Biosynthesis of castor oil: Effect of polyamines on the acylation of lysophosphatidic acid at the sn-2 position with ricinoleic acid

Article abstract of DOI:10.1271/bbb.70044

Polyamines with diamine structures of chain length longer than 3C were essential for the synthesis of phosphatidic acid (PA) from ricinoleoyl-CoA and lysophosphatidic acid (LPA) by the castor LPA acyltransferase reaction, suggesting that polyamines modulate enzyme affinity for the acyl-CoA substrate in vivo.

Full text of DOI:10.1271/bbb.70044

Biosci. Biotechnol. Biochem., 71 (8), 2052–2056, 2007
Note
Biosynthesis of Castor Oil: Effect of Polyamines on the Acylation
of Lysophosphatidic Acid at the sn-2 Position with Ricinoleic Acid
Mitsuhiro TOMOSUGI,y Akane OHSHIRO, Shin’ichiro HARA, and Ken’ichi ICHIHARA
Department of Biological Chemistry, Faculty of Agriculture, Kyoto Prefectural University,
Shimogamo, Kyoto 606-8522, Japan
Received January 23, 2007; Accepted May 30, 2007; Online Publication, August 7, 2007
[doi:10.1271/bbb.70044]
Polyamines with diamine structures of chain length
longer than 3C were essential for the synthesis of
phosphatidic acid (PA) from ricinoleoyl-CoA and lyso-
phosphatidic acid (LPA) by the castor LPA acyltrans-
ferase reaction, suggesting that polyamines modulate
enzyme affinity for the acyl-CoA substrate in vivo.
stress and control of senescence and morphogenesis.6,7)
Polyamines are also essential in seed development.8)
To elucidate the interaction between polyamines and
the LPA acyltransferase reaction, we examined the
dependence of the reaction on the chain length of carbon
and the positive charge of polyamines. We also
determined the acylation of LPA with different hydroxy
fatty acids in the presence of polyamine. The discussion
is focused on the relationship between the structure of
polyamines and the LPA acyltransferase reaction for
ricinoleoyl-CoA in castor bean.
Key words: polyamine; lysophosphatidic acid acyltrans-
ferase; 2-ricinoleoyl phosphatidic acid
Castor oil, a natural oil obtained from the seeds of
the castor bean (Ricinus communis L.), is unique in
composition among all fats and oils in that it is
composed of approximately 90% of a hydroxy unsat-
urated fatty acid, ricinoleic acid (12-hydroxy-cis-9-
octadecenoic acid). This hydroxy fatty acid is a valuable
raw material used in a variety of applications, such as
lubricants, paints, dyes, and coatings.
1-Oleoyl-G3P was enzymatically prepared from 1,2-
dioleoyl phosphatidylcholine, and acyl-CoAs were pre-
pared by a mixed anhydride method.9,10) Castor bean
plants (Ricinus communis L.) were grown in a field at
Kyoto Prefectural University, Kyoto, Japan. Maturing
castor seeds were harvested 28–30 days after flowering,
when lipids accumulate in the seeds. Microsomes were
prepared by the method described previously.3) Protein
was determined by the method of Lowry et al.11)
Enzyme assay was followed spectrophotometrically by
measuring the rate of increase in absorbance at 412 nm
at 30 ꢀC by the procedure described previously.3) The
typical reaction mixture contained 0.05 M Tris/HCl (pH
8.5), 0.4 M sorbitol, 0.25 mM EDTA, 0.2 mM 5,50-
dithiobis-(2-nitrobenzoic acid)(DTNB), 25 mM 1-oleoyl
G3P, 20 mM acyl-CoA, and 50 mg protein of the 20,000 g
precipitate in a final volume of 1 ml. After preincubation
of all other components at 30 ꢀC for 3 min, the reaction
was initiated by the addition of microsomes, and was
followed continuously at 30 ꢀC for 1 min. The values of
specific activity given in the figures represent the initial
rates of phosphatidic acid synthesis, which are expressed
in nanomoles of acyl-CoA acylated per milligram of
microsomal protein. Values are the averages of
ꢁ standard deviations of results from experiments
performed in triplicate.
Triacylglycerol (TAG) is synthesized de novo from
glycerol 3-phosphate and acyl-CoAs by a combination
of acylation and dephosphorylation.1) In general, glyc-
erol-3-phosphate (G3P) acyltransferase and diacylglyc-
erol acyltransferase, which catalyze the first and third
acylations respectively, have broad acyl-CoA specific-
ities.2) Generally, lysophosphatidic acid (LPA) acyl-
transferase, which catalyzes the acylation of LPA at the
sn-2 position, has strict specificity for unsaturated acyl-
CoAs.2) Unsaturated acyl-CoAs are good substrates for
the LPA acyltransferase from castor seeds.3) We have
reported that ricinoleoyl-CoA was not utilized for the
acyltransferase reaction without polyamines, although
the hydroxy fatty acid is an unsaturated fatty acid.
Interestingly, activity for ricinoleoyl-CoA appeared with
the addition of polyamines, including putrescine, sper-
midine, and spermine.3) Polyamines are low-molecular-
weight polycations ubiquitous in plant cells.4,5) In the
plant kingdom, these polyamines are involved in many
metabolic processes, such as responses to biological
The ricinoleate concentration at the sn-2 position of
y
To whom correspondence should be addressed. Fax: +81-75-703-5750; E-mail: a0120712@kpu.ac.jp
Abbreviations: G3P, glycerol 3-phosphate; LPA, lysophosphatidic acid; PA, phosphatidic acid; TAG, triacylglycerol; ER, endoplasmic reticulum;
DTNB, 5,50-dithiobis-(2-nitrobenzoic acid); 12-OH-12:0, 12-hydroxydodecanoic acid; 3-OH-14:0, 3-hydroxytetradecanoic acid; 16-OH-16:0, 16-
hydroxyhexadecanoic acid; 18-OH-18:0, 18-hydroxyoctadecanoic acid; 12-OH-cis-18:1, ricinoleic acid; 12-OH-trans-18:1, trans ricinoleic acid; cis-
13-22:1, erucic acid

Triacylglycerol Synthesis in Castor Bean
2053
data suggest that a carbon chain length longer than 3 and
the flexibility of the carbon chain are important for the
synthesis of 2-ricinoleoyl-PA.
70
60
50
40
30
20
10
0
Since the correlation between the structure of poly-
amines and LPA acyltransferase reaction was evaluated
in Fig. 1, we tested the effects of spermidine on the LPA
acyltransferase reaction for a very long chain fatty acid
moiety and hydroxy fatty acid species with hydroxy
groups at different positions and with various chain
lengths. All the acyl-CoAs tested were utilized as
substrates in the presence of 0.5 mM spermidine
(Fig. 2A), and spermidine at 1 or 2 mM was also found
to increase the incorporation rates of 12-hydroxydode-
canoic acid, 3-hydroxytetradecanoic acid, and 13-doco-
senoic acid (Fig. 2B). In oil-plant species containing
unique fatty acids, such as epoxy, hydroxy, and very
long chain fatty acids, polyamines can affect the
activities of other enzymes engaged in lipid metabolism
and can modulate TAG synthesis, e.g., G3P acyltrans-
ferase from E. coli,12) and both G3P acyltransferase and
LPA acyltransferase from safflower seeds.9,13)
Castor LPA acyltransferase utilized hydroxy fatty
acids with chain lengths of 16C and 18C as substrates,
except for ricinoleic acid, in the absence of spermidine.
Moreever, the trans isomer of ricinoleoyl-CoA served as
a substrate, while LPA acyltransferase did not incorpo-
rate ricinoleic acid with a cis double bond into the sn-2
position of LPA without spermidine. However, there
were no significant differences in the effects of spermi-
dine on the LPA acyltransferase reaction for either cis or
trans ricinoleoyl-CoAs. Polyamines in the LPA acyl-
transferase reaction most likely modulate the affinity of
the enzyme for the substrate.
In vivo, phospholipids, nucleic acids, and acidic
residues of membrane-bound proteins can interact with
polyamines. Modulation of the surface charge by poly-
amines might play an important role in the regulation of
membrane-bond enzymes dealing with charged sub-
strates.14) Several possible mechanisms for the stimula-
tory effect of polyamines on the LPA acyltransferase
reaction have been reported:3) (i) defense against
disruption of endoplasmic reticulum (ER) membranes,
(ii) modulation of the surface charge of ER membranes,
(iii) direct interaction with LPA acyltransferase, (iv) the
existence of isozymes, and (v) the binding of polyamines
to the substrate, LPA or acyl-CoA. Possible mechanism
(i) implies that the production of 2-ricinoleoyl PA
disrupts the ER membrane structure and, that poly-
amines have a stabilizing effect by bridging and
shielding the charged membrane. The LPA acyltransfer-
ase reaction for ricinoleoyl-CoA was performed by
incubation for 5 min, and was followed by further
addition of oleoyl-CoA to the reaction mixture
(Fig. 3). If this possible mechanism is active, LPA
acyltransferase should not be able to utilize additional
oleate. As a consequence, oleate was incorporated into
LPA (experiment 3). These observations indicate that
the production of 2-ricinoleoyl PA does not disrupt ER
None 2
3
4
5
7
8
10 12
Diamine [NH2(CH2)nNH2]
Fig. 1. Effects of Diamines on LPA Acyltransferase Activity.
The acyl acceptor was 1-oleoyl-G3P, and the acyl donor was
oleoyl-CoA (white bars) or ricinoleoyl-CoA (gray bars). Values are
the averages +/ꢂ standard deviations of results from experiments
performed in triplicate. 2, Ethylenediamine; 3, 1,3-diaminopropane;
4, 1,4-diaminobutane (putrescine); 5, 1,5-diaminohexane (cadaver-
ine); 7, 1,7-diaminoheptane; 8, 1,8-diaminooctane; 10, 1,10-diami-
nodecane; 12, 1,12-diaminododecane.
castor bean TAG was higher than those at positions 1
and 3,3) and therefore, LPA acyltransferase was ex-
pected to utilize ricinoleoyl-CoA. However, no activity
was found for ricinoleoyl-CoA at any concentration
tested in vitro. Further research revealed that the rate of
acylation of LPA at the sn-2 position with ricinoleate
was markedly accelerated by the addition of polyamines
(putrescine, spermidine, and spermine), while activities
for other acyl-CoAs were inhibited by polyamines. To
investigate the actions of polyamines in the LPA
acyltransferase reaction, we examined the effects of
various diamines with different carbon chain lengths on
LPA acyltransferase activity (Fig. 1). Ethylenediamine
did not affect the acyltransferase reaction and diamines
with chains lengths longer than 3C enhanced the
incorporation rate of ricinoleate. Diamines longer than
a chain length of 3C showed no remarkable differences
in LPA acyltransferase activity for ricinoleate.
As Fig. 1 suggests, the positive charges of polyamines
are important for incorporation of ricinoleate into
position 2 of LPA, and hence the effects of positive
charges on LPA acyltransferase reaction were examined
using polylysine and chitosan (data not shown). Poly-
lysine stimulated LPA acyltransferase activity for
ricinoleoyl-CoA, whereas chitosan was completely
inactive in the incorporation of both oleate and
ricinoleate. Compounds with heterocycles and aromatic
rings (2,20-dipyridyl, 4,40-bipyridyl, piperazine, 1,4,8,
11-tetraazacyclotetradecane, 1,5-diaminonaphthalene,
and 1,2-bis(4-pyridyl)ethane) had no effect on the LPA
acyltransferase reaction (data not shown). The cyclic
structure or hydroxy groups of chitosan probably
interfere with the incorporation of ricinoleate. These

2054
M. TOMOSUGI et al.
A
100
80
60
40
20
0
B
10
8
6
4
2
0
0
1.0
2.0
Spermidine (mM)
Fig. 2. Very Long Chain Fatty Acyl-CoA and Hydroxy Fatty Acyl-CoA Specificities of LPA Acyltransferase Activity.
The acyl acceptor was 1-oleoyl-G3P. Values are the averages +/ꢂ standard deviations of results from experiments performed in triplicate. A,
Control, white bars; 0.5 mM spermidine, gray bars; oleoyl-CoA, 18:1; 12-OH-12:0, 12-hydroxydodecanoyl-CoA; 3-OH-14:0, 3-hydroxyte-
tradecanoly-CoA; 16-OH-16:0, 16-hydroxyhexadecanoyl-CoA; 18-OH-18:0, 18-hydroxyoctadecanoyl-CoA; 12-OH-cis-9-18:1, ricinoleoyl-
CoA; 12-OH-trans-9-18:1, trans ricinoleoyl-CoA; cis-13-22:1, erucoyl-CoA. B, Symbols: triangles, erucoyl-CoA; squares, 12-hydroxydode-
canoyl-CoA; circles, 3-hydroxytetradecanoly-CoA.
membranes. Even if polyamines interact with ER
membranes, it appears that the interaction does not
influence the enhancing effect of polyamines on LPA
acyltransferase activity. LPA acyltransferase was an
unstable enzyme, and the activity was reduced to about a
half the initial value after 5 min of incubation at 30 ꢀC
even in the absence of acyl-CoA and DTNB (experi-
ments 1, 4, and 5).
the incorporation of unique fatty acids, such as erucic
acid and hydroxy fatty acids, including ricinoleic acid
(Fig. 2). The substrate affinity of LPA acyltransferase is
probably modulated by polyamines, i.e., it is anticipated
that mechanism (iii), direct interaction with LPA
acyltransferase, is active. Based on the data in Figs. 2
and 3, we speculate that the structure of the binding site
for acyl-CoA is altered by the interaction of polyamines
with the enzyme, and that the affinity of LPA acyl-
transferase for acyl-CoA consequently becomes less
Polyamines decreased the incorporation of normal
fatty acids, such as oleic and linoleic acid, and increased

Triacylglycerol Synthesis in Castor Bean
2055
60
50
40
30
20
10
0
5 +5
5 +5
5 +5
5 +5
5 +5
Time(min)
Acyl-CoA
+ +
1
+ +
2
+ +
3
+ +
4
- +
5
DTNB
Experiment
Fig. 3. Effects of the 2-Ricinoeleoyl PA Product on LPA Acyltransferase Activity.
The reaction mixture contained 0.05 M Tris/HCl (pH 8.5), 0.4 M sorbitol, 0.25 mM EDTA, 0.2 mM DTNB, 50 mM 1-oleoyl G3P, 20 mM acyl-
CoA, and 50 mg protein of the 20,000 g precipitate in a final volume of 1 ml. After the reaction mixture containing oleoyl or ricinoleoyl-CoA was
incubated at 30 ꢀC for 5 min, changes in LPA acyltransferase activity were followed by further addition of 100 ml of 200 mM oleoyl or ricinoleoyl-
CoA at 30 ꢀC for 5 min. Values are the averages +/ꢂ standard deviations of results from experiments performed in triplicate.
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specific. Determination of the structure of castor bean
LPA acyltransferase protein should confirm the validity
of this mechanism.
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