Caffeine a well known but little ...

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Opinion
TRENDSin Plant Science Vol.6 No.9 September 2001
407
Caffeine: a well known
but little mentioned
compound in plant
science
→
7-methylxanthine → theobromine → caffeine pathway;
the first, third and fourth steps are catalysed by
N
-methyltransferases that use
S
-adenosyl-
L
-
methionine (SAM) as the methyl donor
13
. A recent
important development has been the cloning and
expression in
E. coli
of a gene from tea leaves that
encodes caffeine synthase, an extremely labile
N
-methyltransferase that catalyses the last two steps in
this pathway
14
. In addition, coffee leaf cDNAs of
theobromine synthase, which catalyses the penultimate
methylation step, have been similarly cloned and
expressed in
E. coli
15,16
. There are also preliminary
reports on the cloning of an
N-
methyltransferase from
coffee that catalyses the initial methylation step in the
pathway
17,18
. These advances in our knowledge of the
metabolism of caffeine and related compounds in plants
and the potential biotechnological applications of
purine alkaloid research are highlighted in this article.
7-methylxanthosine
Hiroshi Ashihara and Alan Crozier
Caffeine, a purine alkaloid, is a key component of many popular drinks, most
notably tea and coffee, yet most plant scientists know little about its
biochemistry and molecular biology. A gene from tea leaves encoding caffeine
synthase, an N-methyltransferase that catalyses the last two steps of
caffeine biosynthesis, has been cloned and the recombinant enzyme
produced in E.coli.Similar genes have been isolated from coffee leaves but
the recombinant protein has a different substrate specificity to the tea
enzyme. The cloning of caffeine biosynthesis genes opens up the possibility of
using genetic engineering to produce naturally decaffeinated tea and coffee.
Caffeine (1,3,7-trimethylxanthine) is one of the few
plant products with which the general public is readily
familiar, because of its occurrence in beverages such as
coffee and tea, as well as various soft drinks. A growing
belief that the ingestion of caffeine can have adverse
effects on health has resulted in an increased demand
for decaffeinated beverages
1
. Unpleasant short-term
side effects from caffeine include palpitations,
gastrointestinal disturbances, anxiety, tremor,
increased blood pressure and insomnia
2,3
. In spite of
numerous publications on the long-term consequences
of caffeine consumption on human health, no clear
picture has emerged, with reports of both protective
and deleterious effects
4
.
Caffeine was discovered in tea (
Camellia sinensis
)
and coffee (
Coffea arabica
) in the 1820s (Ref. 5).
Along with other methylxanthines, including
theobromine (3,7-dimethylxanthine), paraxanthine
(1,7-dimethylxanthine) and methyluric acids (Fig. 1),
caffeine is a member of a group of compounds known
collectively as purine alkaloids. There are two
hypotheses about the role of the high concentrations of
caffeine that accumulate in tea, coffee and a few other
plant species. The ‘chemical defence theory’ proposes
that caffeine in young leaves, fruits and flower buds
acts to protect soft tissues from predators such as insect
larvae
6
and beetles
7
. The ‘allelopathic theory’ proposes
that caffeine in seed coats is released into the soil and
inhibits the germination of other seeds
8
. The potential
ecological role of caffeine is described in Ref. 6.
It is only within the past five years that the
biosynthetic and catabolic pathways that regulate the
build-up of caffeine in the vacuoles of cells of tea and
coffee plants have been elucidated fully. In contrast with
0.8%).
The beans of most cultivars of Arabica coffee
(
C. arabica
) (Fig. 3) contain ~1.0% caffeine, whereas
Coffea canephora
cv. Robusta (1.7%) and cv. Guarini
(2.4%),
Coffea dewevrei
(1.2%) and
Coffea liberica
(1.4%)
contain higher concentrations. By contrast, the caffeine
contents of the seeds of other species, such as
Coffea
eugenioides
(0.4%),
Coffea salvatrix
(0.7%) and
Coffea
racemosa
(0.8%), are lower than that of
C. arabica
.
Young expanding leaves of
C. arabica
plants also
contain caffeine, with traces of theobromine. In model
systems, weak intermolecular complexes form
between caffeine and polyphenols
19
, and it has been
proposed that caffeine is sequestered in the vacuoles
of coffee leaves as a chlorogenic acid complex
20
.
Mature leaves of
C. liberica
,
C. dewevrei
and
Coffea
abeokutae
convert caffeine to the methyluric acids,
theacrine (1,3,7,9-tetramethyluric acid), liberine
[
O
(2),1,9-trimethyluric acid] and methylliberine
[
O
(2),1,7,9-tetramethyluric acid] (Fig. 1).
Purine alkaloids are also present in the leaves of
maté (
Ilex paraguariensis
), which is used in rural areas
of South America, such as the Brazilian Panthanal and
Hiroshi Ashihara
Metabolic Biology Group,
Dept Biology, Faculty of
Science, Ochanomizu
University, Otsuka,
Bunkyo-ku, Tokyo
112-8610, Japan.
e-mail:
ashihara@cc.ocha.ac.jp
Alan Crozier
Plant Products and
Human Nutrition Group,
Division of Biochemistry
and Molecular Biology,
Faculty of Biomedical and
Life Sciences, University
of Glasgow, Glasgow,
UK G12 8QQ.
e-mail:
a.crozier@bio.gla.ac.uk
the widespread medical interest in caffeine as a dietary
component, these developments have received little
attention in the plant literature, with the topic being all
but neglected in recent biochemistry text books
9–12
.
Caffeine is synthesized from xanthosine via a
xanthosine
Distribution of purine alkaloids
Purine alkaloids have a limited distribution within
the plant kingdom. In some species, the main purine
alkaloid is theobromine or methyluric acids rather
than caffeine
13
. Among the purine-alkaloid-
containing plants, most studies have been carried out
with species belonging to the genera
Camellia
and
Coffea.
In
C. sinensis
(Fig. 2), caffeine is found in the
highest concentrations in young leaves of first-flush
shoots of var.
sinensis
(2.8% of the dry weight).
Theobromine is the predominant purine alkaloid in
young leaves of cocoa tea (
Camellia ptilophylla
)
(5.0–6.8%) and
Camellia irrawadiensis
(
408
Opinion
TRENDSin Plant Science Vol.6 No.9 September 2001
O
O
O
O
O
O
CH
3
CH
3
CH
3
H
N
CH
3
H
N
H
3
C
1
7
H
3
C
H
3
C
H
3
C
H
3
C
N
N
N
N
N
HN
N
N
N
N
O
O
O
O
N
N
O
N
N
O
N
N
O
N
N
O
N
N
O
N
N
H
3
C
H
3
C
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
Caffeine
Theobromine
Paraxanthine
Theacrine
Liberine
Methylliberine
TRENDS in Plant Science
Fig. 1.
Structures of the
methylxanthines caffeine,
theobromine and
paraxanthine, and the
methyluric acids
theacrine, liberine and
methylliberine.
the Pampas in Argentina, to produce a herbal tea
Young maté leaves contain 0.8–0.9% caffeine and
0.08–0.16% theobromine. Theobromine is the
dominant purine alkaloid in seeds of cocoa (
Theobroma
cacao
), with cotyledons of mature beans containing
2.2–2.7% theobromine and 0.6–0.8% caffeine. Caffeine
(4.3%) is the major methylxanthine in cotyledons of
guaraná (
Paulliania cupana
), extracts of which are
tree.com/guarana.htm) and which is, in a dilute form,
sold extensively in Brazil as a carbonated drink. Seeds
of cola (
Cola nitida
) also contain caffeine (2.2%)
21
.
Caffeine has recently been detected in flowers of
several citrus species, with the highest concentrations
(0.2%) in pollen
22
, and is also a fungal metabolite, being
the principal alkaloid in sclerotia of
Claviceps
sorhicola
, a Japanese ergot pathogen of
Sorghum
23
.
purine ring of caffeine can be produced exclusively
by this route in young tea leaves
25
. The formation of
caffeine by this pathway is closely associated with
the SAM cycle (also known as the activated-methyl
cycle) because the three methylation steps in the
caffeine biosynthesis pathway use SAM as the
methyl donor (Fig. 4). During this process, SAM is
converted to SAH, which in turn is hydrolysed to
L
-homocysteine and adenosine. The adenosine is
used to synthesize the purine ring of caffeine and
the
L
-homocysteine is recycled to replenish SAM
levels. Because 3 moles of SAH are produced via the
SAM cycle for each mole of caffeine that is
synthesized, this pathway has the capacity to be the
sole source of both the purine skeleton and the
methyl groups required for caffeine biosynthesis in
young tea leaves
25
.
Biosynthesis of purine alkaloids
Origin of the purine ring of caffeine
Caffeine is a trimethylxanthine whose xanthine
skeleton is derived from purine nucleotides that are
converted to xanthosine, the first committed
intermediate in the caffeine biosynthesis pathway.
There are at least four routes from purine
nucleotides to xanthosine (Fig. 4). The available
evidence indicates that the most important routes
are the production of xanthosine from inosine
5′ -monophosphate, derived from
de novo
purine
nucleotide biosynthesis, and the pathway in
which adenosine, released from
S
-adenosyl-
L
-
homocysteine (SAH), is converted to xanthosine
via adenine, adenosine 5′ -monophosphate,
inosine 5
Purine ring methylation
Xanthosine is the initial purine compound in the
caffeine biosynthesis pathway, acting as a substrate
for the methyl group donated by SAM. Tracer
experiments with labelled precursors and leaf discs
from tea and coffee plants have shown that the major
route to caffeine is xanthosine
7-methylxanthosine
7-methylxanthine → theobromine → caffeine,
although alternative minor routes might also
operate
26
. However, as well as entering the caffeine
biosynthesis pathway, xanthosine is also converted to
xanthine, which is degraded to CO
2
and NH
3
via the
purine catabolism pathway
27,28
(Fig. 5).
The first methylation step in the caffeine
biosynthesis pathway, the conversion of xanthosine
-monophosphate and xanthosine
5′ -monophosphate
13,24,25
.
Recently published data indicate that the
conversion of SAH to xanthosine is such that the
Fig. 2.
Commercial tea
plantation in Kenya
(photograph courtesy of
David Werndly, Unilever
Research, Colworth, UK).
Fig. 3.
Ripening beans of Coffea arabica (photograph courtesy of the
All Japan Coffee Association, Tokyo, Japan).
3
Opinion
TRENDSin Plant Science Vol.6 No.9 September 2001
409
NH
3
+
ATP
Tetrahydrofolate
CHCH
2
CH
2
S
CH
3
P P
i
+
P
i
COO
–
5-Methyl
tetrahydrofolate
Methionine
(1)
NH
2
N
(4)
HN
NH
3
+
CH
3
N
N
CHCH
2
CH
2
S
+
CH
2
O
COO
–
OH
OH
NH
3
+
S-adenosyl-
L
-methionine
CHCH
2
CH
2
SH
SAM cycle
Guanylate pool
COO
–
Homocysteine
-
monophosphate
Guanosine-5
NH
2
P
i
N
(2)
(3)
HN
Guanosine
NH
2
NH
3
+
N
N
HN
N
CHCH
2
CH
2
S
CH
2
O
Ribose
N
N
COO
–
CH
3
+
O
HOH
2
C
OH
OH
N
O
S-adenosyl-
L
-homocysteine
HN
O
N
H
N
Caffeine biosynthesis
7-Methylxanthosine
7-Methylxanthine
Theobromine
Caffeine
OH
OH
HOH
2
C
O
(2,11)
Adenosine
OH
OH
Xanthosine
ATP
(5)
(10)
P
i
ADP
Ribose
(7)
NH
2
O
O
NH
3
NH
2
PRPP
P P
i
N
N
NAD
+
NADH
N
N
HN
HN
N
N
N
N
N
(6)
N
(8)
N
(9)
O
N
H
N
H
N
P
OH
2
C
P
OH
2
C
P
OH
2
C
O
O
O
Adenine
OH
OH
OH
OH
OH
OH
Adenosine
Inosine
Xanthosine
5
′
-monophosphate
5
-monophosphate
5
-monophosphate
Adenylate pool
De novo
purine synthesis
TRENDS in Plant Science
Fig. 4.
Proposed new major pathway for the biosynthesis of purine alkaloids in which adenosine derived from the S-adenosyl-
L
-methione (SAM)
cycle is metabolized to xanthosine, which is converted to caffeine by a route that involves three SAM-dependent methylation steps. In addition,
xanthosine is synthesized from inosine 5
-triphosphate; NAD
+
,
nicotinamide adenine dinucleotide; NADH, reduced NAD; PRPP, 5-phosphoribosyl-1-diphosphate. Enzymes: (1) SAM synthetase; (2) SAM-
dependent N-methyltransferases; (3) S-adenosyl-
L
-homocysteine hydrolase; (4) methionine synthase; (5) adenosine nucleosidase; (6) adenine
phosphoribosyltransferase; (7) adenosine kinase; (8) adenine 5
-diphosphate; ATP, adenosine 5
-monophosphate deaminase; (9) inosine 5
-monophosphate dehydrogenase;
(10) 5
-nucleotidase; (11) 7-methylxanthosine nucleosidase.
-monophosphate produced by de novopurine synthesis. Small amounts of xanthosine might also be
derived from the guanylate and adenylate pools. Abbreviations: ADP, adenosine 5
 410
Opinion
TRENDSin Plant Science Vol.6 No.9 September 2001
O
H
N
Ribose
O
labile, therefore achieving even partial purification
has proved a difficult task
29
. However, the purification
of MXS from coffee leaves has been achieved
17,18
. The
pH optimum of the purified enzyme was 7.0 and the
K
m
values for xanthosine and SAM were 22 µ
HN
HN
N
Xanthosine
O
N
H
N
NSD
O
N
H
N
M
and
Xanthine
HOH
2
C
O
M
, respectively. The next enzyme, methylxanthine
nucleosidase, which has been partially purified
from tea leaves, catalyses the hydrolysis of
7-methylxanthosine to 7-methylxanthine
30
.
The activities of the
N-
methyltransferases that
catalyse the conversions of 7-methylxanthine to
theobromine and theobromine to caffeine were first
shown in crude extracts of tea leaves by Takeo Suzuki
and Ei-ichi Takahashi, 26 years ago
31
. However, like
MXS, the activity is extremely labile and it was not
until 1999 that an enzyme from young tea leaves
was purified to apparent homogeneity
32
. This
N
-methyltransferase, caffeine synthase (CS), is
monomeric, has an apparent molecular mass of
41 kDa and displays a sharp pH optimum of 8.5. It
exhibits
N
-3- and
N
-1
-
methyltransferase activities,
and a broad substrate specificity, showing high
activity with paraxanthine, 7-methylxanthine and
theobromine, and low activity with 3-methylxanthine
and 1-methylxanthine (Table 1). Furthermore, the
enzyme has no MXS activity towards either
xanthosine or xanthosine-5′ -monophosphate
32
. The
V
ma
÷
XDH
Uric acid
OH
OH
Purine
catabolism
pathway
Caffeine
biosynthesis
pathway
Allantoin
SAM
MXS
Allantoic acid
SAH
O
CH
3
+
N
HN
CO
2
+ NH
3
7-Methylxanthosine
O
N
H
N
HOH
2
C
O
OH
OH
MXN
H
2
O
Ribose
O
CH
3
HN
N
O
N
H
N
7-Methylxanthine
K
m
value of tea CS is highest for paraxanthine
and so paraxanthine is the best substrate for CS
(Ref. 32). However, there is limited synthesis of
endogenous paraxanthine from 7-methylxanthine
and therefore,
in vivo
, paraxanthine is not an
important methyl acceptor
28
.
The effects of the concentration of SAM and several
methyl acceptors on the activity of CS show typical
Michaelis–Menten kinetics, and there is no feedback
inhibition by caffeine. It is therefore unlikely that
allosteric control of the CS activity is operating in tea
leaves. One of the major factors affecting the activity of
CS
in vitro
appears to be a product inhibition by SAH.
CS is inhibited completely by low concentrations of
SAH. Therefore, control of the intracellular SAM:SAH
ratio is one possible mechanism for regulating the
activity of CS
in vivo.
CS is a chloroplast enzyme but
CS activity is not affected by light
in situ
and caffeine
is synthesized in the darkness
33
.
SAM
CS
SAH
O
CH
3
HN
N
O
N
N
Theobromine
CH
3
SAM
CS
SAH
O
CH
3
H
3
C
N
N
Caffeine
O
N
N
CH
3
TRENDS in Plant Science
Cloning of caffeine synthase and related genes
Using 3′ rapid amplification of cDNA ends with
degenerate gene-specific primers based on the
N
-terminal residues of purified tea CS, a 1.31 kb
sequence of cDNA has been obtained
14
. The 5
Fig. 5.
Biosynthesis of caffeine from xanthosine and the conversion of xanthosine to xanthine and
its breakdown to CO
2
and NH
3
via the purine catabolism pathway. Abbreviations: CS, caffeine
synthase; MXS, methylxanthosine synthase; MXN, methylxanthosine nucleotidase; NSD,
inosine–guanosine nucleosidase; SAH, S-adenosyl-
L
-homocysteine; SAM, S-adenosyl-
L
-methione;
XDH, xanthine dehydrogenase.
′
untranslated sequence of the cDNA fragment was
isolated by 5
rapid amplification of cDNA ends. The
total length of the isolated cDNA, termed
TCS1
(GenBank Accession No. AB031280), is 1438 bp and it
encodes a protein of 369 amino acids. The deduced
amino acid sequence of TCS1 shows low homology
with other
N
-,
S-
and
O
-methyltransferases from
plants and microorganisms, with the exception of
to 7-methylxanthosine, is catalysed by an
N
-methyltransferase, 7-methylxanthosine synthase
(MXS). MXS has been extracted from tea and coffee
leaves, and exhibits high substrate specificity for
xanthosine as the methyl acceptor and for SAM as
the methyl donor. It has low activity and is extremely
15
Opinion
TRENDSin Plant Science Vol.6 No.9 September 2001
411
Table 1. Substrate specificity of native and recombinant N-methyltransferases from tea and coffee
a
Source
Substrate (methylation position)
7-mX (N-3) 3-mX (N-1) 1-mX (N-3) Tb (N-1)
Tp (N-7)
Px (N-3)
X (N-3)
XR
Refs
Tea leaves (native)
100
17.6
4.2
26.8
TR
210.0
TR
ND
32
Tea leaf TCS1 (recombinant)
100
1.0
12.3
26.8
TR
230.0
*
ND
14
Coffee leaf CTS1 (recombinant)
100
ND
ND
ND
ND
1.4
ND
ND
15
Coffee leaf CTS2 (recombinant)
100
ND
ND
ND
ND
1.1
ND
ND
15
Coffee leaf CaMXMT (recombinant)
100
ND
ND
ND
ND
15.0
ND
ND
16
a
Enzyme activities of each source are presented as a percentage of the activity when 7-mX is used as the substrate.
Abbreviations: 1-mX, 1-methylxanthine; 3-mX, 3-methylxanthine; 7-mX, 7-methylxanthine; *, not determined; ND, not detected; Px, paraxanthine; Tb, theobromine;
Tp, theophylline; TR, trace; X, xanthine; XR, xanthosine.
salicylic acid
O
-methyltransferase
34
, with which it
shares 41.2% sequence homology. To determine
whether the isolated cDNA encoded an active CS
protein,
TCS1
was expressed in
E. coli
and lysates of
the bacterial cells were incubated with a variety of
xanthine substrates in the presence of SAM, which
served as a methyl donor. The substrate specificity of
the recombinant enzyme was similar to that of
purified CS from young tea leaves (Table 1). The
recombinant enzyme mainly catalysed
N
-1- and
N
-3-methylation of mono- and dimethylxanthines. No
7-
N
-methylation activity was observed when xanthosine
was used as the methyl acceptor. These results provide
convincing evidence that
TCS1
encodes CS.
Recently, four
CS
genes from young coffee leaves
have been cloned
15
. The predicted amino acid
sequences of these genes showed ~40% homology
with that of TCS1. Two of the coffee genes,
CTS1
and
CTS2
, were expressed in
E. coli.
The substrate
specificity of the recombinant coffee enzymes was
much more restricted than that of recombinant tea
CS because they used only 7-methylxanthine as a
methyl acceptor, converting it to theobromine
(Table
specificity of the recombinant enzyme or about
whether the conversion of xanthosine to
7-methylxanthosine is blocked in transgenic
antisense coffee plants, it cannot yet be concluded
that the cloned gene encodes MXS.
Therefore, coffee
N
-3
-
methyltransferases
are referred to as theobromine synthases.
Independently, another laboratory has cloned similar
genes from coffee leaves
16
. Upon expression in
E. coli
,
one of the genes,
CaMXMT
, was found to encode a
protein possessing
N
-3-methylation activity. The
N
-terminal sequence of CaMXMT shows similarities
(35.8%) to that of tea CS and also shares 34.1%
homology with salicylic acid
O
-methyltransferase.
Catabolism of caffeine
In tea and coffee plants, caffeine is mainly produced
in young leaves and immature fruits, and it continues
to accumulate gradually during the maturation of
these organs. However, it is slowly catabolized by the
removal of the three methyl groups, resulting in the
formation of xanthine (Fig. 6)
35
. Several demethylases
seem to participate in these sequential reactions but
no such enzyme activity has been isolated to date from
higher plants. Xanthine is further degraded by the
conventional purine catabolism pathway to CO
2
and
NH
3
via uric acid, allantoin and allantoate (Fig. 6).
Exogenously supplied [8-
14
C]theophylline is degraded
to CO
2
far more rapidly than [8-
14
C]caffeine, indicating
that the initial step in the caffeine catabolism
pathway, the conversion of caffeine to theophylline,
is the major rate-limiting step. This is not the case in
the low-caffeine-containing leaves of
C. eugenioides
,
which, unlike
C. arabica
, metabolize [8-
14
C]caffeine
rapidly, with much of the label being incorporated into
CO
2
within 24 h (Ref. 36).
C. eugenioides
therefore
appears to have far higher levels of
N
-7-demethylase
activity than
C. arabica
, and thus can efficiently
convert endogenous caffeine to theophylline, which is
rapidly metabolized further.
Several species of caffeine-degrading bacteria
have been isolated, including
Pseudomonas cepacia
,
Pseudomonas putida
and
Serratia marcescens
.
Bacterial degradation is different from that
operating in higher plants because it appears to
involve a caffeine →
Cloning of the MXSgene
The coffee
MXS
gene, which participates in the first
methylation step of the caffeine biosynthesis pathway,
has been cloned
17,18
. The cDNA encoded a protein of
371 amino acids that does not exhibit significant
homology to other known proteins, including CS.
C. arabica
callus independently transformed with
antisense
MXS
secreted caffeine into the incubation
medium in amounts ranging from that produced by
untransformed callus to ~2% of the normal levels
18
.
This indicates that the antisense cDNA can inhibit
caffeine production in coffee callus. However, in the
absence of information, either about the substrate
theobromine →
NH
3
pathway
(Fig. 6). Bacterial
N
-1-demethylase activity
catalysing the metabolism of caffeine to theobromine
has been isolated from
Pseudomonas putida
37
.
xanthine
Future perspectives: biotechnology of caffeine
Genes encoding CS and other
N
-1 and
N
-3
methyltransferases have been cloned. This
development opens up the possibility of using genetic
7-methylxanthine
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