Molecular and biochemical characterization of caffeine synthase and purine alkaloid concentration in guarana fruit

Article abstract of DOI:10.1016/j.phytochem.2014.04.018

Guarana seeds have the highest caffeine concentration among plants accumulating purine alkaloids, but in contrast with coffee and tea, practically nothing is known about caffeine metabolism in this Amazonian plant. In this study, the levels of purine alkaloids in tissues of five guarana cultivars were determined. Theobromine was the main alkaloid that accumulated in leaves, stems, inflorescences and pericarps of fruit, while caffeine accumulated in the seeds and reached levels from 3.3% to 5.8%. In all tissues analysed, the alkaloid concentration, whether theobromine or caffeine, was higher in young/immature tissues, then decreasing with plant development/maturation. Caffeine synthase activity was highest in seeds of immature fruit. A nucleotide sequence (PcCS) was assembled with sequences retrieved from the EST database REALGENE using sequences of caffeine synthase from coffee and tea, whose expression was also highest in seeds from immature fruit. The PcCS has 1083 bp and the protein sequence has greater similarity and identity with the caffeine synthase from cocoa (BTS1) and tea (TCS1). A recombinant PcCS allowed functional characterization of the enzyme as a bifunctional CS, able to catalyse the methylation of 7-methylxanthine to theobromine (3,7-dimethylxanthine), and theobromine to caffeine (1,3,7-trimethylxanthine), respectively. Among several substrates tested, PcCS showed higher affinity for theobromine, differing from all other caffeine synthases described so far, which have higher affinity for paraxanthine. When compared to previous knowledge on the protein structure of coffee caffeine synthase, the unique substrate affinity of PcCS is probably explained by the amino acid residues found in the active site of the predicted protein.

Full text of DOI:10.1016/j.phytochem.2014.04.018

Phytochemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Molecular and biochemical characterization of caffeine synthase
and purine alkaloid concentration in guarana fruit
Flávia Camila Schimpl a, Eduardo Kiyota a, Juliana Lischka Sampaio Mayer b,
José Francisco de Carvalho Gonçalves c, José Ferreira da Silva d, Paulo Mazzafera a,
⇑
a Departamento de Biologia Vegetal-IB, CP 6109, Universidade Estadual de Campinas, 13083-970 Campinas, SP, Brazil
b Departamento de Biologia, Universidade Estadual do Centro-Oeste, Guarapuava, PR, Brazil
c Instituto Nacional de Pesquisas da Amazônia, Manaus, AM, Brazil
d Departamento de Produção Animal e Vegetal, Faculdade de Ciências Agrárias, Universidade Federal do Amazonas, Manaus, AM, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Guarana seeds have the highest caffeine concentration among plants accumulating purine alkaloids, but
in contrast with coffee and tea, practically nothing is known about caffeine metabolism in this Amazo-
nian plant. In this study, the levels of purine alkaloids in tissues of five guarana cultivars were deter-
mined. Theobromine was the main alkaloid that accumulated in leaves, stems, inflorescences and
pericarps of fruit, while caffeine accumulated in the seeds and reached levels from 3.3% to 5.8%. In all tis-
sues analysed, the alkaloid concentration, whether theobromine or caffeine, was higher in young/imma-
ture tissues, then decreasing with plant development/maturation. Caffeine synthase activity was highest
in seeds of immature fruit. A nucleotide sequence (PcCS) was assembled with sequences retrieved from
the EST database REALGENE using sequences of caffeine synthase from coffee and tea, whose expression
was also highest in seeds from immature fruit. The PcCS has 1083 bp and the protein sequence has greater
similarity and identity with the caffeine synthase from cocoa (BTS1) and tea (TCS1). A recombinant PcCS
allowed functional characterization of the enzyme as a bifunctional CS, able to catalyse the methylation
of 7-methylxanthine to theobromine (3,7-dimethylxanthine), and theobromine to caffeine (1,3,7-trim-
ethylxanthine), respectively. Among several substrates tested, PcCS showed higher affinity for theobro-
mine, differing from all other caffeine synthases described so far, which have higher affinity for
paraxanthine. When compared to previous knowledge on the protein structure of coffee caffeine syn-
thase, the unique substrate affinity of PcCS is probably explained by the amino acid residues found in
the active site of the predicted protein.
Received 13 November 2013
Received in revised form 17 February 2014
Available online xxxx
Keywords:
Amazon
Venezuelan guarana
Paullinia cupana var. sorbilis
Sapindaceae
Caffeine
Theobromine
Recombinant protein
Seed
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
2.5% to 6.5% caffeine at dry weight (Oliveira, 2010; Spoladore
et al., 1987). Theobromine (3,7-dimethylxanthine) (2) and theoph-
Guarana is the name given to Paullinia cupana var. sorbilis
Kunth, a native species to the Maués region in the Brazilian Ama-
zon. The variety sorbilis is known as the Brazilian guarana, while
Paullinia cupana var. typica is the Venezuelan guarana (Schimpl
et al., 2013). Except for Paullinia pinnata L., which is also present
in tropical Africa, all other species of the genus are restricted to
tropical and subtropical America (Missouri Botanical Garden,
2012). In this study, guarana will designate P. cupana var. sorbilis,
which is the only commercially exploited species and has been
studied most intensively.
ylline (1,3-dimethylxanthine) (3) are present in smaller quantities
(see structures 1, 2 and 3 in Fig. 1). Indians and natives of the Ama-
zon have consumed guarana for centuries as an aqueous beverage
made from the powder obtained from roasted seeds, whose best
known physiological effect is improved alertness because of caf-
feine (Henman, 1986; Schimpl et al., 2013; Schmidt, 1941).
Caffeine (1) has been reported in 13 plant orders, mostly
eu-dicotyledonous plants. With minor variations, the main route
of its synthesis is highly conserved in the plants studied so far,
mainly coffee (Coffea arabica) and tea (Camellia sinensis)
(Ashihara et al., 2008; Ashihara and Suzuki, 2004; Mazzafera,
2004).
Guarana has the highest caffeine (1,3,7-trimethylxanthine) (1)
concentration reported in plants and its seeds may contain from
Caffeine (1) biosynthesis has three methylation steps and xan-
thosine is the first compound to be methylated. It has been sug-
gested that there are four possible routes in the origin of
⇑
Corresponding author. Tel.: +55 19 3521 6213.
E-mail address: pmazza@unicamp.br (P. Mazzafera).
http://dx.doi.org/10.1016/j.phytochem.2014.04.018
0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Schimpl, F.C., et al. Molecular and biochemical characterization of caffeine synthase and purine alkaloid concentration in
guarana fruit. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.04.018

2
F.C. Schimpl et al. / Phytochemistry xxx (2014) xxx–xxx
Fig. 1. Main metabolic pathway for the biosynthesis and biodegradation of caffeine. SAM = S-adenosyl-
L-methionine, SAH = S-adenosyl-L-homo-cysteine. I = 7-methylxan-
thosine synthase (xanthosine methyltransferase), II = nucleosidase, III = theobromine synthase or caffeine synthase, IV = caffeine synthase.
xanthosine (4) (Ashihara, 2006; Ashihara et al., 2008). In coffee and
tea, the caffeine biosynthesis pathway is: xanthosine (4) ? 7-
methylxanthosine (5) ? 7-methylxanthine (6) ? theobromine
(3,7-dimethylxanthine) (2) ? caffeine (1,3,7-trimethlxanthine)
(1) (Fig. 1) (Ashihara et al., 2008; Mizuno et al., 2003a; Uefuji
bled high similarity to caffeine synthase from cocoa, and the
recombinant protein proved to be a bifunctional caffeine synthase,
being able to methylate 7-methylxanthine (8) and theobromine
(2). In contrast with all caffeine synthases studied so far, which
have paraxanthine (8) as the best substrate, P. cupana caffeine syn-
thase (PcCS) has theobromine as the best substrate and no activity
against paraxanthine. PcCS had higher expression in immature
seeds compared to other guarana tissues.
et al., 2003). S-adenosyl-L-methionine (SAM) is the methyl donor
in caffeine (1) biosynthesis (Suzuki, 1972). The genes coding for
the N-methyltransferase enzymes catalysing the three methyla-
tions have been studied mainly in coffee, and to a lesser extent
in tea (Ashihara et al., 2008, 2011; Kato and Mizuno, 2004).
So far studies on guarana have mostly been restricted to charac-
terization of caffeine (1) and other purine alkaloid concentration in
the seeds. Recently, with the aim of increasing genetic knowledge
of this plant, an EST database was made from guarana fruit at three
different stages of maturation [Rede Amazônia Legal de Pesquisas
Genômicas – Realgene (Ângelo et al., 2008)]. Recently, Figueiredo
et al. (2011) analysed the 15,490 ESTs of the Realgene database
and, among 4697 full-length clones identified, 84 clones were
identified as N-methyltransferases and 18 were sequenced for con-
firmation of their identity. Phylogenetic analyses were carried out
for three clones (with only 0.017% dissimilarity), which were close
to a postulated caffeine synthase of Theobroma cacao (BTS1/BCS1).
However, the recombinant protein from the cocoa sequence and
other sequences resembling caffeine synthase only had 3-N-meth-
yltransferase activity, i.e., theobromine synthase activity
(Yoneyama et al., 2006).
2. Results
2.1. Methylxanthines concentration
Theobromine (2) was the main alkaloid found in leaves, fol-
lowed by theophylline (3), and higher concentrations of both alka-
loids were found in young leaves. Caffeine (1) was detected only in
intermediate and mature leaves. Theobromine (2) concentration
ranged from 3.47% to 4.18% (Fig. 2).
Similar to leaves, apical and basal portions of stems had theo-
bromine (2) as the predominant alkaloid, followed by theophylline
(3). Caffeine (1) was detected only in the basal portion. Among the
tissues analysed, stems were those containing the lowest methyl-
xanthine (8) concentrations (Fig. 3).
Theobromine (2) was also the most abundant alkaloid in the
inflorescences of all cultivars and, for most of them, the theophyl-
line (3) concentration was higher than caffeine (1) (Fig. 4).
In the pericarp, theobromine (2) was also the predominant alka-
loid but, in this tissue, caffeine (1) was more abundant than the-
ophylline (3) (Fig. 5). In general, the alkaloid concentration
In this study, the purine alkaloid concentration in different tis-
sues of five guarana cultivars grown in Brazil was analyzed and, in
one of them, caffeine synthase activity was assayed. Using the
Realgene database, a nucleotide sequence displaying was assem-
Please cite this article in press as: Schimpl, F.C., et al. Molecular and biochemical characterization of caffeine synthase and purine alkaloid concentration in
guarana fruit. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.04.018

F.C. Schimpl et al. / Phytochemistry xxx (2014) xxx–xxx
3
Fig. 2. Levels of theobromine (2) (Tb), caffeine (1) (Cf) and theophylline (3) (Tp) in leaves at three developmental stages of five guarana cultivars. Bars indicate standard
deviation of three replicates.
Fig. 3. Levels of theobromine (2) (Tb), caffeine (1) (Cf) and theophylline (3) (Tp) in apical and basal stems of five cultivars of guarana. Bars indicate standard deviation of three
replicates.
decreased in the pericarp with fruit maturation (Fig. 5). The caf-
feine (1) concentration was higher than theobromine (2) only in
seeds, and the values ranged from 3.3% to 5.8% (Fig. 5). As in leaves
and stems, caffeine concentration was higher in young tissues
(immature seeds). Theophylline (2) concentrations in the pericarp
and seeds were less than 0.04% (Fig. 5).
2.2. Multiple alignment and phylogenetic analysis
The translated amino acid sequence obtained from the PcCS
nucleotide sequence showed high similarity with three sequences
previously assembled from the Realgene EST database (Figueiredo
et al., 2011), whose expression or functionality was not tested
Please cite this article in press as: Schimpl, F.C., et al. Molecular and biochemical characterization of caffeine synthase and purine alkaloid concentration in
guarana fruit. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.04.018

4
F.C. Schimpl et al. / Phytochemistry xxx (2014) xxx–xxx
synthase amino acid sequence was phylogenetically grouped in a
clade with sequences of caffeine synthase of theobromine-accu-
mulating species: C. sinensis (TCS1: AB031280), C. irrawadiensis
(ICS1: AB056108), C. ptilophylla (PCS1: AB207817) and T. cacao
(BTS1, AB096699) (Fig. 7). The other clade grouped sequences of
caffeine synthase, as well as theobromine synthase and 7-methyl-
xanthine synthase from coffee species (C. arabica and C. canephora).
2.3. Gene expression of PcCS in guarana fruit
PcCS is expressed in all tissues of the BRS-Amazonas cultivar
(Fig. 8). Roots and old leaves showed the lowest transcript levels
among the vegetative tissues. The highest expression in fruit tis-
sues was observed in seeds, but in both seeds and pericarps, the
expression level decrease with maturation. PcCS expression in
seeds from green fruit was 7–20 times higher than in the other tis-
sue and stages. The results obtained from RT-qPCR were confirmed
by in situ hybridization analysis, as PcCS was expressed in the cot-
yledon of seeds of immature and intermediate stages of fruit mat-
uration (Fig. 9A and B, see arrows) but not in mature fruits
(Fig. 9D). The probe signal intensity in seed tissues was low prob-
ably because of the presence of many starch granules and other
interfering substances. No positive probe signal was observed in
other parts of the fruit (seed coat, aril and pericarp – not shown).
Fig. 4. Levels of theobromine (2) (Tb), caffeine (1) (Cf) and theophylline (3) (Tp) in
inflorescences of cultivars of guarana. Bars indicate standard deviation of three
replicates.
(Supplementary Fig. S1). PcCS was also very similar to other N-
methyltransferases of caffeine-accumulating plants and presents
the highly conserved domains A, B0, C (specific for binding of S-
adenosylmethionine) and YFFF (Fig. 6). Phylogenetic analysis
showed two very distinct clades (Fig. 7). The guarana caffeine
Fig. 5. Levels of theobromine (2) (Tb), caffeine (1) (Cf) and theophylline (3) (Tp) in seeds and pericarp of fruit at different stages of maturation of five guarana cultivars. Bars
indicate standard deviation of three replicates.
Please cite this article in press as: Schimpl, F.C., et al. Molecular and biochemical characterization of caffeine synthase and purine alkaloid concentration in
guarana fruit. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.04.018

F.C. Schimpl et al. / Phytochemistry xxx (2014) xxx–xxx
5
Fig. 6. Alignment of PcCS translated amino acid sequence with N-methyltransferase sequences from other species and secondary structures moulded from C. canephora
CcDXMT1 (caffeine synthase). The core SAM binding domain is indicated in green and the folding helical domain is indicated in orange. The SAM binding motifs (A, B’ and C)
and YFFF conserved region are shown by black boxes. Access Numbers: CcDXMT: DQ422955; CaDXMT1: AB084125; CCS1: AB086414; TCS1: AB031280; BTS1: AB096699;
CTS2: AB054841; CaMXMT1: AB048794; CaMXMT2: AB084126; PCS1, AB207817; CaXMT1: AB048793; CcXMT1: DQ422954. (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: Schimpl, F.C., et al. Molecular and biochemical characterization of caffeine synthase and purine alkaloid concentration in
guarana fruit. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.04.018

6
F.C. Schimpl et al. / Phytochemistry xxx (2014) xxx–xxx
PcCS
CS
TS
TCS1
100
100
ICS1
96
PCS1
76
BTS1
CcXMT1
CaXMT1
CTS2
100
7XMT
TS
97
CaMXMT2
CaMXMT1
CcDXMT1
CCS1
99
76
100
CS
99
CaDXMT1
Fig. 7. Phylogenetic tree of N-methyltransferase amino acid sequences. Coffea arabica (CaXMT1: AB048793, CTS2: AB054841, CaMXMT1: AB084794, CaMXMT2: AB084126,
CaDXMT1: AB084125, CCS1: AB086414), Coffea canephora (CcXMT1: DQ422954, CcDXMT: DQ422955), Camellia sinensis (TCS1: AB031280), Camellia irrawadiensis (ICS1:
AB056108), Camellia ptilophylla (PCS1: AB207819) and Theobroma cacao (BTS1: AB096699). Functional activities are indicated as 7MXS: 7-methylxanthine synthase, TS:
theobromine synthase, CS: caffeine synthase.
Fig. 8. PcCS expression in vegetative and fruit tissues of the cultivar BRS-Amazonas. Bars indicate standard deviation of three replicates.
2.4. Recombinant caffeine synthase
3. Discussion
PcCS was expressed in Escherichia coli BL21-pRil (Supplemen-
tary Fig. S2) and although most of the recombinant protein was
retained in the pellet, the soluble fraction purified by affinity chro-
matography in Ni–NTA agarose yielded a reasonably pure protein
fraction (lane 5, Supplementary Fig. S2). Studies on this purified
3.1. Caffeine distribution in guarana tissues
In general, caffeine (1) is the main alkaloid in plants containing
purine alkaloids and its biosynthesis is highest in young tissues
(Ashihara et al., 2011). Other plants accumulating purine alkaloids,
such as the tea species C. irrawadiensis (Nagata and Sakai, 1985)
and C. ptilophylla (Ye et al., 1997), have theobromine (2) as the
main alkaloid in young leaves. Theobromine (2) is also the predom-
inant alkaloid in seeds of T. cacao (Senanayake and Wijesekera,
1971). In coffee seeds and leaves, caffeine (1) is the main alkaloid
(Ashihara et al., 2011; Ashihara et al., 2008; Mazzafera, 2004;
Mazzafera and Carvalho, 1991; Mazzafera et al., 1991).
fraction showed a Km = 328
l
M and Vmax = 20.7 nmol minꢀ1 for 7-
M and Vmax = 10.0 nmol minꢀ1
methylxanthine (6) and a Km = 277
l
for theobromine (2). The recombinant PcCS was able to catalyse
the methylations of 7-methylxanthine (6) to theobromine (2) and
of theobromine (2) to caffeine (1) (Table 1). The protein has a the-
oretical molecular mass of 40.1 kDa and theoretical pI of 5.14
(http://ca.expasy.org/).
In this study, analysis of five guarana cultivars showed that the
levels of caffeine (1), theobromine (2) and theophylline (3) in gua-
rana varied significantly among tissues and at different develop-
mental stages. In all cultivars, theobromine (2) was the most
abundant alkaloid in vegetative tissues (leaves and stems), flowers
and pericarp, followed by theophylline (3), while caffeine (1) was
predominant only in seeds. In general, the concentrations of all
alkaloids were higher in young tissues, decreasing with tissue
maturity/aging.
2.5. Caffeine synthase activity
Caffeine synthase activity was determined in protein extracts
from guarana seeds at three stages of maturation. Activity was
higher in seeds of fruit at the green stage using theobromine as a
substrate:
stage = 6.6 1.0 fkat; mature stage = 3.8 0.9 fkat. Caffeine syn-
thase of seeds has Km = 258
M and Vmax = 0.5 nmol minꢀ1
green
stage = 20.9 2.4
fkat;
intermediate
l
.
Please cite this article in press as: Schimpl, F.C., et al. Molecular and biochemical characterization of caffeine synthase and purine alkaloid concentration in
guarana fruit. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.04.018

F.C. Schimpl et al. / Phytochemistry xxx (2014) xxx–xxx
7
A
B
C
D
Fig. 9. In situ hybridization of PcCS in longitudinal sections of guarana fruit using an antisense probe. Arrows indicate the purple coloration resulting from detection of gene
transcripts. A: Cotyledon of immature seed; B: Cotyledon of seed at intermediate maturation stage; C: Control with sense probe (cotyledon of immature seed); D: Cotyledon
of mature seed. Bars = 50 lm.
Table 1
Specificity of recombinant PcCS and other N-methyltransferases to different substrates.
Recombinant enzyme
Substrate/methylation position
References
1-mX/3N
3-mX/1N
7-mX/3N
pX/3N
Tp/7N
Tb/1N
Theobromine synthase
Relative activity (%)
CaMXMT1
CaMXMT2
CTS1
BTS1
ICS1
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
100
100
100
100
100
100
5.0
5.3
1.4
ND
ND
11
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Uefuji et al. (2003)
Uefuji et al. (2003)
Mizuno et al. (2003b)
Yoneyama et al. (2006)
Yoneyama et al. (2006)
Yoneyama et al. (2006)
PCS1
Caffeine synthase
CCS1
CaDXMT1
TCS1
PcCS
Product
0.53
ND
5.2
ND
Tp
0.82
ND
0.4
ND
Tp
24
100
100
100
ND
Cf
ND
ND
0.0
ND
Cf
25
Mizuno et al. (2003b)
Uefuji et al. (2003)
Kato et al. (2000)
1.0
43.5
53
3.8
7.8
100
Cf
Tb
Relative activity presented as percentage of the highest activity (100%), indicated in bold print. ND = not detected; 1-mX = 1-methylxanthine (9); 3-mX = 3-methylxanthine
(8); 7-mX = 7-methylxanthine (6); pX = paraxanthine; Tp = theophylline (3); Tb = theobromine (2); Cf = caffeine (1). The activity of the recombinant PcCS using theobromine
(2) as a substrate was 9 fkat mgꢀ1 protein. The values represent mean values of three replicates. Enzymes of Coffea arabica = CTS1, CCS1, CaDXMT1; Camellia sinensis = TCS1;
Camellia irrawadiensis = ICS1; Camellia ptilophylla = PCS1; Theobroma cacao = BTS1.
Theobromine (2) was also detected as the main alkaloid in P.
cupana leaves by Weckerle et al. (2003), being higher in young
leaves (1.3%) than mature ones (0.03%). On the other hand, in P.
Yoco, the caffeine (1) concentration in the basal portion of stems
(0.45%) is about five times higher than in the apical portion, while
theobromine (2) ranged from 0.048% (apical portion) to 0.006/
0.009% (basal portion) (Weckerle et al., 2003). Theobromine (2)
was the only alkaloid detected in P. Pachycarpa, but at very low lev-
els (adult leaves – 0.025%; apical stem – 0.006%, basal stem –
0.001/0.018%) (Weckerle et al., 2003). We have no knowledge of
any report on N-methylxanthine concentration in guarana flowers.
Coffee (Raju and Gopal, 1979) and tea (Fujimori and Ashihara,
1993) flowers have high levels of caffeine (1). Curiously, Citrus
flowers have caffeine (1), but only in parts of the male organs,
and theophylline (3) also accumulates in minor proportions
(Kretschmar and Baumann, 1999).
Similar to flowers and vegetative tissues of guarana, the fruit
pericarp also had theobromine (2) as the main alkaloid, but
depending on the cultivar and the fruit developmental stage, caf-
feine (1) was present in comparable quantities. Caffeine (1) was
also the main alkaloid in seeds, and at levels much higher than
those found for theobromine (2) in any other guarana tissue. Sev-
eral reports have described caffeine concentration in guarana seeds
ranging from 2.5% to 6% in the seed and from zero to 1% in the peri-
carp (Baumann et al., 1995; Maravalhas, 1965; Oliveira, 2010;
Pires, 1949; Spoladore et al., 1987). It has been suggested that
the prevalence of caffeine (1) in guarana seeds is a defense strategy
against predators (Mithöfer and Boland, 2012; Weckerle et al.,
2003). Baumann et al. (1995) suggested that caffeine (1) helps gua-
rana seed dispersion by birds. Some birds swallow the fruit with-
out breaking the seed, which passes through the digestive tract
intact without releasing caffeine (1), thus avoiding intoxication.
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F.C. Schimpl et al. / Phytochemistry xxx (2014) xxx–xxx
However, the aril, which is free of caffeine (1) and rich in sugars, is
digested.
be an enzyme with dual functionality, similar to TCS1 of tea
(Kato and Mizuno, 2004).
Caffeine (1) can be degraded via theophylline (3) and theobro-
mine (2) (Fig. 1). Usually theophylline (1) is the main route occur-
ring with high efficiency, which explains its low concentration, for
example, in coffee tissues (Mazzafera, 2004; Suzuki and Waller,
1984a,b). Theobromine (2) is a precursor and a product of degrada-
tion of caffeine (1) in most of the species where the metabolism of
this alkaloid was studied (Ashihara et al., 2008; Ashihara and
Suzuki, 2004; Suzuki and Waller, 1984a,b). A transient accumula-
tion of theobromine (2) in young tissues of plants accumulating
caffeine (1) has been related to a high biosynthesis rate of the latter
alkaloid (Ashihara et al., 2008; Ashihara and Suzuki, 2004). How-
ever, transient accumulation of theobromine (2) in C. dewevrei
seems to be related to a more significant participation of the theo-
bromine (2) degradation route (Mazzafera et al., 1994a).
The significant accumulation of theobromine (2) in the vegeta-
tive tissues and flowers of guarana might be a consequence of low
conversion to caffeine (1), as observed in coffee mutants almost
free of caffeine (1) (Silvarolla et al., 2004). Support for this is the
very low expression of PcCS in vegetative tissues of guarana
(Fig. 8). However, based on previous knowledge of other plants,
this is not consistent with the amount of theophylline found in
these tissues, which was higher than caffeine (1). Unless guarana
presents a unique metabolism, in the plants studied so far, most
of the theophylline (3) was found as a product of degradation of
caffeine (Mazzafera, 2004), although the pathway xanthine
(7) ? 3-methylxanthine (8) ? theophylline (3) was found as an
alternative in tea seedlings (Deng and Ashihara, 2010). It may be
speculated that, coupled with low biosynthesis from theobromine
(2), caffeine (1) is low in these guarana tissues due to a higher deg-
radation rate, but theophylline (3) degradation might be slower
than in other plants investigated up to now for caffeine metabo-
lism. Only studies using labelled substrates may explain the meth-
ylxanthine concentrations and their metabolism in guarana
tissues, which was outside the scope of this study.
N-Methyltransferases of methylxanthine-containing plants
have greater similarity to each other within the same genus than
among different species (Ashihara et al., 2008; Figueiredo et al.,
2011; Yoneyama et al., 2006). Thus, theobromine (2) and caffeine
(1) synthases from Coffea arabica have more than 80% identity,
while tea and coffee caffeine synthases have only 34% identity.
The identity of the translated guarana PcCS was greater with theo-
bromine synthases (from 44% to 48%) than with caffeine synthases
(less than 35%) identified in other plants (Fig. 7). The contigs
assembled here using the EST sequences of the Realgene EST data-
base had 1083 bp and were similar to other assemblings carried
out with sequences retrieved from the same database [Supplemen-
tary Fig. S1, Figueiredo et al., 2011]. However, as these sequences
were not assessed for expression or their heterologous proteins
were not produced and assayed for activity, it is not possible to
affirm they were caffeine synthases.
The translated amino acid sequence was aligned with theobro-
mine and caffeine (1) synthases from other species and typical A,
B0, and C motifs and the YFFF region of methyltransferases related
to caffeine metabolism were found (Kato and Mizuno, 2004). A, B
and C motifs are characteristics of SAM-dependent O-methyltrans-
ferases (Joshi and Chiang, 1998). The B0 motif and the YFFF region
are specific features of the methyltransferase family, including caf-
feine synthase (Kato and Mizuno, 2004). Salicylic acid carboxyl
methyltransferase, benzoic acid carboxyl methyltransferase and
jasmonic acid carboxyl methyltransferase are other members of
this family (Ishida et al., 2009; Mizuno et al., 2003a,b; Yoneyama
et al., 2006). In addition to the presence of these motifs in the PcCS,
phylogenetic analysis showed that PcCS has higher identity with
methyltransferase sequences from theobromine-accumulating
species such as tea and cacao (Fig. 7), although these sequences
may code for theobromine (BTS1, ICS1, PCS1 – Yoneyama et al.
2006) or caffeine synthases (TCS1
Yoneyama et al., 2006).
– Kato et al., 2000 and
Consistent with the data of methylxanthines concentration in
seeds (Fig. 5), the expression of PcCS was significantly higher in
seeds of immature fruit (Figs. 8 and 9). Complementing this infor-
mation, attempts to determine caffeine synthase activity were suc-
cessful only using seed protein extracts from immature fruit (data
not shown). In order to prove that the PcCS was a caffeine synthase,
a heterologous protein was produced and activity was tested
against several substrates, including paraxanthine. In contrast with
all other caffeine synthases characterized until now, recombinant
PcCS showed theobromine (2) as the best substrate (caffeine syn-
thase activity), followed by 7-methylxanthine (6) (theobromine
synthase activity) (Table 1). No activity was found against any
other substrate tested, including paraxanthine, the best substrate
identified for other caffeine synthases (Ashihara et al., 2008; Kato
and Mizuno, 2004). Theobromine synthase activity of PcCS was
approximately 50% of the caffeine synthase activity.
Small changes in the amino acid sequence may partially explain
the affinity of PcCS for theobromine instead of paraxanthine. The
translated PcCS was aligned with other methyltransferase
sequences and the secondary structure of the caffeine synthase
de C. canephora CcDXMT1 can be used as reference for comparison
(Fig. 6). McCarthy and McCarthy (2007) studied the crystallo-
graphic structure of caffeine synthase (DXMT) from Coffea canepho-
ra and, based on the characteristics of the 15 amino acid residues
within 5 Å of the theobromine binding site, they suggested that
only three have crucial significance for discriminating the sub-
strate specificity of each enzyme, residues 27, 237 and 266. The
residues identified in theobromine synthase are Ala27, Pro237
and Phe266 and, in caffeine synthase, they are Phe27, Ser237 and
Ile266. It is not clearly understood in which way positions 27
3.2. Caffeine synthase activity and expression in guarana
Biosynthesis of caffeine (1) in plants is mediated by the activity
of three N-methyltransferases responsible for the methylation of
xanthosine (4), 7-methylxanthine (6) and theobromine (2) (3,7-
dimethylxanthine). Genes coding for these enzymes have been
characterized in some caffeine (1)-containing plants, but they were
particularly well characterized in coffee (for a review see Ashihara
et al. (2011) and Kato and Mizuno (2004)). Three genes are known
to code for 3,7-methylxanthine methyltransferase (caffeine syn-
thase – EC 2.1.1.160; CaDXMT1 – AB084125; CCS1 – AB086414;
CtCS7 – AB086415). Regarding CtCS7, it is a tentative caffeine syn-
thase because the heterologous protein has very low activity and
only against paraxanthine (1,7-dimethylxanthine) but not theo-
bromine (2). CaDXMT1 and CCS1 caffeine synthases seem to have
unique characteristics as they differ in regard to their affinity for
theobromine (2) (1200 lM and 157 lM, respectively), and their
expression profile, CaDXMT1, is expressed exclusively in immature
fruits while CCS1 is expressed in all tissues containing caffeine
(Ashihara et al., 2011; Kato and Mizuno, 2004). Curiously, both
enzymes have higher affinity for paraxanthine, a methylxanthine
found in minute amounts in a coffee cell suspension (Baumann
and Frischknecht, 1988; Sartor and Mazzafera, 2000) or in tea
leaves (Kato et al., 1996). However, a key difference between these
two enzymes is the fact that CaDXMT1 has low activity against 7-
methylxanthine (6) and theobromine (2) (1 and 4%, respectively,
considering 100% activity for paraxanthine) while CCS1 has much
higher and comparable activities (16–24% for 7-methylxanthine
(6) and 23–25% for theobromine (2)). CCS1 has been suggested to
Please cite this article in press as: Schimpl, F.C., et al. Molecular and biochemical characterization of caffeine synthase and purine alkaloid concentration in
guarana fruit. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.04.018

F.C. Schimpl et al. / Phytochemistry xxx (2014) xxx–xxx
9
and 237 influence substrate discrimination, while a Phe residue at
position 266 would prevent theobromine binding and, therefore,
theobromine synthase activity. However, while the 266 residue
substitution may be important in coffee, it probably is not in tea
since a Ser is the amino acid present in both theobromine
(Yoneyama et al., 2006) and caffeine synthase (Kato et al., 2000).
In PcCS, the amino acids in these positions are Thr28, Pro238 and
Tyr267.
the cultivars BRS-Amazonas, BRS-Maués, BRS-Luzéia, BRS-CG372,
BRS-CG611. Samples were taken from three different plants and
immediately after collection they were frozen in dry ice and then,
in the laboratory, in a freezer kept at ꢀ80 °C. Part of the material
was dried at 50 °C for methylxanthines determination.
5.2. Methylxanthine quantification
Yoneyama et al. (2006) made site-directed mutagenesis in PCS1
changing the His221 to Arg221 and observed that the recombinant
protein, while still keeping 7-methylxanthine (6) (theobromine
synthase activity) as the best substrate, showed a fivefold increase
of activity against paraxanthine (caffeine synthase activity).
Although the result is not conclusive, they suggested that the
His221 position plays an important role in defining substrate spec-
ificity but other amino acid residues might be involved in substrate
recognition.
The material dried at 50 °C was ground to a powder in a mortar
with a pestle, and 100 mg was extracted with MeOH–H2O (80:20 v/
v) in a water-bath at 50 °C for 2 h, with occasional stirring. After
cooling on the bench, the samples were centrifuged at 15,000g
for 20 min, and diluted in distilled H2O at a ratio of 1:10 (v/v;
seeds) or 1:2 (v/v; other tissues). Caffeine (1), theobromine (2)
and theophylline (3) were quantified by high performance liquid
chromatography (HPLC Shimadzu System) using a photodiode
array detector operating at 272 nm. A C18 reversed-phase column
PcCS is particularly different from the other caffeine synthases
at the active site, especially in the positions Leu236, Pro237 and
Trp238, although it is not possible to conclude how important
these changes are with the little knowledge there is on the protein
structure of caffeine synthases. In these positions, the caffeine syn-
thase (DXMT) from coffee has Asp236, Ser237 and Met238 (C. arab-
ica – CaDXMT) or Ile238 (C. canephora – CcDXMT). Thus, if the
residue at position 238 is not the same for these coffee species,
the residues 236 and 237 might be determinant to define the affin-
ity of PcCS for theobromine instead of paraxanthine. McCarthy and
McCarthy (2007) observed that Ser237 in caffeine synthase
(DMXT) is mutated to a Pro in theobromine synthase (MXMT), sim-
ilar to PcCS. This reinforces the indication that it is not possible to
draw solid conclusions based on the protein sequences from differ-
ent species, as well as that more than one amino acid residue
defines substrate specificity. Only crystallography structural stud-
ies of PcCS and site-directed mutagenesis may explain the unique
specificity of PcCS for theobromine but not for paraxanthine.
(25 cm ꢁ 4.6 mm ꢁ 5
lm; Supelco™) was used to separate the
alkaloids using aqueous 0.5% AcOH in H2O (A) and MeOH (B) as
mobile phases, at a flow rate of 1.2 mL minꢀ1. The gradient used
was: 0 min 10% B, 18 min 45% B, 19 min 100% B and 20–30 min
10% B. Standard curves for methylxanthine calculation were
obtained from known amounts of pure caffeine (1), theobromine
(2) and theophylline (3) (Sigma).
5.3. Analysis of EST database, primer design and PcCS sequencing
The EST database Realgene (Ângelo et al., 2008) was used for
searching caffeine synthase sequences using the tool BLASTx
(Altschul et al., 1990). Caffeine synthase sequences of Coffea arabica
(CaDXMT1, AB084125; CCS1, AB086414), Coffea canephora
(CcDXMT, DQ422955) and Camellia sinensis (TCS1, AB031280) were
used as bait. Among the retrieved sequences, the longest ones with
e-values lower than eꢀ50 were selected and used to assemble a
contig of 1369 bp (CDS = 1083 bp) using the ClustalW tool in the
software BioEdit (Thompson et al., 1994). Total RNA was extracted
(Rezaian and Krake, 1987) from immature seeds, treated with
DNase (Turbo DNA-free, Ambion, Inc.) and the first cDNA strand
was synthesized (SuperScript III First-Strand, Invitrogen) and used
in RT-PCR reactions. Primers at the 50UTR and 30UTR regions and
also ones internal to the CDS were designed for amplification
(see Fig. 10 for primer sets). The two fragments amplified were
separated in 1% agarose gel, purified from the gel (GeneJET™ Gel
Extraction Kit, Fermentas) and inserted in the cloning vector
pGEM-T Easy Vector System (Promega), which was used to trans-
4. Concluding remarks
Guarana is distinguished from other caffeine-accumulating
plants because of high caffeine (1) concentration in its seeds
whereas, in other parts of the plant, theobromine (2) is the major
purine alkaloid. This indicates that guarana presents peculiar char-
acteristics regarding the control of caffeine (1) biosynthesis in dif-
ferent organs. Like coffee, it seems that the genes coding for
methyltransferases of caffeine (1) biosynthesis in guarana form a
large family. In a search in the guarana EST database, several
incomplete methyltransferases sequences with small differences
regarding their similarity were found, thereby supporting this con-
clusion. The affinity of the PcCS isolated in this study, and compar-
ison of its amino acid sequence with other caffeine synthases
already reported in the literature, show that an understanding
what residues in the active site of the enzyme define the best sub-
strate is still not obtained.
form the thermo-competent E. coli DH5a strain (Novagen). Positive
colonies were miniprepped (PureLink Quick Plasmid Miniprep Kit,
Invitrogen), and the plasmid insert was sequenced using the flank-
ing vector primers (T7 and SP6) and the internal primers already
mentioned. The sequencing reactions were performed using the
BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems)
and an ABI PRISM 377 sequencer (Applied Biosystems). The
sequence obtained will hereinafter be referred to as Paullinia
cupana caffeine synthase (PcCS – GenBank accession number
BK008796).
5. Experimental
5.1. Plant material
5.4. PcCS expression analysis
Guarana plant tissue was collected on the farm Agropecuária
Jayoro Ltda, Presidente Figueiredo, Amazonas, Brazil. Fruit (imma-
ture, intermediate and mature immediately separated into seeds
and pericarps), leaves (young, intermediate and mature), inflores-
cences (containing floral buds and open flowers, but with no fruit),
apical stems (a 20-cm portion from the apex) and basal stems (a
20-cm portion from the base) were collected from adult plants of
Total RNA was extracted from different guarana tissues
(Rezaian and Krake, 1987) and first strand cDNA was obtained as
described above. Expression of PcCS was performed by quantitative
RT-PCR (qRT-PCR). The reactions (processed in technical tripli-
cates) were prepared with the QuantiFast SYBR Green PCR Kit (Qia-
gen) and run in an iCycler iQ5 Multicolour Real-Time PCR
Please cite this article in press as: Schimpl, F.C., et al. Molecular and biochemical characterization of caffeine synthase and purine alkaloid concentration in
guarana fruit. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.04.018

10
F.C. Schimpl et al. / Phytochemistry xxx (2014) xxx–xxx
Fig. 10. Contig assembled from guarana EST sequences. Coding region (1083 bp) is underlined from methionine codon (ATG) to the stop codon (TAA). The primers used for
cloning and sequencing which generated two fragments by amplification are identified as ‘‘Sequencing primers pair’’ 1 and 2; the primers used in RT-qPCR analyses are
identified as ‘‘Real time primer’’; and the primers used for cloning PcCS for heterologous expression in pET28a are identified as ‘‘Expression primer’’, to which was added the
restriction sites for the BamHI (forward) and Sall (reverse) restriction site sequences.
Detection System (Bio-Rad) under the following conditions: 95 °C
for 3 min, followed by 40 cycles at 95 °C for 10 s and 60 °C for
ESPript (Gouet et al., 1999) using the 2D structure of the caffeine
synthase from C. canephora (CcDMXT1, DQ422955; PDB ID: 2EFJ)
as reference (McCarthy and McCarthy, 2007).
D
Ct
30 s. The primers used are shown in Fig. 10 and the 2ꢀ
method
(Livak and Schmittgen, 2001) was used for the quantification of rel-
ative expression. Four reference genes were used (Schimpl, 2013):
actin (ACT, forward: 50-ACCATCACCAGAATCCAACACAATACC-30,
reverse: 50-ACCATCACCAGAATCCAACACAATACC-30), ubiquitin 11
(UQT, forward: 50-TAAGGAAGGCATTCCACCTG-30, reverse: 50-TAATCC
GCTCAATGGT GTCA-30), Alcohol dehydrogenase (ADH, forward:
50-GTTGCAGCAT CAGGTCAAGA-30, reverse: 50-ACGTCCATTTTCAGC
ACACA-30), and 40S ribosomal protein S24-1 (S24, forward:
50-TCATCCTGGAAGGCCTAATG-30, reverse: 50-CAACCTTGGTGTCAA
GTCCA-30). PcCS expression was also analysed by in situ hybridiza-
tion. A probe was produced using the 173 bp fragment amplified
with the qRT-PCR primers. All subsequent steps for probe prepara-
tion followed the protocol of Araújo et al. (2012). Immature, inter-
mediate and mature fruits were analysed, and the images were
captured with an Olympus DP71 video camera connected to an
Olympus BX 51 microscope.
5.6. Recombinant PcCS
cDNA from immature seeds was used in RT-PCR to amplify the
whole PcCS sequence (primer sets are shown in Fig. 10), which was
initially cloned into the pGEM-T Easy Vector System (Promega) and
then into the pET28a (Invitrogen) between the BamHI and SalI
sites, containing the recombinant 6X HiS-tag at the N terminus.
The quality of the insertion was verified by sequencing. The
pET28a-PcCS was expressed in the E. coli BL21 (pRil) electrocompe-
tent strain. The transformed cells were grown for 4 h in 50 mL of LB
medium until the OD600 = 0.6 and then induced with 1 mM isopro-
pyl-b- -thiogalactoside at 37 °C, 200 rpm. The cells were collected
D
by centrifugation (5000g at 4 °C) and the bacterial pellet was solu-
bilized in affinity buffer (50 mM sodium phosphate, pH 7.3,
100 mM NaCl, 5% glycerol) and then lysed by sonication (20 cycles
of 3 s, with intervals of 5 s, at 20 kHz – Vibra-Cell, SONICS). The
presence of the recombinant PcCS in the total extract was verified
by 12% SDS–PAGE (Laemmli, 1970). To obtain the purified protein,
the starting medium was scaled up to 2 L culture and the bacterial
pellet obtained by centrifugation was solubilized in 40 mL of affin-
5.5. Sequence alignment, phylogenetic analysis and 2D protein
structure prediction
Translated amino acid sequences of coffee N-methyltransfer-
ases (C. arabica and C. canephora), tea (Camellia sinensis, C. ptilophy-
lla and C. irrawadiensis) and cocoa (Theobroma cacao) were aligned
with PcCS using the software ClustalW (Thompson et al., 1994).
The software MEGA 5 (Tamura et al., 2011) was used to construct
a phylogenetic tree using the neighbour-joining algorithm (Saitou
and Nei, 1987). Gap regions were excluded by manual adjustments
and bootstrap values were obtained after 10,000 repetitions. The
prediction of the PcCS 2D structure was obtained with the software
ity buffer and treated with lysozyme (80 l
g mLꢀ1) for 30 min and
then sonicated as indicated previously. The sonicated extract was
centrifuged (12,000g for 15 m at 4 °C) and 1 mL of Ni–NTA agarose
resin (Qiagen) previously equilibrated with affinity buffer was
added to the supernatant and this was stirred for 30 min. The
extract-resin mixture was packed into a plastic column (1.0 cm
diameter and 5.0 cm long) and the proteins were eluted by addi-
tion of solutions containing increasing concentrations of imidazole
Please cite this article in press as: Schimpl, F.C., et al. Molecular and biochemical characterization of caffeine synthase and purine alkaloid concentration in
guarana fruit. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.04.018

F.C. Schimpl et al. / Phytochemistry xxx (2014) xxx–xxx
11
(20, 50, 100, 200 and 500 mM, 2 mL of each solution). All fractions
Appendix A. Supplementary data
were collected and analysed by 12% SDS–PAGE.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2014.
04.018.
5.7. Plant PcCS activity and recombinant PcCS kinetic study
Plant PcCS was partially purified from immature guarana seeds
of the cultivar BRS-Amazonas. The seeds were ground in a mortar
with a pestle and extracted (1 g) with 5 mL of phosphate buffer
(200 mM, pH 7.3, 4 °C, containing 5 mM EDTA, 10 mM 2-b-mercap-
toethanol, 0.5% ascorbic acid, 0.5% Triton X-100 and 4% polyvinyl-
polypyrrolidone). The supernatant obtained after centrifugation
(15 min at 30,000g, 4 °C) was saturated to 80% with (NH4)2SO4
and pelleted by centrifugation (15 min at 30,000g, 4 °C). The pellet
was solubilized in phosphate buffer and immediately desalted and
concentrated by ultrafiltration (Amicon Ultra-4 Centrifugal Filter
Devices, Millipore). Protein concentration was determined using
the ready-to-use reagent BioRad (Bradford, 1976).
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F.C.S. thanks the Fundação de Amparo à Pesquisa do Estado de
São Paulo (FAPESP) for a MSc fellowship (2011/03266-6), and JFCG
and PM thank the Conselho Nacional de Desenvolvimento Científ-
ico e Tecnológico (CNPq) for research fellowships.
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Please cite this article in press as: Schimpl, F.C., et al. Molecular and biochemical characterization of caffeine synthase and purine alkaloid concentration in
guarana fruit. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.04.018