Activation of acyl condensation reaction of monomeric 6-hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme, by free coenzyme A

Article abstract of DOI:10.1016/S0031-9422(02)00377-1

6-Hydroxymellein (6HM) synthase is a multifunctional polyketide enzyme induced in carrot cells, whose fully active homodimer catalyzes condensation of acyl-CoAs and the NADPH-dependent ketoreduction of the enzyme-bound intermediate. 6HM-forming activity of the synthase was markedly decreased when the reaction mixture pH was adjusted from 7.5 to 6.0. However, under these slightly acidic conditions, the acyl condensation catalyzed by the dissociated monomer enzyme was appreciably stimulated by addition of free coenzyme A (CoA). In contrast, the condensation reaction at pH 6.0 was significantly inhibited in the presence of CoA when the reaction was carried out with the NADPH-omitted dimer synthase. Among the kinetic parameters of the acyl condensation, velocity of the monomer-catalyzing reaction at the acidic pH was appreciably increased upon addition of CoA while Kms did not show any significant change in the presence and absence of the compound. These results suggest that CoA associates with a specific site in the dissociated monomeric form of 6HM synthase, and the velocity of the acyl condensation reaction catalyzed by the CoA-synthase complex appreciably increases in acidic conditions.

Full text of DOI:10.1016/S0031-9422(02)00377-1

Phytochemistry 61 (2002) 597–604
www.elsevier.com/locate/phytochem
Activation of acyl condensation reaction of monomeric
6-hydroxymellein synthase, a multifunctional polyketide
biosynthetic enzyme, by free coenzyme A
Fumiya Kurosaki*, Satoru Mitsuma, Munehisa Arisawa
Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Sugitani, Toyama 930-0194, Japan
Received 24 May 2002; received in revised form 12 August 2002
Abstract
6-Hydroxymellein (6HM) synthase is a multifunctional polyketide enzyme induced in carrot cells, whose fully active homodimer
catalyzes condensation of acyl-CoAs and the NADPH-dependent ketoreduction of the enzyme-bound intermediate. 6HM-forming
activity of the synthase was markedly decreased when the reaction mixture pH was adjusted from 7.5 to 6.0. However, under these
slightly acidic conditions, the acyl condensation catalyzed by the dissociated monomer enzyme was appreciably stimulated by
addition of free coenzyme A (CoA). In contrast, the condensation reaction at pH 6.0 was significantly inhibited in the presence of
CoA when the reaction was carried out with the NADPH-omitted dimer synthase. Among the kinetic parameters of the acyl con-
densation, velocity of the monomer-catalyzing reaction at the acidic pH was appreciably increased upon addition of CoA while Kms
did not showany significant change in the presence and absence of the compound. These results suggest that CoA associates with a
specific site in the dissociated monomeric form of 6HM synthase, and the velocity of the acyl condensation reaction catalyzed by the
CoA-synthase complex appreciably increases in acidic conditions.
# 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Daucus carota; Umbelliferae; Carrot; CoA; Multifunctional polyketide biosynthetic enzymes; Acyl condensation reaction; Acidic condi-
tion; Dissociation of homodimer; Enzyme regulation
1. Introduction
the rate-limiting step in the series of partial reactions
which determines the overall rate of 6HM biosynthesis
(Kurosaki et al., 1999). It has been shown (Kurosaki et
al., 1989; Kurosaki, 1995) that if the reducing co-factor
(NADPH) is either omitted or the active homodimer is
dissociated into the monomer subunits, the ketoredu-
cing reaction is disabled and triacetic acid lactone 2
(TAL) is liberated as a derailment product instead of
6HM (1) (Fig. 1a). We previously demonstrated (Kur-
osaki et al., 1989, 2000) that free coenzyme A (CoA) is
an important allosteric factor stimulating 6HM-biosyn-
thetic activity of the enzyme. It was also shown that the
CoA associated with the dimeric enzyme reduced the
energy barrier of the ketoreducing reaction by altering
the microstructure of the reaction center of the protein
and enhanced 6HM (1) formation and accumulation.
However, the factor(s) affecting the acyl condensation
reaction of 6HM synthase, if any, are not yet known.
Since several catalytic reactions and the structural
organization of 6HM synthase are similar to those of
animal fatty acid synthase (FAS), it has been assumed
that these two enzymes share many common properties
6-Hydroxymellein 1, (6HM) is a direct precursor of
the carrot phytoalexin, 6-methoxymellein (Coxon et al.,
1973), and the enzyme catalyzing the biosynthesis of this
compound, 6HM synthase, is a multifunctional polyke-
tide synthetic enzyme induced in the cells (Kurosaki et
al., 1991). The enzyme catalyzes the condensation of
acetyl- and malonyl-CoAs to form the pentaketomethy-
lene chain, and, as shown in Fig. 1a, an NADPH-depen-
dent ketoreduction of the carbonyl group takes place at
the triketide intermediate stage (Kurosaki et al., 1989).
The active form of 6HM synthase is organized as a
homodimer (Kurosaki, 1995), and two subunits
(approximately 130 kDa each) containing the functional
domains of the ketoreduction and the acyl condensation
are aligned in the head-to-tail direction to form two com-
plete reaction centers (Fig. 1b). The reduction reaction of
the carbonyl group in the synthase-bound intermediate is
* Corresponding author. Fax: +81-76-434-5052.
E-mail address: kurosaki@ms.toyama-mpu.ac.jp (F. Kurosaki).
0031-9422/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(02)00377-1

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F. Kurosaki et al. / Phytochemistry 61 (2002) 597–604
(Wakil and Stoops, 1989; Wakil et al., 1983; Wakil,
1989; Smith, 1994). In animal FAS, it has been shown
(Stoops and Wakil, 1981; Wakil, 1989) that Cys-SH of
the condensation enzyme is unusually more active at
slightly acidic conditions, such as at pH 6.0 and 6.5,
indicating that the thiol most likely exists as a thiolate
ion and therefore is highly susceptible to alkylation with
appropriate SH reagents (McCarthy and Hardie, 1982;
Anderson and Kumar, 1987).
In the present study, we first attempted to characterize
the possible pH-dependency of 6HM synthase-catalyz-
ing reactions, and special attention was focused on the
role of CoA in the acyl condensation of 6HM synthase.
1993). The activity of 6HM synthase was completely
lost after freezing overnight, and more than 80%
decrease in the activity was observed ꢀwhen the prepara-
tion was stored in pH 7.5 buffer at 4 C for 5 days. The
addition of polyalcohols and sucrose into the buffers did
not appear to stabilize the enzyme. It is reasonable to
assume, therefore, that this very unstable protein would
irreversibly lose its catalytic activities by transfer to, and
storage in pH 6.0 buffer. Thus, we first tested whether or
not the synthase activity is restored after treatment of
the enzyme protein with slightly acidic buffer for several
hours. Accordingly, highly purified 6HM synthase
(approximately 10 mg) in 15 ml of pH 7.5 K-Pi buffer
was divided equally into two portions, and one of the
enzyme solutions was transferred into an Amicon cell
(YM-10 membrane). The buffer was replaced by pH 6.0
buffer in an ice bath, and after washing with acidic buf-
fer (2Â2 ml), the volume of the enzyme solution was
adjusted to 5 ml. The enzyme in the pH 6.0 buffer was
then incubated at 37 ꢀC for 4 h, and the acidic buffer was
again replaced by 5 ml of the pH 7.5 buffer by ultra-
filtration. The other half of the enzyme solution at pH 7.5
was similarly concentrated to 5 ml by ultrafiltration, and
2. Results and discussion
2.1. Stability of 6HM synthase in acidic buffer
As reported previously, 6HM synthase is very
unstable even in neutral buffers, like other multi-
functional polyketide enzymes of microbial origin (Beck
et al., 1990; Spencer and Jordan, 1992; Kurosaki et al.,
Fig. 1. Various aspects of 6HM synthase (a) catalytic reaction. 6HM synthase catalyzes the condensation of acetyl- and malonyl-CoAs, and an
NADPH-dependent ketoreduction takes place at the triketide intermediate stage. Further condensation of malonyl-CoA results in the production of
6HM (1). (b) Schematic presentation of the arrangement of the functional domains of homodimeric 6HM synthase. The catalytic domain for keto-
methylene chain elongation by acyl condensation is associated with that of ketoreduction belonging to another subunit, and two equivalent reaction
centers are organized in each molecule of the active synthase. (c) Schematic presentation of the reaction center of 6HM synthase. The acyl groups
transfer from the CoA esters to Ser-OH of transacylase, and they are properly channeled to two SH groups, Cys-SH and ACP-SH, prior to the
initiation of the condensation reactions.

F. Kurosaki et al. / Phytochemistry 61 (2002) 597–604
599
incubated for 4 h at 37 ꢀC. The 6HM biosynthetic
activities were of these two enzyme preparations were
then determined. In repeated experiments, the activity
of the acidic buffer-treated enzyme was slightly
decreased to 83–91% of the controls in the pH 7.5 buf-
fer (data not shown). These results strongly suggest that
the loss of 6HM synthase activities, i.e. for both acyl
condensation and ketoreduction, is essentially negligible
even after treatment with the pH 6.0 buffer for 4 h. This
also implies that the possible difference in the catalytic
activities of 6HM synthase between pH 7.5 and 6.0 con-
ditions were not due to the irreversible and/or nonspecific
alterations of the protein structures caused by treatment
with the acidic buffer, but rather resulted from the pH-
dependent changes in the catalytic properties of the
enzyme. Therefore, all of the following experiments were
done within 4 h after the transfer of 6HM synthase protein
to the pH 6.0 buffer using freshly prepared enzyme.
activity. The association of CoA to the enzyme suggests
an allosteric interaction that activates the ketoreducing
reaction which determines the rate of 6HM (1) bio-
synthesis. In contrast, CoA exhibited a marked inhibi-
tory effect on TAL (2) formation by the acyl
condensation reaction, catalyzed by either the dimeric
synthase without reducing co-factor or the dissociated
monomer enzyme by product inhibition mechanism
under neutral conditions (Kurosaki, 1998). A set of
these experiments was repeated in the present study, and
the same results were obtained reproducibly (Table 1).
Under the slightly acidic conditions (pH 6.0), CoA also
functioned as an activator of 6HM (1) production, and
a 1.3–1.4-fold increase in the activity (from 1.35 to 1.79
pkat and from 1.01 to 1.45 pkat, respectively) was
observed by addition of 0.3 mM CoA as well as in the
neutral pH (Table 1). However, in sharp contrast to 6HM
(1) production, CoA showed quite different effects on the
TAL (2)-synthetic reactions at pH 6.0. Although, as
observed at neutral pH, TAL (2) formation by NADPH-
free dimer was appreciably inhibited in the presence of
CoA (from 0.11 to 0.05 pkat and from 0.10 to 0.06 pkat,
respectively), the production of 6 HM (1) catalyzed by the
dissociated monomer subunits was significantly enhanced
upon CoA addition. In repeated experiments, the relative
activity of the acyl condensation of the monomer was
increased from 0.11 to 0.19 pkat and from 0.11 to 0.22
pkat, respectively, and therefore, the TAL (2)-forming
ability was elevated 1.7–2.0-fold higher levels in the pre-
sence of CoA. The enhanced activity of TAL (2) pro-
duction again decreased to the control level when CoA
was removed from the mixture by dialysis (data not
shown). It appeared, therefore, that the CoA-induced
activation of the acyl condensation reaction by the
monomer synthase is a reversible process, and it is very
likely that CoA molecule non-covalently associates with
the monomer subunits under acidic condition.
2.2. Effect of CoA on 6HM synthase activities under
neutral and acidic condition
The activities of 6HM synthase for 6HM (1) and TAL
biosyntheses were determined at pH 7.5 and 6.0 under
various conditions. As shown in Table 1, transfer of the
enzyme from pH 7.5 to 6.0 significantly decreased 6HM
(1) formation to 24 and 37% of the controls kept in
neutral buffer (from 4.21 to 1.01 pkat, and from 3.66 to
1.35 pkat, respectively). The TAL-producing activities
were also inhibited under acidic condition either when
NADPH is omitted from the reaction mixture or when the
enzyme is dissociated into monomers by addition of NaCl
(see Experimental). The activities decreased to 31–44% of
the controls in the dimer enzyme without the co-factor,
and from 30–38% in the dissociated monomer subunits,
respectively.
As reported previously (Kurosaki et al., 1999, 2000),
addition of submillimolar concentrations of CoA into
the 6HM-producing system under the neutral condition
resulted in appreciable enhancement of biosynthetic
The effect of various concentrations of CoA on the
acyl condensation reaction of 6HM synthase was also
examined. As shown in Fig. 2, TAL (2) production
Table 1
Catalytic activity of 6HM synthase under neutral and acidic conditionsa
Experimental 1
Experimental 2
ÀCoA
+CoA
ÀCoA
+CoA
6HM (1) formation (pkat)
pH 7.5
pH 6.0
4.21
1.01
6.95
1.47
3.66
1.35
6.55
1.79
TAL (2) formation by dimers without NADPH (pkat)
pH 7.5
pH 6.0
0.25
0.11
0.13
0.05
0.32
0.10
0.13
0.06
TAL (2) formation by monomers (pkat)
pH 7.5
pH 6.0
0.29
0.11
0.13
0.19
0.37
0.11
0.21
0.22
a
6HM (1)-forming and TAL (2)-forming activities of 6HM synthase at pH 7.5 and 6.0 were determined in the absence or presence of 0.3 mM CoA.

600
F. Kurosaki et al. / Phytochemistry 61 (2002) 597–604
increased with an increase in CoA concentration when
the reaction was catalyzed by dissociated monomers of
6HM synthase, and the synthetic ability elevated to
approximately 1.7–2.0-fold higher level than that of the
control in the presence of 0.3–1 mM of CoA. Addition
of 1 mM NADPH or NADH to a monomer-catalyzed
reaction did not showany significant change in the
CoA-induced activation (data not shown), suggesting
that the association of NADPH or NADH with the
ketoreducing domain of the monomers did not affect
enhancement of the condensation. In contrast, if the
reaction was catalyzed by the NADPH-free dimer syn-
thase, CoA exhibited significant inhibitory activity on
the condensation processes in a dose-dependent manner
(Fig. 2) as was the case at the neutral pH (Kurosaki et
al., 1999). As in FAS (Smith, 1994), pantetheine func-
tions as an acyl acceptor in 6HM synthase-catalyzed
reaction (Kurosaki and Arisawa, 1999). However, when
CoA was replaced by pantetheine, TAL (2) biosynthesis
was appreciably inhibited in the reactions catalyzed by
either NADPH-deprived homodimer or the monomeric
form of 6HM synthase (Fig. 3). This result implies that the
biochemical properties of CoA as an acyl acceptor is not
responsible for stimulation of the condensation reaction.
possibility was examined that association of CoA under
acidic condition alters the process of substrate entry
into the reaction center of the monomeric 6HM syn-
thase. Accordingly, two SH groups at the reaction cen-
ter of 6HM synthase, Cys–SH of ketoacyl synthase and
cysteamine-SH attached to the acyl carrier protein
(ACP), were specifically alkylated with iodoacetoamide
(IoAA) and chloroacetyl–CoA (ClAc–CoA) to simplify
estimation of the binding ability of the enzyme toward
its co-substrates (Kurosaki, 1996a). In this partially
masked protein, it is expected that only Ser-OH of the
transacylase structure, the primary binding site of the
co-substrates, should function (Fig. 1c). It has been
confirmed (Kurosaki, 1996a) that the enzyme does not
lose either substrate-binding or substrate-channeling
activity even after modification with these reagents. This
chemically modified protein was incubated with radi-
olabeled acetyl- and malonyl-CoAs, and the ratio of the
synthase-bound forms of the acyl groups was deter-
mined. Since purity of the synthase in each batch of
enzyme preparations varied, besides the enzyme being
very unstable (Kurosaki, 1996a,b), estimation of the
chemical stoichiometry of the synthase-bound acyl
groups toward the enzyme was difficult. Results were,
therefore, expressed as relative values in which the
amount of acetyl group bound to the NADPH-free
dimer enzyme without CoA was taken as 1 in each set of
experiments as reported previously (Kurosaki, 1996a).
As shown in Table 2, addition of 0.3 mM CoA resulted
in a marked decrease in the ratio of enzyme-bound ace-
tyl and malonyl groups when these two substrates were
added independently. The enzyme-bound forms of the
2.3. Effect of CoA on substrate entry into 6HM
synthase under acidic condition
Association of NADPH with the ketoreducing
domain of 6HM synthase enhances the substrate entry
into the enzyme protein to elevate the activity of 6HM
(1) biosynthesis (Kurosaki, 1998). Therefore, the
Fig. 2. Effect of CoA on TAL (2)-forming reaction catalyzed by 6HM
synthase. The acyl condensation reaction catalyzed by 6HM synthase
was determined either in the dissociated monomer subunits (ꢁ) or in
the NADPH-free homodimeric form (~) of the enzyme in the pre-
sence of various concentrations of CoA. The results were expressed as
percentages in which the acyl condensation activities determined
without CoA (0.19 and 0.38 pkat for the dissociated monomer, and
0.22 and 0.43 pkat for NADPH-omitted dimer, respectively) were
taken as 100%.
Fig. 3. Effect of pantetheine on TAL (2)-forming reaction catalyzed
by 6HM synthase. The condensation reaction catalyzed by 6HM syn-
thase was determined either in the dissociated monomers (ꢁ) or in the
NADPH-omitted dimeric form (~) in the presence of various con-
centrations of pantetheine. The results were expressed as percentages
in which the activities without pantetheine (0.24 and 0.36 pkat for the
monomer, and 0.29 and 0.40 pkat for the NADPH-omitted dimer,
respectively) were taken as 100%.

F. Kurosaki et al. / Phytochemistry 61 (2002) 597–604
601
acyl groups decreased to roughly 30–60% of the con-
trols, and similar results were also obtained if the two
substrates were added in a 1:1 mixture (Table 2) indi-
cating that CoA functions as an inhibitor of substrate
entry into the dissociated monomer. It is concluded,
therefore, that this early step of acyl condensation is not
the process responsible for CoA-induced stimulation of
the reaction under acidic condition.
In the next experiment, velocities of the acyl con-
densation were determined under various conditions. As
described above, 6HM synthase preparation in the pre-
sent experiments did not consist of a homogenous pro-
tein though highly purified, and the purity and specific
activities of the synthase varied in each batch of enzyme
preparation. In addition, due to the instability of the
enzyme, part of the synthase activity was lost during
purification (Kurosaki, 1998). Consequently, V values
for 6HM synthase in TAL (2) production varied con-
siderably in repeated experiments (0.7–4.6 nkat/g pro-
tein at pH 7.0), and hence direct comparison of Vs
obtained from independent experiments was not possi-
ble. However, the relative ratio of the V values for the
TAL (2)-producing reactions catalyzed by 6HM syn-
thase was found to be almost constant irrespective of
the specific activity of the enzyme preparations. There-
fore, in order to compare the velocities of TAL (2) -
forming reaction, the results were expressed as relative
values in each set of the experiments in which Vmax of
TAL (2) production catalyzed by the NADPH-free
dimer enzyme without CoA at pH 7.5 was taken as 1.
As shown in Table 3, the two TAL (2) -producing reac-
tions under neutral conditions appreciably decreased in
the presence of CoA (from 1 to 0.75 and 1.12 to 0.71).
At pH 6.0, the relative V value of the TAL (2)-forming
reaction by NADPH-free synthase markedly decreased
(from 0.42 to 0.22) as well as in the reactions at the
neutral pH. However, in sharp contrast, the V of TAL
(2) formation catalyzed by dissociated monomers at pH
6.0 appreciably increased upon association with CoA,
and the value was elevated to an approximately 2.3-fold
higher level than that without CoA (from 0.39 to 0.90).
These results strongly suggest that elevation of the
velocity of the acyl condensation reaction should be the
main reason for the CoA-induced activation of the TAL
2.4. Kinetic parameters of monomeric 6HM synthase
under acidic condition
The CoA-induced activation of acyl condensation by
6HM synthase was further characterized by determining
kinetic parameters of the reactions at pH 6.0 in the
absence and presence of 0.3 mM CoA (Table 3). Several
kinetic values of the synthase have already been deter-
mined at pH 7.5 (Kurosaki, 1998, Kurosaki et al.,
2000). In the present study, almost identical values were
obtained. In the monomer-catalyzed TAL (2) formation
at pH 6.0, Km for acetyl-CoA was slightly increased by
addition of CoA (from 383 to 423 mM), and the values
for malonyl-CoA were almost comparable in the
absence and presence of CoA (51 and 48 mM,
respectively). As shown in Table 3, Kms obtained for the
monomeric synthase were essentially similar to those
estimated for the NADPH-free dimer synthase in acidic
pH. In addition, these values in the TAL (2)-forming
reactions at pH 6.0, either in the presence or absence of
CoA, were essentially comparable to the figures
obtained for the reactions at neutral pH (Table 3).
Therefore, it is likely that alteration of affinity of the
monomeric 6HM synthase to co-substrates upon pre-
sumed association of the enzyme to CoA is not respon-
sible for activation of the condensation reaction of the
enzyme at acidic pH.
Table 2
Substrate-binding activity of 6HM synthase under acidic conditionsa
Experiment 1
Experiment 2
ÀCoA
+CoA
ÀCoA
+CoA
Acetyl-CoA
Dimers without NADPH
Dissociated monomers
Dimers without NADPH
Dissociated monomers
1b
0.45
0.51
0.60
0.41
1b
0.61
0.39
0.55
0.33
0.84
1.12
0.89
1.20
0.81
1.09
Malonyl-CoA
Acetyl-CoA plus malonyl-CoA
Dimers without NADPH
Dissociated monomers
Acetyl
0.64
0.51
0.47
0.59
0.31
0.22
0.24
0.32
0.68
0.44
0.57
0.41
0.27
0.31
0.24
0.23
Malonyl
Acetyl
Malonyl
a
Two SH groups at the reaction center of 6HM synthase were blocked by alkylation, and binding ability of the modified enzyme against acyl-
CoAs was examined in the absence or presence of 300 mM of CoA.
b
Results were expressed as relative values in which the amounts of acetyl group bound to the dimeric form of the modified enzyme without the
reducing co-factor were taken as 1 in each set of the experiments.

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F. Kurosaki et al. / Phytochemistry 61 (2002) 597–604
Table 3
Kinetic parameters of condensation reaction catalyzed by 6HM synthase
ÀCoA
+CoA
Km for acetyl-CoA (mM)
Km for malonyl-CoA (mM)
Relative Vmaxb
pH 7.5a
pH 6.0
pH 7.5
pH 6.0
pH 7.5
pH 6.0
Dimers without NADPH
Dissociated monomers
Dimers without NADPH
Dissociated monomers
Dimers without NADPH
Dissociated monomers
Dimers without NADPH
Dissociated monomers
Dimers without NADPH
Dissociated monomers
Dimers without NADPH
Dissociated monomers
284Æ43
318Æ144
417Æ81
383Æ83
43Æ16
468Æ52
524Æ24
344Æ46
423Æ68
41Æ18
40Æ13
46Æ15
57Æ11
49Æ18
51Æ8
48Æ7
1
0.75Æ0.30
0.71Æ0.27
0.22Æ0.14
0.90Æ0.21
1.12Æ0.15
0.42Æ0.11
0.39Æ0.14
a
Km values of 6HM synthase at neutral pH have been reported previously, however, the set of the experiments was repeated in the present study.
Since almost identical Kms were reproducibly obtained, the previously reported values are presented for reference (Kurosaki et al., 1999).
b
Vs of TAL-producing reactions catalyzed by 6HM synthase were expressed as relative values in which 6HM (1) formation by the homodimer
enzyme at pH 7.5 without CoA was taken as 1. Km and Vmax values were presented as means and S.D. obtained from four independent experiments.
(2)-forming reaction catalyzed by the monomer subunits
of 6HM synthase under acidic condition.
this class of multifunctional proteins that catalyze acyl
condensation and ketoreduction by association with
CoA might be potentially an important phenomenon.
Further studies to elucidate the mechanisms of the
CoA-induced stimulation of acyl condensation
catalyzed by 6HM synthase are in progress.
In the present study, it has been shown that associ-
ation of CoA molecule with 6HM synthase monomer
subunits results in activation of the acyl condensation
reaction to liberate TAL (2) as product. We reported
previously (Kurosaki et al., 1999, 2000) that the ketor-
educing process, the rate-limiting step of 6HM (1) bio-
synthesis, is appreciably activated by a certain allosteric
interaction when CoA associates with the homodimer
form of 6HM synthase. One possible explanation for
these results is that there are two CoA-binding sites in
6HM synthase, and the first site would function only in
the homodimeric form to activate the reducing reaction
upon association with CoA (Fig. 1c). Another CoA-
binding site might accept CoA molecule only in the dis-
sociated monomer form to stimulate the acyl condensa-
tion reaction. Alternatively, it is also possible that the
same site for CoA-binding is involved with the mono-
mer and dimer (plus NADPH) but with different out-
comes in the two different reactions. CoA-induced
activation of the acyl condensation catalyzed by mono-
meric 6HM synthase was specifically observed under the
acidic condition (Table 2); however, at present, it is not
clear why stimulation of the monomer-catalyzed con-
densation by CoA is observed only at slightly acidic pH.
Although no evidence is available to explain this obser-
vation, it is possible that a key amino acid residue
involved in monomer-specific CoA-binding site exhibits
affinity against CoA only in the acidic condition.
3. Experimental
3.1. Chemicals
6HM (1) was prepared by demethylating 6-methoxy-
mellein isolated from fungal-infected carrot roots with
BBr3 in anhydrous CH2Cl2 as reported in detail (Kur-
osaki et al., 1989). TAL (2) was synthesized from dehy-
droacetic acid (nacalai tesque) as reported (Kurosaki et
al., 1989), and ClAc–CoA was prepared according to
the method of Kawaguchi et al. (1981).
2-Chloroethylphosphonic acid, acetyl-CoA, malonyl-
CoA, NADPH, NADH, CoA, pantetheine and bovine
serum albumin were purchased from Sigma, while
dithiothreitol (DTT) and IoAA were from Wako Pure
Chemicals. [2-14C]Acetyl-CoA (2.1 GBq/mmol) and
[2-14C]malonyl-CoA (2.2 GBq/mmol) were obtained
from Perkin Elmer. All other chemicals were reagent
grade.
3.2. Induction, purification and assay of 6HM synthase-
catalyzing reactions
In animal FAS, it was demonstrated (Strom et al.,
1979; Strom and Kumar, 1979) that the activity of enoyl
reductase was markedly stimulated in the presence of
submillimolar levels of CoA, and an almost 5-fold
increase in the activity was observed. Although the cat-
alytic domain corresponding to enoyl reductase does
not exist in 6HM synthase, regulation of the activities of
6HM synthase was induced in carrot cells by treat-
ment of root tissues with 2-chloroethylphosphonic acid
(Kurosaki et al., 1989). The synthase was purified
according to methods described previously (Kurosaki et
al., 1993). Protein concentrations were determined by
the method of Bradford (1976). The purity of the

F. Kurosaki et al. / Phytochemistry 61 (2002) 597–604
603
synthase was assessed by a densitometric scan after
separation by sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS–PAGE) according to the
method of Laemmli (1970), and the results were repor-
ted previously (Kurosaki et al., 1993). The standard
assay mixture for the determination of 6HM-producing
activity of the enzyme consisted of 10 mM K-Pi (pH
7.5), acetyl-CoA (100 mM), [2-14C]malonyl-CoA (50
mM) 3.7 kBq), enzyme preparation (2–5 pkat), DTT (2
Mm) and NADPH (1 mM) in a total volume of 100 ml
unless otherwise noted. Formation of TAL (2) by 6HM
synthase was achieved by employing either the dis-
sociated monomer subunits of the synthase or the
homodimer without NADPH. For dissociation of the
homodimeric to the monomer subunits, the enzyme was
incubated with 2 M NaCl at 37 ꢀC for 5 min prior to the
assay, and the TAL (2)-forming reaction was carried
out in the presence of the same NaCl concentration
(Kurosaki, 1995). The assay mixtures were incubated at
reaction was run for 2 min at 37 ꢀC, and was terminated
by the addition of TCA (500 ml, 2 M). Bovine serum
albumin (100 mg) was then added to the reaction mix-
ture as a carrier, and the precipitated proteins were
recovered by centrifugation (700 X g, 5 min). The sam-
ples were denatured and subjected to SDS–PAGE, and
the position of 6HM synthase subunits was determined
with the molecular weight markers (Bio-Rad) after
staining with Coomassie Brilliant Blue (Kurosaki et al.,
1991). The gels containing the enzyme were excised with
a blade, and the radioactivities were determined as
described (Kurosaki, 1996a).
3.4. Determination of kinetic parameters of 6HM
synthase
In order to determine the kinetic parameters of 6HM
synthase under various reaction conditions, the assay
was carried out with a series of concentrations of each
of the substrates. Km values for acetyl-CoA were esti-
mated by a set of enzyme reactions with a fixed con-
centration of malonyl-CoA (50 mM) and 20–100 mM of
acetyl-CoA, while, for estimation of malonyl-CoA, the
reactions were run with 100 mM of acetyl-CoA and 3–10
mM of malonyl-CoA (Kurosaki, 1996b), respectively.
The results were analyzed by the double reciprocal plots
with the method of the least squares.
ꢀ
37 C for 30 min, and the reactions were terminated by
the addition of 50% (v/v) acetic acid (50 ml). The pro-
ducts were extracted with EtoAc (200 ml), and 50 ml-ali-
quots were applied onto a silica gel TLC plate. After
development, the radioactivities co-migrating with
authentic 6HM (1) or TAL (2) were determined as repor-
ted previously in detail (Kurosaki et al., 1989, 1993).
3.3. Chemical modification and substrate-binding assay
of 6HM synthase
Acknowledgements
Two SH groups at the reaction center of 6HM syn-
thase, Cys-SH and ACP-SH, were irreversibly blocked
by alkylation with IoAA and ClAc–CoA according to
the method described previously (Kurosaki, 1996a). In
brief, DTT was removed from the synthase preparation
by dialysis, and the enzyme was ꢀincubated with IoAA (5
Mm) ClAc–CoA (1mM) at 37 C for 15 min to block
Cys- and ACP-SHs, respectively (McCarthy and
Hardie, 1982; Anderson and Kumar, 1987). After
alkylation, DTT (7mM) was added to the mixture to
quench excess SH inhibitors, and the sample was dia-
lyzed against 20 mM K-Pi buffer containing 5 mM DTT
(pH 7.5) to remove these reagents. When necessary, the
enzyme preparation was dialyzed against the buffer
containing 2 M NaCl for the dissociation of the homo-
dimers to the monomer subunits. The partially masked
6HM synthase was then incubated with [14C]-labeled
acyl-CoAs to estimate the binding properties toward the
latter substrates according to the method described
previously (Kurosaki, 1996a). The assay mixture con-
sisted of 10 mM K- Pi (pH 7.5), [14C]acetyl-CoA or
[14C]malonyl-CoA (10 mM, 7.4 kBq), NADPH (1 mM),
5 mg proteins of the enzyme preparation (approximately
50 pkat/assay) and DTT (5mM) in a total volume of 100
ml. In some experiments, non-radiolabeled substrates
were appropriately added to the assay mixture. The
This work was supported in part by a Grant-in-Aid
for Scientific Research from the Ministry of Education,
Science and Culture, Japan.
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