4-Coumarate:Coenzyme A Ligase Has the Catalytic

Capacity to Synthesize and Reuse Various

(Di)Adenosine Polyphosphates1

Małgorzata Pietrowska-Borek, Hans-Peter Stuible, Erich Kombrink, and Andrzej Guranowski*

Katedra Biochemii i Biotechnologii, Akademia Rolnicza, ul. Wołyńska 35, 60–637 Poznań, Poland (M.P.-B.,
A.G.); and Max-Planck-Institut für Züchtungsforschung, Abteilung Biochemie, Carl-von-Linné-Weg 10,
50829 Köln, Germany (H.-P.S., E.K.)

4-Coumarate:coenzyme A ligase (4CL) is known to activate cinnamic acid derivatives to their corresponding coenzyme A
esters. As a new type of 4CL-catalyzed reaction, we observed the synthesis of various mono- and diadenosine polyphos-
phates. Both the native 4CL2 isoform from Arabidopsis (At4CL2 wild type) and the At4CL2 gain of function mutant
M293P/K320L, which exhibits the capacity to use a broader range of phenolic substrates, catalyzed the synthesis of
adenosine 5⬘-tetraphosphate (p4A) and adenosine 5⬘-pentaphosphate when incubated with MgATP⫺2 and tripolyphosphate
or tetrapolyphosphate (P4), respectively. Diadenosine 5⬘,5⵮,-P1,P4-tetraphosphate represented the main product when the
enzymes were supplied with only MgATP2⫺. The At4CL2 mutant M293P/K320L was studied in more detail and was also
found to catalyze the synthesis of additional dinucleoside polyphosphates such as diadenosine 5⬘,5⵮-P1,P5-pentaphosphate
and dAp4dA from the appropriate substrates, p4A and dATP, respectively. Formation of Ap3A from ATP and ADP was not
observed with either At4CL2 variant. In all cases analyzed, (di)adenosine polyphosphate synthesis was either strictly
dependent on or strongly stimulated by the presence of a cognate cinnamic acid derivative. The At4CL2 mutant enzyme
K540L carrying a point mutation in the catalytic center that is critical for adenylate intermediate formation was inactive in
both p4A and diadenosine 5⬘,5⵮,-P1,P4-tetraphosphate synthesis. These results indicate that the cinnamoyl-adenylate
intermediate synthesized by At4CL2 not only functions as an intermediate in coenzyme A ester formation but can also act
as a cocatalytic AMP-donor in (di)adenosine polyphosphate synthesis.

4-Coumarate:CoA ligase (4CL; EC 6.2.1.12) repre-
sents the branch point enzyme of plant phenylpro-
panoid metabolism, catalyzing the activation of
4-coumaric acid and various other hydroxylated and
methoxylated cinnamic acid derivatives to the corre-
sponding CoA esters in a two-step reaction. During
the first step (Eq. 1), coumarate and ATP form a
coumaroyl-adenylate intermediate with the simulta-
neous release of pyrophosphate (PPi), a reaction
which is reversible (Eq. 2).
E⫹coumarate ⫹ ATP 3 E:coumaroyl⬃pA ⫹ PPi
(1)
E:coumaroyl⬃pA ⫹ PPi 3 ATP ⫹ coumarate ⫹ E
(2)
In the second step (Eq. 3), the coumaroyl group is
transferred to the sulfhydryl group of CoA, and AMP
1
This work was supported by the Polish State Committee for
Scientific Research (project no. PBZ–KBN– 059/T09/2001 to
M.P.-B. and A.G.) and by the Max-Planck-Society (to E.K. and
H.-P.S.).
* Corresponding author; e-mail guranow@au.poznan.pl; fax 48 –
61– 8487146.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.011684.
Plant Physiology, March 2003, Vol. 131, pp. 1401–1410, www.plantphysiol.org © 2003 American Society of Plant Biologists
is released (Knobloch and Hahlbrock, 1975; Becker-
André et al., 1991).
E:coumaroyl⬃pA ⫹ CoA 3 coumaroyl-CoA ⫹
AMP ⫹ E (3)
On the basis of the presence of a highly conserved
peptide motive, the putative nucleotide-binding do-
main, 4CLs, luciferases, fatty acyl-CoA synthetases,
acetyl-CoA synthetases, and non-ribosomal peptide
synthetases have been grouped in one superfamily
of adenylate-forming enzymes (Fulda et al., 1994). A
second, independent group of adenylate-forming
proteins make up the aminoacyl-tRNA synthetases
(Pavela-Vrancic et al., 1994). In the presence of ATP
and Mg2⫹, all enzymes listed above form an enzyme-
bound acyl-adenylate intermediate, which is esterified
with either CoA (4CL, acetyl-CoA synthetases,
and fatty acyl-CoA synthetases), or the enzyme-bound
cofactor 4⬘-phosphopantetheine (non-ribosomal pep-
tide synthetases), or tRNAs (aminoacyl-tRNA
synthetases).
Several of the enzymes forming an acyl-adenylate
intermediate with concomitant release of PPi can in
a second type of reaction also catalyze the formation
of uncommon mononucleoside and dinucleoside
polyphosphates such as adenosine 5⬘-tetraphosphate
(p4A; Eq. 4), adenosine 5⬘-pentaphosphate (p5A;
1401
Pietrowska-Borek et al.
Eq. 5), diadenosine 5⬘,5⬙-P1,P4-tetraphosphate (Ap4A;
Eq. 6), and diadenosine 5⬘,5⬙-P1,P5-pentaphosphate
(Ap5A; Eq. 7; for reviews, see McLennan, 2000; Sillero
and Günther Sillero, 2000).
E:acyl⬃pA ⫹ P3 3 p4A ⫹ acid ⫹ E (4)
where P3 is tripolyphosphate.
E:acyl⬃pA ⫹ P4 3 p5A ⫹ acid ⫹ E (5)
where P4 is tetrapolyphosphate.
E:acyl⬃pA ⫹ ATP 3 Ap4A ⫹ acid ⫹ E (6)
E:acyl⬃pA ⫹ p4A 3 Ap5A ⫹ acid ⫹ E (7)
Such capacity was demonstrated for certain
aminoacyl-tRNA synthetases (Plateau et al., 1981;
Goerlich et al., 1982; Plateau and Blanquet, 1982;
Jakubowski, 1983), some acetyl-CoA and fatty acyl-
CoA synthetases (Guranowski et al., 1994; Fontes et
al., 1998), firefly luciferase (Guranowski et al., 1990),
and more recently also for a non-ribosomal peptide
synthetase from Bacillus brevis (Dieckmann et al.,
2001). Corresponding to this broad range of enzymes
catalyzing reactions that in vitro lead to the synthesis
of (di)nucleoside polyphosphates, appreciable
amounts of these compounds, pnN and NpnN⬘, have
been found to also naturally occur in a variety of
tissues and organisms, including bacteria, fungi, and
animal cells (Garrison and Barnes, 1992; Kisselev et
al., 1998; McLennan, 2000). They presumably occur
ubiquitously in all organisms, however, their pres-
ence in plants has not yet been demonstrated.
Although (di)nucleoside polyphosphate synthesis
is typically promoted by the absence or an insufficient
concentration of the respective end acceptor (e.g. CoA,
4⬘-phosphopantetheine, and tRNAs) the (di)adenosine
polyphosphate product spectra observed are specific
for each enzyme. For example, most aminoacyl-tRNA
synthetases can catalyze the formation of Ap4A,
whereas mammalian aminoacyl-tRNA synthetases
with specificity for Trp only produce Ap3A (Kisselev
et al., 1998). Adenosine 5⬘-polyphosphates (e.g. p4A)
and diadenosine 5⬘-polyphosphates (e.g. Ap3A and
Ap4A) have been considered to play important roles
in regulating cellular processes such as sporulation,
stress responses, cell proliferation, and apoptosis in
various biological systems (Kisselev et al., 1998;
Nishimura, 1998). In Brewer’s yeast (Saccharomyces
cerevisiae), p4A and p5A accumulate during sporula-
tion, whereas these compounds were not detectable
during its vegetative growth (Jakubowski, 1986). Fur-
ther studies demonstrated that yeast acetyl-CoA syn-
thetase can synthesize p4A and p5A and therefore
might be the enzyme responsible for the accumula-
tion of these adenosine 5⬘-polyphosphates (Gu-
ranowski et al., 1994). In bacteria (Escherichia coli,
Synechococcus sp.), yeast (S. cerevisiae), and animal
cells (fruitfly [Drosophila melanogaster] and Artemia
franciscana) the concentrations of diadenosine poly-
1402
phosphates (ApnA) rapidly increased in response to
heat shock, oxidative, or heavy-metal ion stress, sug-
gesting that these compounds could act as alarmones
(Brevet et al., 1985; Baltzinger et al., 1986; Miller and
McLennan, 1986; Coste et al., 1987; Johnstone and
Farr, 1991; Pàlfi et al., 1991). Several proteins were
identified to directly bind diadenosine polyphos-
phates, including the stress-related proteins DnaK
and GroEL from E. coli (Johnstone and Farr, 1991). In
higher eukaryotes, especially mammalian cells, it has
been observed that appreciable physiological and
pathological effects are associated with changes of
diadenosine polyphosphate levels, in particular with
alterations of the Ap3A to Ap4A ratio (Kisselev et al.,
1998). In agreement with the suggested importance
of the Ap3A to Ap4A ratio, the human Ap3A hydro-
lase, Fhit, appeared to be involved in the protection
of cells against tumorigenesis (Siprashivili et al.,
1997).
Phenylalanyl- and seryl-tRNA synthetases from
yellow lupin seeds are the only plant enzymes known
to synthesize Ap3A and Ap4A (Jakubowski, 1983).
However, the existence of highly specific enzymes
that catalyze the hydrolysis of (di)nucleoside
polyphosphates, such as (asymmetrical) dinucleo-
side tetraphosphatase (EC 3.6.1.17), dinucleoside
triphosphatase (EC 3.6.1.29), and nucleoside 5⬘-
tetraphosphatase (EC 3.6.1.14), indicates that these
nucleotide derivatives may exist and have a func-
tion in planta (Jakubowski and Guranowski, 1983;
Guranowski et al., 1996, 1997). In this report, we
demonstrate that Arabidopsis 4CL2, a key enzyme
of plant secondary metabolism, can catalyze the
synthesis of various mono- and dinucleoside
polyphosphates. The basic requirements and kinetic
parameters of these reactions are presented, and
their possible biological roles are discussed.
RESULTS
Synthesis of Adenosine 5ⴕ-Polyphosphates
Having identified At4CL2 as a member of the large
family of adenylate-forming enzymes (Stuible et al.,
2000), we wanted to test whether At4CL2 and two
mutant variants derived from this enzyme are also
capable to catalyze the synthesis of p4A or p5A. The
respective enzymes were incubated with either P3 or
P4 as potential adenylate acceptors in the presence of
their cognate phenolic acid substrate (a cinnamic acid
derivative), ATP and Mg2⫹, whereas CoA was omit-
ted from the incubation mixture. For product analy-
sis, aliquots were sampled at different time points,
subjected to thin layer chromatography and subse-
quently visualized under short wavelength UV. Var-
ious At4CL2 variants (the wild-type enzyme and mu-
tants derived thereof) support the synthesis of p4A
and p5A in the presence of P3 or P4, respectively. In
Figure 1, this is demonstrated for the mutant M293P/
K320L. The identity of the reaction products, p4A and
Plant Physiol. Vol. 131, 2003
 (Di)Adenosine Polyphosphate Synthesis by 4-Coumarate:Coenzyme A Ligase

Figure 1. Synthesis of p4A and p5A by the

At4CL2 mutant M293P/K320L from Arabidop-
sis. Product synthesis from ATP and P3 or P4 in
the absence (no FA) and presence of 62 ␮M
ferulic acid (⫹FA) was monitored by withdraw-
ing 3-␮L aliquots of the reaction mixtures at the
times indicated. After 120 min, the reactions
were stopped by heating (3 min at 100°C) and
further incubated at 30°C in the presence of 1 ng
of scPPX1 (exopolyphosphatase from Brewer’s
yeast), and two additional aliquots were with-
drawn at the times indicated. Samples were sub-
jected to thin-layer chromatography in solvent
system I.

p5A, was verified by their comigration with authentic
standards and their susceptibility to yeast exopoly-
phosphatase, scPPX1, which degrades nucleoside
polyphosphates to ATP and orthophosphate (Gu-
ranowski et al., 1998). Addition of inorganic pyro-
phosphatase to the reaction mixture had no signifi-
cant effect on the rates of p4A and p5A production
and was therefore not included in the standard assay.
For the At4CL2 wild-type enzyme, nucleoside
polyphosphate synthesis was strictly dependent on
the presence of a cinnamic acid derivative, such as
coumarate or caffeate, which are efficiently con-
verted to their corresponding thiol-esters by the en-
zyme in the standard 4CL reaction (Ehlting et al.,
1999). In the presence of 100 ␮m coumarate, a specific
activity of 6.2 nkat mg⫺1 protein for p4A synthesis
was determined, whereas in its absence p4A was
undetectable. Ferulate and cinnamate, in contrast to
coumarate and caffeate, are known to represent only
poor substrates for the At4CL2 wild-type enzyme
and correspondingly were considerably less efficient
in stimulating p4A synthesis (Table I). Sinapate,
which is not converted to the CoA ester by At4CL2,
does also not support p4A synthesis. In Table I, the
kinetic properties of the At4CL2 wild-type catalyzed
reactions leading to the synthesis of p4A or the CoA
esters of various cinnamic acid derivatives are com-
pared. Interestingly, apparent Km values for couma-
rate and caffeate are about 10-fold lower in the reac-
tion leading to p4A in comparison with the
corresponding values for thiol-ester formation.
The At4CL2 mutant enzyme carrying two amino
acid substitutions, M293P and K320L, was analyzed
in detail for its capacity to catalyze adenosine 5⬘-
polyphosphate formation. The At4CL2 mutant
M293P/K320L is a gain of function variant of
At4CL2, which in contrast to the wild-type enzyme is
capable to efficiently activate ferulate and cinnamate
due to its enlarged and more hydrophobic substrate
binding pocket (Stuible and Kombrink, 2001; K.
Schneider, K. Witzel, E. Kombrink, and H.-P. Stuible,
unpublished data). In addition to its broader sub-
strate utilization spectrum, the double mutant exhib-
its higher conversion rates than the wild-type en-

Table I. Kinetic properties of recombinant Arabidopsis At4CL2 wild-type enzyme

Enzyme activities, i.e. synthesis of p4A or CoA esters of cinnamic acid derivatives, were determined as described in “Materials and Methods”
with the indicated phenolic substrate. n.d., Not determined, activity too low. n.c., No conversion detectable.
Synthesis of p4A Synthesis of CoA Ester
Phenolic Substrate
a b b a
Specific Activity Vmax Km Specific Activity Vmaxb K mb
⫺1 ⫺1
% of maximum nkat mg ␮M % of maximum nkat mg ␮M
Cinnamate 2 n.d. n.d. 2.9 190 ⫾ 27 6,350 ⫾ 700
Coumarate 100 6.3 ⫾ 1.0 22 ⫾ 3.5 50 310 ⫾ 33 233 ⫾ 20
Caffeate 12 0.8 ⫾ 0.2 1.4 ⫾ 0.3 100 158 ⫾ 9 24 ⫾ 1.5
Ferulate 1.2 n.d. n.d. 1.1 n.d. n.d.
Sinapate n.c. n.d. n.d. n.c. n.d. n.d.
None n.c. n.d. n.d. n.c. n.d. n.d.
a
Specific activities (based on protein) were determined under standard conditions with 100 ␮M phenolic substrate. The highest activity for
each reaction was set to 100%, corresponding to 6.2 and 137 nkat mg⫺1 for p4A and caffeoyl-CoA synthesis, respectively. b
Vmax and Km
values were determined by linear regression of v against v/s (Eadie-Hofstee plot) with at least six data points and at least three repetitions. In the
assays concerning p4A synthesis, the concentrations of the phenolic substrates varied between 0 and 250 ␮M.

Plant Physiol. Vol. 131, 2003 1403

Pietrowska-Borek et al.

zyme, with several cinnamic acid derivatives as

substrates during CoA ester formation. In accordance
with these properties, the double mutant also dis-
played high in vitro activity of (di)adenosine
polyphosphate synthesis. Under optimal conditions
and in the presence of 62 ␮m ferulate the specific
activities for p4A and p5A synthesis were 7.9 and 0.8
nkat mg⫺1, respectively. Moreover, without added
ferulate, a very low but detectable amount of p4A
was synthesized, accumulating at a 25-fold lower rate
than in the presence of ferulate (Fig. 1; Table II). The
rates of p4A and p5A synthesis were constant over a
time period of at least 40 min when accumulation of
radiolabeled products originating from [3H]ATP was
quantified by scintillation counting (Fig. 2). When
different cinnamic acid derivatives were compared,
ferulate and coumarate were found to be the best
inducers of p4A synthesis, whereas caffeate and cin-
namate were less efficient (Table II). In contrast, si-
napate, Phe, and Tyr, which are no substrates for
thiol-ester formation by the At4CL2 mutant M293P/
K320L, also did not support p4A synthesis. Table II
summarizes the kinetic properties of p4A synthesis
by the At4CL2 mutant M293P/K320L and relates
these values to the kinetic properties of thiol-ester
formation with CoA. As observed for the wild-type
enzyme, the apparent Km values for the different
cinnamic acid derivatives were at least 10-fold lower
for p4A synthesis in comparison with CoA ester for-
mation. However, the relative reaction rates with
each of the phenolic substrates were comparable for
both reactions.
The Km values of all other components participat-
ing in p4A formation by the At4CL2 mutant M293P/
K320L were determined under standard conditions,
with fixed concentrations of ferulate (62 ␮m), P3 (10
mm), and ATP (5 mm), respectively. The Km for the
adenylate donor ATP was 150 ␮m when determined
with [3H]ATP concentrations ranging from 37 to 310
␮m. This value is of the same order of magnitude as
the Km observed for the 4CL-catalyzed standard re-
Figure 2. Time course of p4A and p5A synthesis catalyzed by the
At4CL2 mutant M293P/K320L from Arabidopsis. Product accumula-
tion was monitored by thin-layer chromatography as described in
“Materials and Methods.” Synthesis of p4A was monitored in the
presence of 5 mM [3H]ATP and 10 mM P3 and either in the presence
of 62 ␮M ferulic acid (Œ) or in its absence (F). Synthesis of p5A
proceeded in the presence of 5 mM [3H]ATP, 10 mM P4, and 62 ␮M
ferulate (f). The chromatogram was developed in solvent system I
and radioactivity in p4A or p5A spots determined by scintillation
counting.
action (CoA ester formation), typically ranging from
50 to 250 ␮m for enzymes from different plants (Kno-
bloch and Hahlbrock, 1975; Voo et al., 1995; Stuible et
al., 2000). The Km for the adenylate acceptor P3 was
4.9 mm, as estimated from measurements with P3
concentrations varying from 0.62 to 10 mm.
To get some insight into the reaction mechanism
leading to (di)adenosine polyphosphate synthesis by
At4CL2, we extended our analysis to the mutant
enzyme K540N. The Lys residue 540 has been sug-
gested to function as the active site of adenylate
formation by stabilizing the negatively charged pen-
tavalent transition state (Conti et al., 1997). In agree-
ment with this assumption, the At4CL2 variant car-
rying the K540N substitution was incapable of
activating cinnamic acid derivatives to the corre-
sponding CoA esters (Stuible et al., 2000). Likewise,

Table II. Kinetic properties of recombinant Arabidopsis At4CL2 mutant M293P/K320L

Enzyme activities, i.e. synthesis of p4A or CoA esters of cinnamic acid derivatives, were determined as described in “Materials and Methods”
with the indicated phenolic substrate. n.d., Not determined, activity too low. n.c., No conversion detectable.
Synthesis of p4A Synthesis of CoA Ester
Phenolic Substrate
a b b
Specific Activity Vmax Km Specific Activity Vmaxb K mb
⫺1 ⫺1
% of maximum nkat mg ␮M % of maximum nkat mg ␮M
Cinnamate 45 3.8 ⫾ 0.8 2.7 ⫾ 0.8 27 285 ⫾ 10 292 ⫾ 50
Coumarate 92 7.4 ⫾ 2.5 2.0 ⫾ 0.5 81 226 ⫾ 30 27 ⫾ 9
Caffeate 69 5.7 ⫾ 1.0 6.5 ⫾ 1.3 56 119 ⫾ 33 48 ⫾ 6
Ferulate 100 8.0 ⫾ 1.2 2.7 ⫾ 0.5 100 270 ⫾ 50 46 ⫾ 7
Sinapate 4 n.d. n.d. n.c. n.d. n.d.
None 4 n.d. n.d. n.c. n.d. n.d.
a
Specific activities (based on protein) were determined under standard conditions with 100 ␮M phenolic substrate. The highest activity for
each reaction was set to 100%, corresponding to 7.9 and 147 nkat mg⫺1 for p4A and feruloyl-CoA synthesis, respectively. b
Vmax and Km
values were determined by linear regression of v against v/s (Eadie-Hofstee plot) with at least six data points and at least three repetitions. In the
assays concerning p4A synthesis the concentrations of the phenolic substrates varied between 0 and 250 ␮M.

1404 Plant Physiol. Vol. 131, 2003

 (Di)Adenosine Polyphosphate Synthesis by 4-Coumarate:Coenzyme A Ligase

this mutant is also incapable of catalyzing the syn-
thesis of (di)adenosine polyphosphates (not shown),
indicating that adenylate formation is an essential
step during both CoA ester formation and (di)ade-
nosine polyphosphate synthesis.
Special Requirements for Adenosine
5ⴕ-Polyphosphate Synthesis
The pH dependence of p4A synthesis catalyzed by
At4CL2 was monitored in various buffers, including
acetate, MES, HEPES, CHES, covering a pH range
from 3.6 to 9.5. Maximum activity was observed be-
tween pH 6 and 8, with half-maximal activity at pH
4.5 and 9 (results not shown). 4CL-catalyzed reac-
tions converting cinnamic acid derivatives to CoA
esters typically have a pH optimum ranging from pH
7.5 to 8.5 (Knobloch and Hahlbrock, 1975; Knobloch
and Hahlbrock, 1977; Voo et al., 1995). Various diva-
lent cations were tested for their capacity to support
p4A synthesis, and maximum activity was obtained
with 5 mm Mg2⫹ (100%), whereas lower rates were
observed with Mn2⫹ (54%), Co2⫹ (52%), Ni2⫹ (47%),
and Zn2⫹ (15%), all tested at 5 mm. Ca2⫹ had no
stimulating effect.
Synthesis of Dinucleoside Polyphosphates
In additional experiments, we analyzed the capac-
ity of the At4CL2 mutant M293P/K320L to support
the synthesis of dinucleoside polyphosphates, such
as Ap3A, Ap4A, dAp4dA, and Ap5A. When the en-
zyme was supplied with 5 mm ATP, which served as
both adenylate donor and acceptor, 5 mm MgCl2 and
62 ␮m ferulate, accumulation of Ap4A could be ob-
served by thin-layer chromatography (Fig. 3A). The
identity of the product was confirmed by comigra-
tion with the authentic standard, its resistance to
alkaline phosphatase, and its susceptibility to Ap4A
hydrolase from L. angustifolius, which converts
Ap4A to ATP plus AMP (Fig. 3). Maximum rates of
Ap4A synthesis were observed between pH 6 and 7
(not shown). The kinetic constants of Ap4A synthesis
were estimated with [3H]ATP concentrations ranging
from 0.3 to 5 mm and yielded an apparent Km for ATP
of 4.1 mm and Vmax of 1.2 nkat mg⫺1. When ATP was
replaced by dATP, the At4CL2 mutant M293P/
K320L also catalyzed the synthesis of dAp4dA at a
comparable rate (Fig. 3B). With p4A as the only nu-
cleotide present in the reaction mixture, the enzyme
additionally supported the synthesis of Ap5A (not
shown). However, no Ap3A synthesis could be de-
tected in a reaction mixture containing 5 mm ADP as

Figure 3. Synthesis of diadenosine tetraphosphate (Ap4A) and di(deoxyadenosine) tetraphosphate (dAp4dA) by the At4CL2
mutant M293P/K320L from Arabidopsis. Product accumulation was monitored by withdrawing 3-␮L aliquots of the reaction
mixtures at the times indicated. After 16 h, the reactions were stopped by heating (3 min, 100°C) and further incubated at
30°C in the presence of 2 units of alkaline phosphatase (AP) from calf intestine and after its inactivation, with 50 ng of
(asymmetrical) Ap4A hydrolase (Ap4A-ase) from Lupinus angustifolius, and additional aliquots were withdrawn at the times
indicated. Samples were subjected to thin-layer chromatography in solvent system II as described in “Materials and
Methods.” A and dA at the arrows represent adenosine and deoxyadenosine, respectively.

Plant Physiol. Vol. 131, 2003 1405

Pietrowska-Borek et al.
potential adenylate acceptor, in addition to the stan-
dard ingredients (5 mm ATP, 5 mm MgCl2, and 62 ␮m
ferulate).
The At4CL2 wild-type enzyme also catalyzed the
synthesis of Ap4A, albeit at a 10-fold lower rate than
the At4CL2 double mutant. The specific activity de-
termined in the presence of 100 ␮m coumarate as
activator was 0.12 nkat mg⫺1.
Mono- and Dinucleoside Polyphosphates as
AMP-Moiety Donors
In the following experiments, we analyzed whether
(di)nucleoside polyphosphates can also serve as ad-
enylate donors in At4CL2-catalyzed reactions,
thereby leading to regeneration of ATP at the ex-
pense of the unusual nucleotide derivatives. In fact,
these reactions are the reverse of the reactions char-
acterized above (compare Eq. 2 with Eqs. 4–7). As
shown in Figure 4, the At4CL2 mutant M293P/K320L
efficiently catalyzed the transfer of the AMP moiety
from p4A onto PPi resulting in the accumulation of
ATP. Ap4A and Ap5A could also serve as adenylate
donors, when polyphosphates such as PPi, P3, and P4
were supplied as acceptors. Incubation of the At4CL2
mutant M293P/K320L with [3H]Ap4A and P3 led to
the accumulation of both p4A and ATP (Fig. 5). The
Km for Ap4A in this reaction was 64 ␮m and the Vmax
0.53 nkat mg⫺1; the pH optimum was between pH 5.5
and 6.5, and the optimum MgCl2 concentration, 7.5
mm (not shown). The transfer of the AMP moiety
from [3H]Ap4A to the acceptors PPi or P4, yielding
two molecules of either ATP or ATP plus p4A, oc-
curred with lower efficiency (not shown). The rela-
tive reaction rates determined in the presence of 10
Figure 4. Synthesis of ATP from p4A and PPi catalyzed by the At4CL2
mutant M293P/K320L from Arabidopsis. Time course of the reaction
was monitored as described in “Materials and Methods.” The chro-
matogram was developed in solvent system I.
1406
Figure 5. Synthesis of p4A and ATP from Ap4A and P3 by the At4CL2
mutant M293P/K320L from Arabidopsis. Time course of the reaction
was monitored as described in “Materials and Methods.” The chro-
matogram was developed in solvent system II.
mm each of PPi, P3, or P4 were 100:5.3:1. All of the
above reactions were strictly dependent on the pres-
ence of a suitable phenolic compound as cocatalyst
and hence were tested in the presence of 62 ␮m
ferulate.
Finally, the AMP moiety originating from Ap4A
could also be transferred to orthophosphate (Pi). Us-
ing [3H]Ap4A as adenylate donor and 5 mm potas-
sium phosphate as acceptor, equal amounts of radio-
activity appeared in the products ADP and ATP that
were generated by At4CL2 mutant M293P/K320L in
the presence of 62 ␮m ferulate. This result clearly
shows that the reaction proceeds via a feruloyl-
adenylate intermediate from which the AMP moiety
is transferred onto Pi. A symmetric, Pi-independent
cleavage of Ap4A resulting in two ADP molecules
can therefore be excluded.
When incubated with Ap5A, instead of Ap4A, and
the same oligophosphate acceptors as above (e.g. PPi,
P3, and P4), the At4CL2 mutant M293P/K320L cata-
lyzed the synthesis of the corresponding products:
Two molecules of p4A were generated from Ap5A
and P3 (Fig. 6), whereas p4A and p5A originated from
Ap5A and P4 (not shown).
DISCUSSION
Here, we demonstrate that 4CL2 from Arabidopsis,
in addition to its bona fide function of generating
CoA esters of cinnamic acid derivatives, is also capa-
ble to catalyze the in vitro synthesis of (di)adenosine
polyphosphates from ATP and (nucleoside) polypho-
sphates. This novel reaction strictly depends on the
presence of at least traces of an authentic phenolic
substrate. The assumption that a cinnamoyl-
adenylate intermediate acts as AMP donor during
catalysis is strengthened by the observation that the
Plant Physiol. Vol. 131, 2003
 (Di)Adenosine Polyphosphate Synthesis by 4-Coumarate:Coenzyme A Ligase
Figure 6. Synthesis of p4A from Ap5A and P3 by the At4CL2 mutant
M293P/K320L from Arabidopsis. Time course of the reaction was
monitored as described in “Materials and Methods.” The chromato-
gram was developed in solvent system I.
At4CL2 mutant enzyme K540N, which carries a point
mutation in the catalytic center responsible for ade-
nylate formation (Stuible et al., 2000), is incapable to
synthesize (di)adenosine polyphosphates. Similar re-
sults were recently obtained by studying the enzy-
matic properties of the heterologously expressed ad-
enylation domain of tyrocidine synthetase A, which
also efficiently catalyzes the formation of various
mono- and dinucleoside polyphosphates (Dieck-
mann et al., 2001). The adenylation domain of this
non-ribosomal polypeptide synthetase shares signif-
icant structural and sequence similarities with
At4CL2 (Stuible et al., 2000).
Formation of (di)adenosine polyphosphates has
been reported for several, but not all, enzymes that
perform catalysis via an adenylate intermediate. For
example, highly purified acetyl-CoA synthetases
from E. coli (kindly donated by Dr. Alan J. Wolfe),
bovine heart, and lupin seeds (A. Guranowski and A.
Biryukov, unpublished data) did not catalyze the
synthesis of p4A, Ap4A, or Ap3A, although all prep-
arations were fully active to carry out the acetylation
of CoA. On the other hand, acetyl-CoA synthetases
purified from yeast (A. Guranowski, unpublished
data) and some, but not all, commercially available
preparations of yeast acetyl-CoA synthetase (A. Gu-
ranowski, M.A. Günther Sillero, and A. Sillero, un-
published data) were able to synthesize p4A, Ap3A,
and Ap4A. These examples indicate that the capacity
of an enzyme to synthesize (di)nucleoside polypho-
sphates is difficult to predict and therefore has to be
verified experimentally in each single case.
On the basis of their different requirements for low
Mr AMP donors functioning as cocatalytic factor,
such as fatty acids, luciferin, amino acids or cinnamic
acid derivatives, adenylate-forming enzymes exhib-
Plant Physiol. Vol. 131, 2003
iting the capacity to synthesize (di)adenosine
polyphosphates synthesis can be further subclassi-
fied. One group of enzymes, including most of
aminoacyl-tRNA synthetases (Plateau et al., 1981;
Goerlich et al., 1982) and firefly luciferase (Gu-
ranowski et al., 1990) strictly require their cognate
acid substrate for diadenosine polyphosphate syn-
thesis. In contrast, members of the second group,
composed of lysyl-tRNA synthetase from E. coli
(Zamecnik et al., 1966; Goerlich et al., 1982), seryl-
tRNA and phenylalanyl-tRNA synthetases from yel-
low lupin (Jakubowski, 1983), and fatty acyl-CoA
synthetase from Pseudomonas fragi (Fontes et al.,
1998), can synthesize measurable amounts of (di)a-
denosine polyphosphates in the absence of their pri-
mary AMP acceptor, such as fatty acids or amino
acids. However, even with the latter group of en-
zymes, a stimulation of p4A synthesis by cognate
acid substrates seems to be possible, as previously
demonstrated for acetyl-CoA ligase from Brewer’s
yeast (Guranowski et al., 1994).
From the above observations, the existence of two
alternative molecular mechanisms for (di)adenosine
polyphosphate synthesis can be proposed, one oper-
ating via an enzyme-bound acyl⬃adenylate (afore-
mentioned Eqs. 1 and 4–7), the other operating via an
enzyme⬃adenylate (Eqs. 8 and 9)
E ⫹ ATP 3 E⬃pA ⫹ PPi (8)
E⬃pA ⫹ P3 3 p4A ⫹ E (9)
Adenosine 5⬘-polyphosphate synthesis catalyzed
by the At4CL2 wild-type enzyme obviously func-
tions according to the mechanism described for ex-
ample by Equations 1 and 4, because its activity was
absolutely dependent on the presence of a cinnamic
acid derivative.
Although p4A is the most prominent nucleotide
derivative synthesized by At4CL2 wild-type and mu-
tant M293P/K320L, a significant production of dif-
ferent dinucleoside polyphosphates has also been ob-
served. The At4CL2 variant M293P/K320L catalyzes
the synthesis of Ap4A and dAp4dA at comparable
rates (Fig. 3). The capacity to synthesize these nucle-
otide derivatives at comparable rates has previously
been observed for the lysyl-tRNA synthetase from E.
coli (Plateau and Blanquet, 1982), firefly luciferase
(Günther Sillero et al., 1991), and acyl-CoA syn-
thetase from P. fragi (Fontes et al., 1998). At4CL2
wild-type and the mutant M293P/K320L were also
able to catalyze the synthesis of Ap5A. In this reac-
tion, p4A either acts as an acceptor of adenylate do-
nated by ATP, or alternatively, functions both as an
AMP-donor and acceptor when representing the only
available nucleotide. ADP is obviously not recog-
nized as an AMP acceptor because neither enzyme
variant produced Ap3A, thereby resembling proper-
ties of firefly luciferase, which synthesized Ap4A but
not Ap3A (Günther Sillero et al., 1991). In contrast,
1407
Pietrowska-Borek et al.
several aminoacyl-tRNA synthetases produced Ap3A
(Ap3N) and Ap4A (Ap4Ns) at comparable rates
(Zamecnik et al., 1966; Plateau and Blanquet, 1982;
Jakubowski, 1983; Kitabatake et al., 1987).
Finally, we observed that At4CL2 was also capable
to catalyze reverse reaction, i.e. the (re)use of the
adenylate moieties originating from adenosine 5⬘-
polyphosphates and diadenosine polyphosphates for
the generation of ATP. This activity may connect the
degradation of unusual nucleotide derivatives with
the regeneration of ATP. Comparable enzymatic ac-
tivities have previously been reported for some
aminoacyl-tRNA synthetases (Zamecnik et al., 1966;
Plateau et al., 1981; Goerlich et al., 1982; Led et al.,
1983; Guédon et al., 1987) as well as for firefly lucif-
erase (Momsen, 1978; Günther Sillero et al., 1991;
Ortiz et al., 1993). The functional significance of these
reactions remains to be demonstrated but it could be
related to (a) recycling the energy stored in the PPi
linkages of (di)nucleoside polyphosphates back to
ATP, (b) necessary degradation of important signal-
ing molecules, or (c) efficient removal of otherwise
toxic compounds that accumulate as byproducts of
adenylate-transfer reactions.
4CL represents a key enzyme of the general phe-
nylpropanoid metabolism, which connects primary
metabolism with various product-specific pathways
by supplying appropriate mixtures of substrates for
these subsequent reactions (Dixon and Paiva, 1995;
Douglas, 1996). Due to its position at a metabolic
branch point, 4CL represents a prime target for reg-
ulatory control mechanisms. In this context, the ob-
servation that At4CL2 is capable to synthesize and
reuse unusual nucleotide derivatives, which may
function as intracellular messengers is obviously in-
triguing. Interestingly, 4CL can support the synthesis
of Ap4A but does not transfer the AMP-moiety onto
ADP to yield Ap3A. This is contrasted by properties
of some other plant enzymes, such as phenylalanyl-
tRNA synthetase from yellow lupin, which can pro-
duce both Ap3A and Ap4A (Jakubowski, 1983).
Whether these differences in catalytic properties of
different adenylate-forming enzymes contribute to
the Ap3A to Ap4A ratio in plant cells and whether
this ratio has a regulatory function or determines cell
fate, such as apoptosis versus proliferation as previ-
ously suggested for some mammalian model systems
(Vartanian et al., 1997), remains to be elucidated.
Nevertheless, the idea that unusual nucleotide deriv-
atives may have a regulatory function in plant me-
tabolism is strengthened by the recent identification
of Arabidopsis enzymes involved in (p) ppGpp syn-
thesis. Characterization of the respective proteins
suggest a function of (p) ppGpp in mediating stress-
induced defense responses in plants (van der Biezen
et al., 2000).
In conclusion, the data obtained in this study
clearly show that, in the absence of CoA, 4CL can
function as a pnA and ApnA synthetase, provided
1408
that alternative adenylate acceptors such as oligo-
phosphates (Pn) or adenosine oligophosphates (pnA)
are present at sufficient concentrations. In fact, 4CL
represents the first plant enzyme, in addition to
aminoacyl-tRNA synthetases, for which the capacity
to synthesize these unusual mono- and diadenosine
polyphosphates has been demonstrated. Thus, our
results shine new light on an old enzyme, 4CL, which
may have an important function beyond the well-
established role in phenylpropanoid metabolism.
MATERIALS AND METHODS
Chemicals and Enzymes
Coenzyme A, mono- and dinucleotides, buffers, and salts were from
Sigma-Aldrich (St. Louis), [2,8-3H]ATP (25 Ci mmol⫺1) was from ICN
(Irvine, CA), and [3H]Ap4A (20 Ci mmol⫺1) was from Moravek Biochemi-
cals (Brea, CA).
Recombinant At4CL2 proteins, the wild-type form, and the two mutants
M293P/K320L and K540N were expressed and purified as previously de-
scribed (Stuible et al., 2000; Stuible and Kombrink, 2001). The following
enzymes were used as analytical tools: yeast inorganic pyrophosphatase
(Sigma-Aldrich), calf intestinal alkaline phosphatase (Promega, Madison,
WI), recombinant exopolyphosphatase from yeast, scPPX1 (kindly supplied
by Dr. S. Liu, Department of Biochemistry, Beckman Center, Stanford, CA;
Guranowski et al., 1998), and recombinant Ap4A hydrolase from Lupinus
angustifolius (kindly donated by Drs. K. Gayler and D. Maksel, University of
Melbourne; Maksel et al., 2001).
Chromatographic Media and Solvent Systems
Thin-layer chromatography was performed with aluminum sheets pre-
coated with silica gel containing fluorescent indicator (catalog no. 5554, E.
Merck, Darmstadt, Germany). For monitoring of the synthesis of mono-
nucleoside polyphosphates, the chromatograms were developed in dioxane:
ammonia:water mixed at the ratio 6:1:6 (v/v/v), (system I) and for moni-
toring of the synthesis of dinucleoside polyphosphates that ratio was 6:1:4
(v/v/v, system II).
Enzyme Assays
The synthesis of adenosine 5⬘-polyphosphates p4A and p5A was deter-
mined in an assay mixture (50 ␮L final volume) containing 100 mm HEPES/
KOH (pH 7.0), 5 mm MgCl2, 5 mm ATP, 10 mm polyphosphate (P3 or P4),
and either 100 ␮m coumarate and 12.5 ␮g of At4CL2 wild-type enzyme or 62
␮m ferulate and 3.75 ␮g of the ferulate-preferring At4CL2 mutant M293P/
K320L. Incubations were carried out at 30°C. At appropriate times, usually
10 to 120 min, 3-␮L aliquots were withdrawn and spotted onto thin-layer
plates. Chromatograms were developed for 90 min in solvent system I,
dried, and visualized/photographed under short wave UV light. For quan-
titative assays, the mixture was supplied with [3H]ATP (2.5 ␮Ci); at appro-
priate times 5-␮L aliquots were withdrawn for analysis by thin-layer chro-
matography, and radioactivity in p4A or p5A spots was determined by
scintillation counting. For estimation of kinetic constants, pH optima, etc.,
the conditions varied appropriately.
The synthesis of diadenosine 5⬘,5⵮-P1,P4-tetraphosphate, Ap4A, was as-
sayed in an assay mixture (50 ␮L final volume) containing 100 mm HEPES/
KOH (pH 7.0), 5 mm MgCl2, 5 mm ATP, 62 ␮m ferulic acid, and 7.5 ␮g of the
At4CL2 mutant M293P/K320L. Incubations were carried out at 30°C and
lasted up to several hours. Five-microliter aliquots were withdrawn and
spotted onto thin-layer plates, and chromatograms were developed in sol-
vent system II. To monitor the synthesis of dAp4dA or Ap5A, dATP or p4A
substituted ATP. When the activity of the At4CL2 wild-type was deter-
mined, 100 ␮m coumarate was used instead of ferulate.
The transfer of the adenylate moiety from mono- or dinucleoside 5⬘-
polyphosphates onto various acceptors, was monitored in a reaction mix-
tures (50 ␮L final volume) containing 100 mm HEPES/KOH (pH 7.0), 5 mm
Plant Physiol. Vol. 131, 2003
 (Di)Adenosine Polyphosphate Synthesis by 4-Coumarate:Coenzyme A Ligase
MgCl2, 62 ␮m ferulate, an adenylate donor (1 mm p4A, 0.5 mm Ap4A, or 1
mm Ap5A), an adenylate acceptor (10 mm PPi, 5 mm P3, or 5 mm P4), and 7.5
␮g of the At4CL2 mutant M293P/K320L. The time course of product accu-
mulation in these reactions was monitored as above on thin-layer chromato-
grams, which were developed either in system I or II.
Synthesis of CoA esters of different cinnamic acid derivatives by At4CL2
was determined by the spectrophotometric assay as previously described
(Ehlting et al., 1999; Stuible et al., 2000).
Received July 25, 2002; returned for revision November 10, 2002; accepted
December 5, 2002.
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