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7-Methylxanthine Methyltransferase of Coffee Plants: Gene Isolation and Enzymatic
Properties
Mikihiro Ogawa, Yuka Herai, Nozomu Koizumi, Tomonobu Kusano and Hiroshi Sano*
Research and Education Center for Genetic Information, Nara Institute of Science and
Technology, Nara 630-0101, Japan
The nucleotide sequences reported in this paper have been submitted to the
GenBank/EMBL/DDBJ with accession numbers AB039725 (CaMTL1), AB048792
(CaMTL2), AB048793 (CaMTL3) and AB048794 (CaMXMT).
* To whom correspondence should be addressed: phone 81-743-72-5650; fax 81-743-72
-5659; e-mail sano@bs.aist-nara.ac.jp
The abbreviations used are: AdoMet, S-adenosyl-L-methionine; CS, caffeine synthase;
GFP, green fluorescent protein; 7mX, 7-methylxanthine
Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on December 6, 2000 as Manuscript M009480200 by guest on A
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(Abstract)
Caffeine is synthesized through sequential three-step methylation of xanthine
derivatives at positions 7-N, 3-N and 1-N. However, controversy exists as to the number
and properties of the methyltransferases involved. Using primers designed on the basis
of conserved amino acid regions of tea caffeine synthase and Arabidopsis hypothetical
proteins, a particular DNA fragment was amplified from a mRNA population of coffee
plants. Subsequently, this fragment was used as a probe, and four independent clones
were isolated from a cDNA library derived from coffee young leaves. Upon expression
in E. coli, one of them was found to encode a protein possessing 7-methylxanthine
methyltransferase activity, and designated as CaMXMT. It consists of 378 amino acids
with a relative molecular mass of 42.7 kDa, and shows similarity to tea caffeine
synthase (35.8%) and salicylic acid methyltransferase (34.1%). The bacterially
expressed protein exhibited an optimal pH for activity ranging between 7 and 9, and
methylated almost exclusively 7-methylxanthine with low activity towards paraxanthine,
indicating a strict substrate specificity regarding the 3-N position of the purine-ring. Km
values were estimated to be 50 µM and 12 µM for 7-methylxanthine and S-adenosyl-L-
methionine, respectively. Transcripts of CaMXMT could be shown to accumulate in
young leaves and stems containing buds and GFP-fusion protein assays indicated
localization in cytoplasmic fractions. The results suggest that, in coffee plants, caffeine
is synthesized through three independent methylation steps from xanthosine, in which
CaMXMT catalyzes the second step to produce theobromine.
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(Introduction)
Among more than 50,000 secondary metabolites of plants, 12,000 are alkaloids. Their
physiological roles are considered to be chemical defense against invertebrate
herbivores. Caffeine, a typical purine alkaloid, is found in seeds and leaves of coffee
(Coffea arabica), cola (Cola nitida), maté (Ilex paraguariensis) and tea (Camellia
sinensis) at concentrations up to 1 mg per 1g dry weight (1, 2). It exhibits a lethal effect
on tobacco horn worm (Manduca sexta) by inhibiting phosphodiesterase activity which
hydrolyzes cAMP (3).
The biosynthetic pathway of caffeine has been intensively studied, and it is now
established that it is successively synthesized from adenine nucleotides through multiple
steps catalyzed by several enzymes (4-6). The final series of steps involve methylation
of xanthosine by N-methyltransferase, yielding 7-methylxanthosine, whose ribose
residue is removed by 7-methylxanthosine nucleosidase. The resulting 7-
methylxanthine (7mX) is methylated at the 3-N position by N-methyltransferase,
producing 3,7-dimethylxanthine (theobromine), which is again methylated at the 1-N
position to give 1,3,7-trimethylxanthine (caffeine) (Fig. 1). All reactions require S-
adenosyl-L-methionine (AdoMet) as a methyl donor. Some bypass pathways, for
example featuring paraxanthine, have also been suggested, but in coffee and tea plants,
it was confirmed that the major pathway is through theobromine (5, 6).
At least three N-methyltransferases are considered to contribute to this pathway;
these catalyze methylation of xanthosine (the first), methylation of 7mX (the second)
and methylation of theobromine (the third). Their isolation and characterization have
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attracted a good deal of attention and enzymes catalyzing the second and the third steps
were first identified in crude extract of tea leaves (7). Since then a dozen surveys
describing their purification and characterization in coffee and tea plants have been
published (2, 8-13). However, it was found that the enzymes are extremely labile,
making it difficult even to distinguish each activity. Indeed, it is not clear yet whether
the activities are catalyzed by independent or multifunctional proteins (2, 12). Despite
such difficulties, a caffeine synthase (CS) was recently isolated successfully from tea
leaves (14). The enzyme has a native molecular mass of 61 kDa and exhibits 3- and 1-
N-methyltransferase activities towards substrates such as 7mX, theobromine and
paraxanthine (14). It was thus concluded that, at least in tea leaves, a single enzyme has
dual functions in caffeine synthesis. Subsequently, the gene encoding this CS was
isolated (TCS1), and the predicted amino acid sequence was found to show considerable
similarity with salicylic acid O-methyltransferase (15). Whether or not a similar
enzyme(s) functions in coffee plants has not been hitherto determined. Although a
coffee gene encoding xanthosine methyltransferase (XMT), was reported in a patent
(16), the details remain to be clarified.
In this communication, we document isolation of a gene encoding an enzyme that
catalyzes methylation of 7mX from coffee plants. In contrast to tea CS, the enzyme
features strict substrate specificity towards methylation only at the 3-N position of the
purine ring. It is suggested that, in coffee plants, caffeine synthesis is mediated by three
methylation steps catalyzed by distinct enzymes, including the presently-identified 7mX
methyltransferase.
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EXPERIMENTAL PROCEDURES
Plant Materials
Coffee plants (Coffea arabica L. var. caturra) were cultivated in a greenhouse.
Preparation of the probe for isolating caffeine synthase cDNA
Two degenerated oligonucleotides, 5’-GGITGYDSIDSIGGICCIAAYAC-3’ (forward)
and 5’-ARIYKIYYRTRRAAISWICCIGG-3’ (reverse) which correspond to the amino
acid sequences of GC(A/S)(A/S)GPNT and PGSF(H/Y)(G/K)(R/N)LF, respectively,
were synthesized based on conserved regions among TCS1 (15, accession number
AB031280) and two Arabidopsis hypothetical proteins (Z99708 and AC008153). PCR
was performed in a 25 µl of reaction mixture containing C. arabica cDNA and the pair
of primers mentioned above under the conditions of 94°C for 1 min, 30 cycles of
denaturation at 94°C for 30 sec, annealing at 52°C for 30 sec and extension at 72°C for
1 min, followed by a final extension at 72°C for 7 min. A 255 bp fragment was
amplified and the one of the deduced amino acid sequences from its DNA sequence
showed 34 % identity to that of TCS1. This fragment was used to screen the C. arabica
cDNA library.
cDNA Library Construction
Total RNA was extracted by the CTAB method (17) with a slight modification and
poly(A+) RNA was purified using an mRNA purification kit (Pharmacia) according to
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the manufacturer’s instructions and converted into double-stranded cDNA using a
ZAPII cDNA synthesis kit (Stratagene). The cDNA was ligated with Uni-ZAP XR
vector arms and packaged using a Gigapack III kit. The titre of the library was 3 x 107
plaque-forming units.
Production of GST fusion proteins
The open-reading frame (ORF) regions of clones 1, 6, 35 and 45 sandwiched with SmaI
and NotI restriction sites were subcloned into the pGEX 4T-2 vector (Pharmacia)and
Escherichia coli JM109 cells were transformed with the resulting plasmids. When the
A600 of the E. coli cell culture reached 0.5, 1 mM of isopropyl-1-thio-β-galactoside
(IPTG) was added for production of glutathione S-transferase (GST) fusion proteins
followed by further incubated at 18 ˚C for 6 h. The bacterial cells were collected by
centrifugation, resuspended in a sonication buffer and disrupted by a sonicator. Fusion
proteins were purified from the clear lysates as described earlier (18).
Measurement of N-methyltransferase activity
The enzyme activity was determined by an established procedure (14) with a slight
modification. The reaction mixture of 100 µl containing 100 mM Tris-HCl (pH 8.3),
200 µM substrate, 4 µM S-adenosyl-L-[methyl-14C] methionine (2.15 GBq/mmol,
Amersham), 200 µM MgCl2 and 200 ng purified recombinant protein was incubated at
27°C for 2h. The reaction was terminated by addition of 1 ml chloroform and the
organic phase was recovered, dried at 60°C and dissolved in 10 µl of 50% methanol.
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This fraction was separated by a thin-layer (Silica gel 60 F254, Merck) chromatography
with a solution of H2O: acetic acid: n-butanol (2:1:4, v/v/v). Radioactive images were
detected with an image analyzer (Fuji BAS2000).
Stoichiometric analysis
Data from at least 3 replicate experiments in each case were pooled and analyzed by
non-linear least squares regression fitting to the Hill equation (Eqn 1) with the Anemona
program (19).
v = Vmax [S]h / (K+ [S]h) (Eqn 1)
Where v is the rate of reaction (rate of product formation), Vmax is maximum rate, K is
the rate constant, [S] is the substrate concentration, and h is the Hill number.
High performance liquid chromatography (HPLC)
A reaction mixture of 100 µl containing 100 mM Tris-HCl (pH 7.5), 200 µM substrate,
50 µM AdoMet, 200 µM MgCl2 and 200 ng purified GST fusion protein was incubated
at 27°C for 2 h and extracted with 1 ml chloroform. The chloroform phase was dried,
resolved in 12% acetonitrile and separated by HPLC using a column (Shodex Rspak
DS-613, Showa Denko) with a flow rate of 1 ml/min of 12% acetonitrile, then
monitored for absorbance at 254 nm.
Reverse Transcription(RT)-PCR
Total RNAs were isolated from various C. arabica tissues and reverse-transcribed by
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SuperScript II (Gibco BRL). The first-strand cDNAs were used as a template for RT-
PCR analysis, performed as follows; 96°C for 20 sec, 30 cycles of [96°C for 20 sec,
60°C (55°C in case of XMT) for 30 sec, 72°C for 30 sec], followed by further extension
at 72°C for 7 min. The primers used were CaMXMT-Fw (5’-
CCAGTAAGATCCCATGAACAAAT-3’), CaMXMT-RV (5’-
TTATTACGAATACAAAACGACAATACC-3’), XMT-Fw (5’-
AGCACATTCGGACTCTCCAG-3’), XMT-RV (5’-
TACCGAGTTAAGCGATGCAC-3’), CaMTL1/2-Fw (5’-
CCATTCCCCAGAATACAGCG-3’), CaMTL1/2-RV (5’-
CCCCGTATCAGAAAACAAACC-3’), CaMTL3-Fw (5’-
GGCTTCTCTATTGACGATGAACATAT-3’) and CaMTL3-RV (5’-
CACTTATTCCTTTCCCCAACAC-3’),
Construction of GFP-Fusion Plasmid and Fluorescence Microscopy
The CaMXMT-entire coding region fragments sandwiched with XbaI and KpnI sites
were subcloned into pGFP2 (provided by Drs. Chua and Spielhofer), resulting in
pCaMXMT::GFP. Thin sections of onion bulbs cut into 9 cm2 squares were biolistically
bombarded as described (Hara et al. 1999), with gold particles (Bio-Rad) coated with
the plasmids pGFP2, pCaMXMT::GFP. After bombardment, they were incubated for 12
h at 25 ˚C in darkness and then viewed using epifluorescence microscopy (20).
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Chemicals
All chemicals were purchased from Sigma unless otherwise described. S-Adenosyl-L-
[methyl-14C] methionine (2.15 GBq/mmol) was purchased from Amersham.
RESULTS
Isolation of candidate cDNA clones encoding 7mX methyltransferase
In order to isolate genes for caffeine synthase of coffee plants, a 255 bp fragment
amplified by PCR with degenerated primers was used for screening a phage library. A
total of 35 randomly selected plaques hybridized to the probe were converted into
phagemids. They were classified into 4 groups by physical mapping and by partial DNA
sequencing. The longest cDNAs of each group, clones 1, 6, 35 and 45 were selected,
who their DNA sequences determined, and the deduced products aligned (Fig. 2).
Pairwise identities between clone 45 product and those of clones 1, 6 and 35 were
80.8%, 81.3% and 84.7%, respectively. Clones 1 and 6 showed 95.8% identity with
each other.
Production of GST-fusion proteins and measurement of N-methyltransferase activity
The GST-fusion proteins of clones 6, 35 and 45 were produced in E. coli, purified on a
glutathione-Sepharose column (Fig. 3A), and N-methyltransferase activity was assayed.
The product of clone 45 catalyzed conversion of 7mX to theobromine and that of
paraxanthine to caffeine (Fig. 3B). Identification of the product as theobromine was
performed by high performance liquid chromatography (Fig. 3C). The protein catalyzes
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methylation either of 7mX or of paraxanthine at the 3-N position, and has a 5-fold
preference for 7mX as opposed to paraxanthine as the substrate (Table 1). Substrate
specificity of the clone 45 product is distinct from that of tea CS, which prefers
paraxanthine to 7mX (14). The cDNA of clone 45 was, therefore, designated as
CaMXMT (C. arabica 7-methylxanthine methyltransferase). The deduced amino acid
sequence showed identity to TCS1 of 35.8%, to salicylic acid methyltransferase
(SAMT) of 34.1%, and to benzoic acid carboxyl methyltransferase (BAMT) of 34.2%
(Fig. 4). Although the products of clones 1, 6 and 35 showed high similarity to
CaMXMT, they had no methyltransferse activity for the substrates tested (data not
shown). These clones were designated as CaMTL1 (C. arabica methyltransferase-like 1,
clone 1), CaMTL2 (clone 6) and CaMTL3 (clone 35), respectively.
Catalytic properties of CaMXMT
The optimal pH for 7mX methyltransferase activity of CaMXMT ranged between 7 and
9, with the peak at 7.5 (Fig. 5A). The effects of 7mX and AdoMet concentrations on the
reaction velocity of GST-CaMXMT protein were determined (Fig. 5B). The Km values
for 7mX and AdoMet were 50 µM and 11.9 µM, respectively, and apparent Vmax values
were estimated to be 7.14 and 7.94 pmol theobromine/min/µg protein upon
measurement with the variable amounts of 7mX and AdoMet, respectively.
Tissue specificity
Accumulation of CaMXMT transcripts was estimated by RT-PCR together with
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CaMTL1, CaMTL2, and CaMT3 in various tissues including roots, stems containing
buds, old leaves and young leaves of C. arabica (Fig. 6A). The level of transcripts for
XMT, which catalyzes the conversion of xanthosine to 7-methylxanthosine, was also
tested. Transcripts of CaMXMT were detected in stems and young leaves, but not in
roots and old leaves, similar to the expression pattern for XMT. Transcripts of CaMTL1
and CaMTL2 were present in all tissues at high levels, whereas CaMTL3 transcripts
were abundant in stems and young leaves and also in roots and old leaves at a lower
level.
Subcellular localization
In order to identify the cellular localization of CaMXMT, the cDNA fragment covering
the entire coding region of CaMXMT was fused to pGFP2, and the resulting plasmid
was introduced into the onion epidermal layer by a biolistic bombardment. Green
fluorescence was detected in the cytoplasm (Fig. 6B).
DISCUSSION
This report documents isolation of a gene encoding 7mX methyltransferase from coffee
plants, and characterization of the bacterially expressed recombinant enzyme. Screening
a coffee cDNA library with the probe constructed from a conserved amino acid region
of TCS1 and similar sequences derived from Arabidopsis EST clones, four distinct
cDNA clones were isolated. The protein encoded by one of them showed 7mX
methyltransferase activity when expressed as a fusion protein with GST in E. coli, and
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was designated as CaMXMT. Proteins encoded by other clones (CaMTL1, 2 and 3) did
not show any methyltransferase activity on substrates examined for CaMXMT. The
deduced amino acid sequence of CaMXMT showed rather low similarity to tea CS
(35.8%), and high similarity to CaMTLs (more than 80%) (Fig. 4B). This result
indicates CaMXMT is not close in an evolutionary sense to TCS1. In other words,
caffeine biosynthetic pathway in coffee and tea might have evolved independently,
consistent with different catalytic properties of the enzymes involved (see below).
CaMXMT also showed low similarity to salicylic acid O-methytransferase
(SAMT) from Clarkia breweri (21), and bezoic acid carboxyl methyltransferase
(BAMT) isolated from snapdragon (Antirrhinum sp.) flowers (22). In addition, we
found several related sequences in the EST of Arabidopsis. Although more than 120
methyltransferases have so far been reported from various organisms (23),
methyltransferases of this type are not well characterized. Their structures appear to be
unique, with little similarities to other methyltransferases, suggesting a new class.
However, it has been pointed out that SAMT contains domains similar to motifs I and
III found in plant O-methyltransferases (21). Those are proposed to be involved in
AdoMet binding, and conserved in SAMT, BAMT, TCS and CaMXMT (Fig.4A), even
though TCS1 and CaMXMT are N-methyltransferases. The motifs are also found in
CaMTLs, making it highly probable that they possess methyltransferase activity,
although they do not participate in caffeine biosynthesis. The major difference in amino
acid sequence between CaMXMT and CaMTLs is Val159-His160-Try161 (VHW),
which is present in TCS1 and CaMXMT, but absent in CaMTLs. It is tempting to
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speculate that substrate specificity of this class is determined by a few particular amino
acids, and further investigations with point mutated proteins are needed to clarify this
point.
Despite the similar pH optimum for activity, the substrate specificities of
CaMXMT and TCS1 are clearly different. Whereas both native and recombinant TCS1
equally show catalytic activity towards the 1-N and 3-N-sites of the purine ring,
CaMXMT catalyzes only 3-N-methylation (Table 1). In a crude extract of coffee fruits,
the capacity of 1-N-methylation of theobromine to caffeine was detected (8), and we
have confirmed this with crude extracts of young leaves (unpublished observation).
Since recombinant CaMXMT did not show any 1-N-methylation activity, it is obvious
that, in coffee plants, 3-N- and 1-N-methylation is catalyzed by different enzymes. This
is consistent with findings that the apparent Km for xanthine derivatives markedly differs
among enzymes. Crude enzymes exhibit Km values for both 7-methylxanthine and
theobromine ranging between 100 µM and 500 µM (13). This is also the case for
purified tea CS, except that it has much higher affinity for paraxanthine with a Km of 24
µM (14). Such differential Km values suggest that, in spite of apparent multi-functional
properties, each enzyme may be able to select their correct substrate. CaMXMT
methylates predominantly 7mX with a Km of 50 µM, a much higher affinity than for any
other enzymes reported. The observations suggest that enzymes involved in caffeine
synthesis may possess rather strict substrate preference, and that this arises from
diversity in a few amino acids.
The transcript accumulation profiles of CaMXMT, XMT and CaMTLs were analyzed
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by RT-PCR with specific primers for each to avoid cross-hybridization between
CaMXMT and CaMTLs. Transcripts of CaMXMT and XMT accumulated in young
leaves and stems containing buds, suggesting that biosynthesis of caffeine occurs
mainly in those tissues in coffee plants. This is consistent with the fact that theobromine
and caffeine are primarily found in their buds and young leaves (5). It should be noted
that the transcript accumulation profile of CaMTL3 is similar to that of CaMXMT and
XMP, suggesting its involvment in the metabolism of caffeine-related compounds.
Examination of the subcellular localization of CaMXMT using the fusion protein of
CaMXMT and GFP demonstrated an existence predominantly in the cytoplasm of onion
epidermal cells. The PSORT program with the deduced amino amino acid sequence also
predicted a high possibility of cytoplasmic localization for CaMXMT (unpublished
observation). It can thus be concluded that caffeine biosynthesis occurs in the cytoplasm
of cells in buds and young leaves.
It is worthy of mention that CaMXMT may have practically applications. To cope
with occasional health problems caused by caffeine, decaffeinated coffee is currently
produced by chemical treatment of coffee beans. Recombinant DNA technology using
CaMXMT may remove the need for this by creating caffeine-less coffee plants.
Furthermore, the opposite approach may also be applicable to important crops in such a
way as to produce caffeine derivatives as insect repellants.
Acknowledgements
The authors wish to thank Drs. H. Ashihara, M. Kato (Ochanomizu University) and T.
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Fujimura (Tsukuba University) for providing the plasmid pTCS1. They also appreciate
to Drs. N.-H. Chua (The Rockefeller University) and P. Spielhofer (Berne University)
for supplying the plasmid pGFP2. We also thank Dr. M. Moore (Intermal) for critical
reading of the manuscript. This work was supported in part by grants from the Japan
Society for Promotion of Science (JSPS-RFTF 1997R16001) and from New Energy and
Industrial Technology Development Organization.
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(18) Ikeda, Y., Koizumi, N., Kusano, T. and Sano, H. (1999) Plant Physiol., 121: 813-
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(19) Hernandez,A., and Ruiz, M.T. (1998) Bioinformatics 14: 227-228.
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Figure legends
Fig. 1 Proposed biosynthetic pathway of caffeine in Coffea plants.
Abbreviations, AMP adenosine monophosphate; GMP guanosine monophosphate;
XMT xanthosine 7-N methyltransferase.
Fig. 2 Amino acid alignment of the products deduced from cDNAs of clone 45, clone 1,
clone 6 and clone 35. Dashes and colons indicate gaps and identical amino acid residues,
respectively.
Fig. 3 Purification of GST-fusion products and their methyltransferase activities
towards xanthosine derivatives. (A) The purified GST fusion proteins derived from the
cDNAs of clones 6, 35 and 45 were separated by SDS-PAGE and stained by Coumassie
Brilliant Blue. (B) Thin-layer chromatographic analysis of the reaction products from
incubation with the recombinant proteins shown in (A). Substrates used were
xanthosine (X), 7-methylxanthine (7mX), theobromine (Tb), paraxanthine (Px) and
theophylline (Tp). Cf and Tb in the right margin indicate the corresponding positions of
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caffeine and theobromine, and the asterisk indicates a chloroform-extractable
contaminant present in radioactive AdoMet. (C) Elution profiles of HPLC using a DE-
613 column. Standard samples were a mixture of 7mX, Tb, Px and caffeine (Cf). The
reaction mixture (see Materials and methods) containing 7mX and the recombinant
protein of clone 45 were incubated for 2h (Reaction) at 27°C. The control was a sample
without incubation (time 0).
Fig. 4 Amino acid alignment of CaMXMT and related methyltransferases from higher
plants. (A) Amino acid alignment. CaMXMT (this paper), TCS1 (15), SAMT
(AAF00108) and BAMT (AAF98284). Conserved residues in three out of four
sequences are squared. Motifs I and III are supposed to be AdoMet-binding sites (20).
(B) Phylogenetic relationships among the enzymes.
Fig. 5 Catalytic properties of CaMXMT. (A) Effects of pH on CaMXMT activity. The
buffer system was 0.1 M MES-NaOH (closed boxes), 0.1 M Tris-HCl (closed circles),
0.1 M Glycine-NaOH (open circles). (B, C) Stoichiometric analyses for 7mX (B) or for
AdoMet (C). The ordinate indicates velocity expressed in nmoles of theobromine
synthesized per nmole protein per min.
Fig. 6 Tissue specificity of CaMXMT expression and the intracellular localization of
CaMXMT. (A) Tissue specific transcript accumulation of CaMXMT, XMT, CaMTL1,
CaMTL 2 and CaMTL3 was analyzed by RT-PCR. (B) Onion bulbs were bombarded
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with gold particles coated with pGFP2 (a, b) and pCaMXMT::GFP (c, d) plasmids. The
proteins were transiently expressed and individual cells are observed by differential
interference contrast imaging (a, c) and corresponding epifluorescence microscopy (b,
d).
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clone45 MELQEVLHMNEGEGDTSYAKNASYN-LALAKVKPFLEQCIRELLRANLPN 49
clone1 ::::::::::G:::EA:::::S:F:Q:V::::::V::::V:::::::::: 50
clone6 ::::::::::G::::A:::::S:F:Q:V::::::V::::VG::::::::: 50
clone35 :::::::R::G::::::::::SA::Q:V::::::V::::V:::::::::: 50
clone45 INKCIKVADLGCASGPNTLLTVRDIVQSIDKVGQEEKNELERPTIQIFLN 99
clone1 ::::::::::::::::::::::W:T:::::::K::M::::::::::V::T 100
clone6 ::::::::::::::::::::::::::::::::R::M::::::::::V::T 100
clone35 :::::::::::::::::::::::::::::::::::K:::::::::::::: 100
clone45 DLFQNDFNSVFKLLPSFYRKLEKENGRKIGSCLISAMPGSFYGRLFPEES 149
clone1 :::::::::::M::::::::::::::::::::::A::::::H:::::::: 150
clone6 :::::::::::M::::::::::::::::::::::A::::::H:::::::: 150
clone35 :::P::::::::::::::::::::::::::::::::::::::S::::::: 150
clone45 MHFLHSCYSVHWLSQVPSGLVIELGIGANKGSIYSSKGCRPPVQKAYLDQ 199
clone1 ::::::S::LQF:::::::::T::::T:::R::::::ASP:::::::::: 200
clone6 ::::::S::LQF:::::::::T::::T:::R::::::ASP:::::::::: 200
clone35 ::::::::CLQ::::::::::T::::ST:::::::::AS:L::::::::: 200
clone45 FTKDFTTFLRIHSKELFSRGRMLLTCICKVDEFDEPNPLDLLDMAINDLI249
clone1 ::::::::::MR:E::L::::::::::::G::C:G::TM:::E::::::V 250
clone6 :::::::::::R:E::L::::::::::::G::::G::TM:::E::::::V 250
clone35 :::::::::::::E::::H::::::::::GE:L:AR:AI:::E::::::V 250
clone45 VEGLLEEEKLDSFNIPFFTPSAEEVKCIVEEEGSCEILYLETFKAHYDAA 299
clone1 A::R:G::::::::V:IY:A:V:::::M::::::F:::::Q:::LR:::G 300
clone6 :::H::::::::::V:IYAA:V::L:::::::::F:::::::::LR:::G 300
clone35 :::H::::::::::L:VYI:::::::::::::::F:::::::::VL:::G 300
clone45 FSIDDDYPVRSH------EQIKAEYVASLIRSVYEPILASHFGEAIMPDL 343
clone1 ::::::CQ::::SPVYSD:HAR:AH:::::::::::::::::::::I::I 350
clone6 ::::::CQ::::SPEYSD:HAR:AH::::L:::::::::N::::::I::I 350
clone35 :::::EH-------------::::::::SV:A::::::::::::::I::I 337
clone45 FHRLAKHAAKVLHMGKGCYNNLIISLAKKPEKSDV 378
clone1 :::F:TN::::IRL:::F::::::::::::::::I 385
clone6 :::F:TN::::IRL:::F::::::::::::::::I 385
clone35 :::F::::::::PL:::F::::::::::::::::: 372
Fig. 2 Ogawa et al.
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Mikihiro Ogawa, Yuka Herai, Nozomu Koizumi, Tomonobu Kusano and Hiroshi Sanoproperties
7-Methylxanthine methyltransferase of coffee plants: gene isolation and enzymatic
published online December 6, 2000J. Biol. Chem.
10.1074/jbc.M009480200Access the most updated version of this article at doi:
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