lipid and fatty acid analyses were performed as described in
the in vivo labeling experiments. All assays are the average
of three samples.
S. foetida Cyclopropane Synthase Is a Seed-expressed Membrane
Protein--
To determine the level and pattern of
SfCPA-FAS expression, total proteins were extracted from
S. foetida leaves, developing seeds, and transgenic tobacco
cells expressing SfCPA-FAS. SfCPA-FAS is highly
expressed in S. foetida developing seeds but not detected in
leaves (Fig. 1A). In tobacco
suspension cells, SfCPA-FAS was detected in cell lines
expressing SfCPA-FAS cDNA under a constitutive promoter
(1) but not in a control line carrying an empty vector. Based on
immunoblot analysis, the expression level of SfCPA-FAS in
tobacco cells was estimated at less than one-tenth that found in
S. foetida developing seeds.
SfCPA-FAS is an enzyme of 864 amino acids with a predicted
molecular mass of 98 kDa. Analysis of SfCPA-FAS amino
acid sequence does not reveal any predicted trans-membrane
domain; however, this polypeptide appeared to be associated with
membranes (1). To characterize further the enzyme localization, protein
extracts from cells expressing SfCPA-FAS and developing
seeds were centrifuged at 30,000 × g for 35 min. The
supernatants contained most of the soluble proteins, and pellets were
primarily composed of insoluble membrane proteins. SfCPA-FAS
was found almost exclusively in the insoluble pellet from both tobacco
suspension cells and S. foetida developing seeds (Fig.
1B). Thus SfCPA-FAS is a membrane-associated protein in both its native system and after transgenic expression. To
determine the nature of the membrane association, the enriched membrane
pellets were washed with 0.5 M NaCl. After the salt
treatment, the majority of SfCPA-FAS remained in the
insoluble membrane fraction indicating that the protein was not likely
associated by electrostatic bonds (Fig. 1C). In contrast
when Triton X-100 was added to the pellet to a final concentration of
0.1%, the SfCPA-FAS was completely released from the
membrane pellet (Fig. 1C). These results indicate that
SfCPA-FAS is either an integral membrane protein or it is strongly associated with membranes by non-electrostatic interactions.
SfCPA-FAS Is a Microsomal Localized Enzyme--
To clarify the
subcellular location of the cyclopropane synthase, organelle
fractionation was performed with tobacco suspension cells expressing
SfCPA-FAS. The filtered homogenate was first centrifuged at
5000 × g for 2 min to sediment intact plastids. The
biotin carboxyl carrier protein subunit of the heteromeric acetyl-CoA
carboxylase is located exclusively in plastids, and therefore, an
anti-biotin antibody was used to determine the enrichment of plastids
(23). Indeed, the majority of biotin carboxyl carrier protein was found
in the 5,000 × g pellet (Fig.
2). The supernatant was further
centrifuged at 20,000 × g for 5 min to sediment intact mitochondria, and this was confirmed with monoclonal antibodies against
the mitochondrial pyruvate dehydrogenase Phosphatidylcholine Is the Direct Substrate of SfCPA-FAS--
In
previous in vitro labeling experiments with cell-free
extracts of S. foetida developing seeds, most
[14C]dihydrosterculate was found on PC suggesting that
oleoyl-PC might be a substrate for SfCPA-FAS in
vivo (1). Tobacco suspension cells expressing SfCPA-FAS
provide a more tractable system to conduct tracer labeling studies
because the system is available year round, precursors can rapidly
enter the cells, and the tissue is uniform and constant throughout the
labeling period. To examine further whether PC serves as a direct
substrate for SfCPA-FAS in vivo, suspension cells
were labeled with
L-[methyl-14C]methionine under
normal growth conditions. Samples were removed at 3-min intervals from
3 to 24 min, and total lipids from each time point were divided in
half. Half of the sample was used to prepare FAMEs, and the other half
was used to isolate PC (Fig. 3A, 9-min time point). FAMEs
from both PC and total lipid at each time point were fractionated in
parallel by C18 reversed-phase TLC. As shown in Fig.
3B (9-min time point), DHSA was the only radioactive fatty
acid esterified to PC, whereas FAME samples from total lipids revealed
other radioactive bands (most likely sterols) in addition to DHSA. The
percentage of DHSA found in PC versus that from total lipids
was calculated and plotted versus assay time (Fig.
3C). At early time points (3 and 6 min), ~99% of DHSA was
esterified to PC. At later times some labeled DHSA appeared in other
lipids, but the amount in PC stabilized at approximate 85% after 20 min of labeling. This kinetics of appearance of DHSA strongly suggests
that PC is a direct substrate for SfCPA-FAS (Fig.
3C).
SfCPA-FAS Could Also Be Readily Assayed in Vitro--
Assays were
standardized by incubating a range of protein concentrations (from
30,000 × g pellet of tobacco cells expressing SfCPA-FAS) with 0.05 mM
S-adenosyl[methyl-14C]methionine
and 0.05 mM oleoyl-CoA at 30 °C for 1 h.
Radioactive DHSA product increased approximately linearly with proteins
up to 1 mg/ml (Fig. 4A),
whereas higher protein concentrations saturated the assays under the
conditions used. As shown in Fig. 4B, after a 10-20-min
lag, DHSA product accumulated linearly for at least 105 min at a rate
of 0.34 µmol/h/mg protein. Based on these experiments, subsequent in vitro assays were conducted with 100 µg of
protein from the 30,000 × g pellet at 30 °C for
1 h, unless otherwise specified.
To further confirm PC as a direct substrate for SfCPA-FAS by
an alternative method, in vitro assays were performed with
the 30,000 × g pellet using
S-adenosyl[methyl-14C]methionine
as the methylene donor. Upon the termination of the reaction, the
samples were split equally between two tubes. The reaction in one tube
was terminated by adding 0.5 ml of 10% KOH and 1 ml of ethanol. After
saponification, fatty acids were extracted and FAMEs were prepared. In
the other tube, the reaction was stopped by lipid extraction, and lipid
classes were separated by TLC (Fig. 5,
top panel). PC and other radioactive lipid bands were
recovered and used for FAME preparation. Along with the FAMEs from the
other half of the reaction, FAMEs from PC and other radioactive bands were separated by C18 reverse-phase TLC (Fig. 5,
bottom panel). As shown in Fig. 5, DHSA was only detected in
PC and not in any other lipid. In addition, the amount of radioactive
DHSA in PC was approximately the same as that found in the total FAMEs
prepared through KOH saponification. This result was consistent with
in vivo labeling and further supports the hypothesis that PC
is a direct substrate of SfCPA-FAS.
We also considered 18:1-CoA as a potential substrate for
SfCPA-FAS. To test this possibility, the 30,000 × g pellet was washed three times with assay buffer to remove
acyl-CoAs and then assayed with or without 50 µM
oleoyl-CoA. Proteins from control cells (transformed with empty vector)
did not possess any cyclopropane synthase activity with no DHSA
synthesized as shown in the control lane (Fig.
6A). In contrast, DHSA was
produced by the 30,000 × g pellet from tobacco cells
expressing SfCPA-FAS (Fig. 6A). However, the
biosynthesis of DHSA from assays with oleoyl-CoA and that without
oleoyl-CoA was almost identical based on three independent experiments
(Fig. 6B), indicating that the addition of exogenous oleoyl-CoA had no effect on the activity of SfCPA-FAS;
therefore, oleoyl-CoA is not likely a substrate of
SfCPA-FAS.
The Addition of Methylene Group Takes Place on the sn-1 Position of
PC--
Although Figs. 3 and 5 indicate that PC is the direct
substrate for SfCPA-FAS, the modification could take place
on oleic acid esterified to the sn-1, sn-2, or
both positions. To clarify further the reaction mechanism, in
vivo labeling was conducted with tobacco suspension cells with
SfCPA-FAS using
L-[methyl-14C]methionine as
radioactive tracer. Samples were taken at 5, 10, 15, 20, and 25 min,
and PC was purified and treated with position-specific phospholipase
A2 to cleave the fatty acids at the sn-2
positions. The resultant free fatty acids and lyso-PC were separated by
TLC. As shown in Fig. 7A, top
panel, radioactivity was only associated with lyso-PC, whereas no
radioactivity could be detected in the free fatty acid fraction
released from sn-2. Because the head group of lyso-PC can
also be labeled by
L-[methyl-14C]methionine, FAMEs
were prepared from the labeled lyso-PCs and fractionated by
C18 reverse-phase TLC. Only one radioactive spot corresponding to DHSA could be found on the reverse TLC (Fig. 7A,
bottom panel). Radioactivity from total lipids, PC, lyso-PC, and
DHSA were plotted versus labeling time (Fig. 7B).
The accumulation of radioactivity in total lipids increased linearly
during the 25-min labeling period; however, there was a 10-min lag for
the accumulation of radioactivity in PC, lyso-PC, and DHSA.
As shown in Fig. 7, the in vivo experiment suggested that,
in contrast to most acyl modification reactions of PC in plants, the
synthesis of DHSA in tobacco cells occurred preferentially on
sn-1 instead of the sn-2 position. This
conclusion was also supported by analysis of non-radioactive acyl
groups from the sn-1 and sn-2 positions of PC
from tobacco cells expressing SfCPA-FAS and from
S. foetida developing seeds. As shown in Fig.
8, ~90% of DHSA found on PC (from
tobacco cells expressing SfCPA-FAS) was located on the
sn-1 position, indicating relatively little acyl migration.
A similar distribution pattern of DHSA was obtained from PC of S. foetida developing seeds (data not shown). Thus, both in short
term labeling and during steady-state accumulation, DHSA is
predominantly associated with sn-1 of PC.
To confirm this observation further, PC was purified from the in
vitro assay after using
S-adenosyl[methyl-14C]methionine
as methylene donor. The purified PC sample was split equally into two
parts; one part was treated with phospholipase A2, and the
resulting free fatty acids and lyso-PC were separated by TLC along with
the other half of untreated PC sample. No radioactivity was detected
from the free fatty acids derived from the sn-2 position of
PC (Fig. 9A). The
radioactivity, equal to the untreated PC, was exclusively associated
with lyso-PC (Fig. 9A). FAMEs were prepared from radioactive
lyso-PC and PC and fractionated with C18 reverse-phase TLC.
The amount of radioactivity in DHSA derived from lyso-PC was the same
as that derived from PC as measured in Fig. 9B. These
in vitro assay data further demonstrated that the addition
of the methylene group to oleic acid to form DHSA occurred at the
sn-1 position of PC.
Cyclopropane synthases have been extensively studied in two
systems, namely E. coli and Mycobacterium
tuberculosis. E. coli CFA-FA synthase is a soluble
enzyme found in the cell cytoplasm loosely associated with the inner
membrane (29). The substrates of E. coli CFA synthase are
phospholipids (most likely PE) containing unsaturated fatty acids whose
double bond must be positioned 9-11 carbon units from the ester
linking to the glycerol backbone. It is still unresolved whether this
soluble enzyme gains access to the double bond deep within the
hydrophobic core of the bilayer structures of the membranes or if acyl
groups must be removed from the membrane prior to cyclopropanation
(18). In Mycobacteria, data related to the enzymology of
cyclopropane synthases are not available due to the unavailability of
substrate acyl chains, and it is still not clear whether
cyclopropanation occurs during chain elongation or after (30). But
mycobacterial cyclopropane synthases are believed to be soluble
proteins because they were purified in buffers without any detergent
(21). In plants, S. foetida cyclopropane synthase
is the only plant enzyme studied by in vitro assays. Western
blots (Fig. 1A) indicate the enzyme is relatively abundant
in S. foetida developing seeds, consistent with the 0.4%
abundance of ESTs found in the seed cDNA library (1). One of the
differences between SfCPA-FAS and bacterial cyclopropane
synthases is that SfCPA-FAS is a more strongly associated membrane protein than its bacterial counterparts (Fig. 1, B
and C). SfCPA-FAS contains a 400-amino acid
N-terminal extension when compared with bacterial counterparts that
could be responsible for membrane anchorage. The finding that
SfCPA-FAS is enriched in microsomal fraction is expected
because most fatty acid modification enzymes, which yield unusual fatty
acids, are located within the endoplasmic reticulum (31).
In several bacteria, PE is the likely direct substrate for cyclopropane
synthases in vivo. In the protozoan Crithidia
fasciculata, CPA-FAs are found only in PE (32). From experiments
with crude enzyme extracts from Serratia
marcescens and C. butyricum, Zalkin et al. (33) observed that CPA-FAs were synthesized only with the presence of phospholipids, particularly PE. This observation was
later confirmed by Chung and Law (34) with purified enzymes. Most
importantly, a synthetic diether analog of PE was found to be an
effective substrate for bacterial cyclopropane synthases, which ruled
out the possibility of removal of the acyl group from PE before
synthesis of CPA-FAs (35). Other phospholipids might be used as
substrate as well; especially worthy of mention is PC for
Agrobacterium tumefaciens (22). In the case of
Mycobacterium, the crystal structures of three cyclopropane
synthases revealed a hydrophobic patch very similar to known acyl-ACP
utilization enzymes and is believed to be an ACP-binding site. This
observation leads to the suggestion that acyl-ACP might be the
substrate of cyclopropane synthase from Mycobacterium (21).
In plants, because SfCPA-FAS has no plastid targeting
sequence, it can be assumed that acyl-ACP does not serve as a potential
substrate for SfCPA-FAS. In vivo labeling results
(Fig. 3C) indicated that at early time points the newly
synthesized radioactive DHSA is exclusively associated with
PC. In vitro assays with the membrane fraction of tobacco cells with SfCPA-FAS also confirmed that all the radioactive
DHSA was esterified to PC. In addition, oleoyl-CoA failed to enhance the rate of reaction. Based upon these observations, we conclude that
PC is the direct substrate of plant cyclopropane synthase, although
transfer of the acyl group to another carrier for cyclopropanation cannot be completely ruled out.
In PE of E. coli and S. marcescens and PE and PC
of A. tumefaciens, CPA-FAs are predominantly in the
sn-2 position (22), which is also the predominant location
of unsaturated fatty acids in most non-photosynthetic bacteria. One
exception is the PE of C. butyricum in which unsaturated and
CPA-FAs are found in greater abundance at the sn-1 position
(22). Craven and Jeffrey (36) showed through x-ray studies that
CPA-FAs have a similar shape and crystal structure to the
corresponding monoenoic acids. In addition, phospholipids
containing either unsaturated or CPA-FAs are similar in their
solubility characteristics in polar solvents and in their ability to
form stable micellar dispersions (37, 38). Therefore, in many respects
CPA-FAs have both similar physical properties and positional
distribution as monounsaturated fatty acids in phospholipids. In plant
phospholipids (except phosphatidylglycerol), saturated fatty
acids in general show preferential esterification at sn-1,
and unsaturated fatty acids are more abundant at sn-2. Furthermore, the most common substrates for microsomal fatty acid modification enzymes are acyl groups on the sn-2 position of
PC. In particular, although both sn-1 and sn-2
bound acyl groups can serve as substrates, for most desaturases (39,
40, 42) and for the biosynthesis of ricinoleic acid in endosperm of
castor (41) modifications occur predominantly on the
sn-2 position of PC. To date, we are not aware of any report
of plant fatty acid modification enzymes that specifically act on the
acyl group esterified to the sn-1 of PC. The
SfCPA-FAS clearly has strong selectivity to the oleic acid
on the sn-1 position of PC. This feature of sn-1
specificity of SfCPA-FAS not only makes it unprecedented as
a plant microsomal fatty acid modification enzyme but also presents
interesting questions for the downstream desaturation. In particular it
will be of interest to determine whether the cyclopropane desaturase
also acts on the sn-1 position of PC. In the PC fraction of
S. foetida developing seeds, 5% of total fatty acids was
DHSA of which 91% was located on sn-1 and 9% on sn-2 position (data not shown). In contrast, ~37% of
total fatty acids of PC was sterculic acid which was equally
distributed between sn-1 and sn-2 position. These
data imply that the DHSA synthesized on sn-1 of PC may be
removed from the sn-1 before desaturation and subsequently
re-incorporated into PC. The positional distribution of CPE-FAs in
triacylglycerols of S. foetida seeds indicates that for
triacylglycerols containing only one CPE-FA, CPE-FAs are more abundant
at sn-1 or 3 positions, but for triacylglycerols containing two CPE-FAs, CPE-FAs are enriched at the sn-2 position (43). These data suggest that the metabolism of cyclopropene fatty acids in
seeds oils may involve several acylation steps.
A number of plant DNA sequences are related to the
SfCPA-FAS. In Arabidopsis, there are two genes
that locate in tandem on chromosome 3 which have ~60% amino acid
identity with Sterculia cyclopropane synthase and 95% with
each other. (Due to annotation errors, At3g23500 and At3g23510 are
actually one gene with 854 aa, and At3g23520 and At3g23530 is a second
gene with 867 aa). Although CPA-FAs and CPE-FAs have not been reported
in Arabidopsis so far, these structures might be further
modified or used to synthesize complex lipids that are not easily
analyzed or extracted. Alternatively, the Arabidopsis
enzymes might catalyze the formation of related products that
originated from the common intermediate as CPA-FAs. Therefore, it will
be beneficial to understand the catalytic property of the gene family,
which is related to SfCPA-FAS. When SfCPA-FAS is
compared with its bacterial counterparts, it possesses a >400-aa-long
N-terminal extension. This N-terminal portion has some features of
oxidases and contains a conserved FAD-binding motif. At present, the
function(s) of the N-terminal extension are not clear. Interestingly,
homologs of the N-terminal portion exist in several
cyclopropane-producing bacteria, and they locate at very close
proximity to cyclopropane synthase genes. In Mycobacteria,
the hypothetical protein Rv0449c is homologous to the N-terminal
portion of SfCPA-FAS and is separated from the cyclopropane
synthase (ufaA1) by only 200 bp. In Agrobacteria, AGR-C-3599p (N-terminal homolog to SfCPA-FAS) and
AGR-C-3601p (C-terminal homolog to SfCPA-FAS) are distinct
genes separated by only 802 bp. This striking coincidence between two
proteins with adjacent genomic locations in two bacteria and their
fusion to form one protein in plants suggests that the N-terminal
domain in plants and its homologs in bacteria may play a role(s)
related to cyclopropanation. From the evolution point of view, it will be interesting to know how the cyclopropane synthase evolved from E. coli (homologous to C-terminal portion) to
Agrobacteria and Mycobacteria (two separated
proteins, one homologous to N-terminal and the other to C-terminal
portion), and eventually to plants (one fused protein). In
Mycobacteria, there are at least three genes that encode
cyclopropane synthases, but there is only one gene that shares homology
with the N-terminal half of Sterculia enzyme. If the
hypothetical protein (Rv0449c homologous to N-terminal half) is indeed
involved in cyclopropanation, drugs that inhibit this protein may
provide new strategies for treatment for tuberculosis.
In conclusion, the SfCPA-FAS is a microsomal localized
membrane enzyme, which catalyzes the addition of a methylene group derived from S-adenosyl-L-methionine across the
double bond of oleic acid esterified to the sn-1 position of
PC. Further studies will focus on the function(s) of the N-terminal
domain, such as its membrane association, substrate recognition, or
whether it is an authentic FAD-containing protein.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Developing seeds of
Sterculia foetida L. were obtained from Montgomery Botanical
Center (Miami, FL). Frozen developing seeds were used for lipid
analysis and immunoblotting. Tobacco suspension cells (Nicotiana
tabacum L. cv. Bright yellow 2) containing the vector pE1776 or
SfCPA-FAS were maintained as described by Bao et
al. (1).
L-[methyl-14C]Methionine (55 mCi/mmol) was purchased from American Radiolabeled Chemicals, Inc. (St.
Louis, MO), and
S-adenosyl-L-[methyl-14C]methionine
(59 mCi/mmol) was from Moravek Biochemicals (Brea, CA). Phospholipase
A2 (from Crotalus atrox venom) was
purchased from Sigma.
20 °C.
Frozen gel slices of partial SfCPA-FAS were sent to Cocalico
Biologicals, Inc. (Reamstown, PA), for antibody preparation in New
Zealand White rabbits.
-mercaptoethanol, and 5 mM
4-(2-aminoethyl)benzenesulfonyl fluoride and filtered through two
layers of Miracloth into centrifuge tubes on ice. Protein was
quantitated using a dye-binding assay (Bio-Rad) with bovine serum
albumin as the standard. Total protein extracts from transgenic tobacco
cell line expressing SfCPA-FAS and developing seeds of
S. foetida were centrifuged at 30,000 × g
for 35 min. Supernatants were transferred to new tubes, and the pellets
were resuspended in extraction buffer. SfCPA-FAS-enriched membranes (pellet) from tobacco suspension cells were resuspended to 2 mg of protein ml
1 in 20 mM HEPES-KOH, pH 8.0, 10% (v/v) glycerol, 1 mM dithiothreitol, 1 mM
-amino-n-caproic acid, 0.5 mM
benzamidine, 0.5 mM EDTA. Resuspended membranes were washed
by 0.5 M NaCl or 0.1% (v/v) Triton X-100. Samples were
then vortexed for 30 s and incubated on ice for 10 min. Washed
membranes were collected by centrifugation (10 min at 14,000 × g), and supernatant was removed and placed into a fresh
tube. Protein samples were denatured in SDS-PAGE sample buffer and
loaded onto 10% SDS gel for electrophoresis. Proteins were transferred
to nitrocellulose, probed with antibodies against SfCPA-FAS
(1:1000 dilution), and detected as described previously by Thelen
et al. (23).
-subunit (26)
were used to assay mitochondria enrichment.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (28K):
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Fig. 1.
Western blot analysis of
SfCPA-FAS. A, total proteins (100 µg) were loaded to each lane (lane 1, leaves of S. foetida; lane 2, developing seeds of S. foetida; lane 3, tobacco with empty vector pE1776;
lane 4, tobacco expressing SfCPA-FAS).
B, the total protein extracts from transgenic tobacco
cell line expressing SfCPA-FAS and developing seeds of
S. foetida were centrifuged at 30,000 × g
for 35 min. 200 µg of proteins from tobacco cells (lane 1,
pellet; lane 2, supernatant) and 100 µg from seeds
(lane 3, pellet; lane 4, supernatant) were loaded
to each lane. C, the 30,000 × g pellet
from tobacco cells with SfCPA-FAS was washed by 0.5 M NaCl or 0.1% (v/v) Triton X-100, then vortexed for
30 s, and incubated on ice for 10 min. After centrifugation (10 min at 14,000 × g), proteins from supernatant
and pellet were resolved in SDS-PAGE. Lane 1, pellet of NaCl
wash; lane 2, supernatant of NaCl wash; lane 3,
pellet of Triton X-100 wash; and lane 4, supernatant of
Triton X-100 wash.
-subunit (26) (Fig. 2). The
20,000 × g supernatant was centrifuged at 100,000 × g for 60 min to collect low density membrane particles
derived from microsomes. The SfCPA-FAS was greatly enriched
in this fraction (Fig. 2). These results demonstrate that
SfCPA-FAS is a microsomal localized enzyme and suggested
that CPA-FAs are synthesized outside of plastids.
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Fig. 2.
Subcellular localization of
SfCPA-FAS. Organelles of tobacco cells expressing
SfCPA-FAS were sequentially fractionated. Plastids were
sedimented by centrifuging at 5,000 × g for 2 min,
mitochondria at 20,000 × g for 5 min, and the
microsomal membranes at 100,000 × g for 60 min.
Enrichment of organelle fractions was estimated by immunoblot analyses
using antibodies against plastid and mitochondrial marker proteins.
Anti-biotin antibodies detected the biotin carboxyl carrier protein
(BCCP) subunit of plastid acetyl-CoA carboxylase and
monoclonal antibodies to the mitochondrial pyruvate dehydrogenase
-subunit (PDH-
) estimated mitochondrial
(mito) enrichment. The distribution of SfCPA-FAS
was determined by antibodies against SfCPA-FAS.
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Fig. 3.
Appearance of DHSA in PC versus
total lipids after in vivo labeling. Cell
suspension cultures (100 ml) of tobacco expressing SfCPA-FAS
were labeled with 20 µCi of
L-[methyl-14C]methionine. Samples
were collected at 3-min intervals followed by lipid extraction. For
each time point, total lipids were divided equally in two parts.
A, one part was used for lipid separation (9-min time
point shown). B, FAMEs were prepared from purified PC
(lane 1) and the other half of total lipids (lane
2), and fractionated with C18 reverse-phase TLC (9-min
time point). C, finally, the percentages of radioactive
DHSA found in PC to that in total lipids were plotted with time based
on three independent experiments.
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Fig. 4.
Linear range of SfCPA-FAS in
in vitro assays. A, radioactive
DHSA formed versus total protein extracted from
SfCPA-FAS tobacco cells in 200-µl reaction volume for
1 h. B, time course of SfCPA-FAS from
100 µg of total protein in 200-µl reaction volume for 1 h.
Each time point is calculated from three determinations.
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Fig. 5.
Association of DHSA with radioactive lipids
synthesized in in vitro assays. Standard in
vitro assays as described under "Experimental Procedures."
After the assay, half the reaction was used to prepare FAMEs directly;
the other half was used for lipid separation shown on the
top. FAMEs were prepared from each radioactive spot and
fractionated along with that from the other half on C18
reverse-phase TLC on the bottom. DHSA was only recovered
from the PC band. Absence of radioactive recovery from other bands
occurred if the radioactive product is water-soluble after FAME
preparation.
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Fig. 6.
Influence of oleoyl-CoA on
SfCPA-FAS in in vitro assays.
A, FAMEs separated on TLC after in vitro
assays. pE1776, control (lane 1); SfCPA-FAS
assay with addition of 0.05 mM oleoyl-CoA (lane
2); SfCPA-FAS assay without addition of oleoyl-CoA
(lane 3). B, SfCPA-FAS activities from three
determinates.
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Fig. 7.
Position analysis of DHSA on PC after
in vivo labeling of
L-[methyl-14C]methionine.
A, PC was purified from each time point, treated with
phospholipase A2, and free fatty acids and lyso-PC were
separated by TLC (left top). FAMEs were prepared from
lyso-PC and fractionated with C18 reverse-phase TLC
(bottom). B, the accumulation of
radioactivity in different classes is plotted versus time.
Radioactivity from lyso-PC was slightly lower than that from PC at
given time point, which was due to losses during recovery from TLC and
phospholipase A2 treatment.
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Fig. 8.
Fatty acid composition of
sn-1 and sn-2 positions of PC from
tobacco cells expressing SfCPA-FAS. PC was isolated from the total
lipids of tobacco cells expressing SfCPA-FAS and then
treated with phospholipase A2. FAMEs were prepared from
both free fatty acids (from sn-2 of PC) and lyso- PC (from
sn-1 of PC), and followed by gas chromatography-mass
spectrometry analysis.
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Fig. 9.
Position analysis of DHSA in PC from in
vitro assays. A, lane 1, PC
purified from in vitro assay and treated with phospholipase
A2, and lane 2, equal amount of PC without
phospholipase A2 treatment. B, DHSA from
corresponding lyso-PC (lane 1) and PC (lane 2) in
the upper panel.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We thank Mike Pollard for helpful discussions.
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FOOTNOTES |
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* This work was supported by The Dow Chemical Co., Dow AgroSciences, National Science Foundation Grant MCB9817882, and the Michigan Agricultural Experiment Station.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Biological Sciences, University of
Missouri, Columbia, MO 65211.
§ To whom correspondence should be addressed: Dept. of Plant Biology, Michigan State University, East Lansing, MI 48824. Tel.: 517-353-0611; Fax: 517-353-1926; E-mail: ohlrogge@pilot.msu.edu.
Published, JBC Papers in Press, January 31, 2003, DOI 10.1074/jbc.M212464200
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ABBREVIATIONS |
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The abbreviations used are: CPA-FAs, cyclopropane fatty acids; FAME, fatty acid methyl ester; CPE-FA, cyclopropene fatty acid; SfCPA-FAS, Sterculia foetida cyclopropane fatty-acid synthase; DHSA, dihydrosterculic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; aa, amino acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; ACP, acyl carrier protein; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid.
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