1

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

pril 7, 2018http://w

ww

.jbc.org/D

ownloaded from

2

(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.

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

3

(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

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

4

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.

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

5

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

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

6

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.

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

7

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

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

8

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).

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

9

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

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

10

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

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

11

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

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

12

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

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

13

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

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

14

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.

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

15

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.

REFERENCES

(1) Roberts, M.F., and Wink, M. (1998) Alkaloids: Biochemistry, Ecology, and Medical

Applications. Plenum Press, New York

(2) Baumann, T.W., Koetz, R., and Morath, P. (1983) Plant Cell Rep. 2: 33-35.

(3) Croteau, R., Kutchan, T.M., and Lews, N.G. (2000) Natural Products. In

Biochemistry and Molecular Biology of Plants, eds. Buchanan, B.B., Gruissem, W., and

Jones, R.L. American Society of Plant Physiologists, Rockville, MD. pp.1250-1318.

(4) Ashihara, H., Kato, M., and Chuang-xing, Y. (1998) J. Plant Res. 111: 599-604.

(5) Ashihara, H., Monteiro, A.M., Gillies, F.M.m and Crozier, A. (1996) Plant Physiol.

111: 747-753.

(6) Ashihara, H., and Crozier, A. (1999) Adv. Bot. Res. 30: 117-205.

(7) Suzuki, T., and Takahashi, E. (1975) Biochem. J. 146: 87-96.

(8) Roberts, M.F., and Waller, G.R. (1979) Phytochem. 18: 451-455.

(9) Negishi, o., Ozawa, T., and Imagawa, H. (1985) Agric. Biol. Chem. 49: 887-890.

(10) Mozzafera, P., Wingsle, G., Olsson, O., and Sandberg, G. (1994) Phytochem. 37:

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

16

1577-1584.

(11) Kato, M., Kanehara, T., Shimizu, H., Suzuki, T., Gillies, F.M., Crozier, A., and

Ashihara, H. (1996) Physiol. Plantarum 98: 629-636.

(12) Mosli Waldhauser, S.S., Gillies, F.M., Crozier, A., and Baumann, T.W. (1997)

Phytochem. 45: 1407-1414.

(13) Mosli Waldhauser, S.S., Kretschmar, J.A., Baumann, T.W. (1997) Phytochem. 44:

853-859.

(14) Kato, M., Mizuno, K., Fujimura, T., Iwama, M., Irie, M., Crozier, A., and Ashihara,

H. (1999) Plant Physiol. 120: 579-586.

(15) Kato, M., Mizuno, K., Crozier, A., Fujimura, T., and Ashihara, H. (2000) Nature

406: 956-957.

(16) Stiles, J. I. and Moisyadi, I. (1997) Patent, International publication number WO

97/35960.

(17) Hatanaka, T., Choi, Y.E., Kusano, T., and Sano, H. (1999) Plant Cell Rep.19: 106-

110.

(18) Ikeda, Y., Koizumi, N., Kusano, T. and Sano, H. (1999) Plant Physiol., 121: 813-

820.

(19) Hernandez,A., and Ruiz, M.T. (1998) Bioinformatics 14: 227-228.

(20) Hara, K., Yagi, M., Kusano, T. and Sano, H. (2000) Mol. Gen. Genet., 263: 30-37.

(21) Ross, J.R., Nam, K.H., D’Auria, J.C., and Pichersky, E. (1999) Arch. Biochem.

Biophys. 367: 9-16.

(22) Dudareva, N., Murfitt, L.M., Mann, C.J., Gorestein, N., Kolosava, N., Kish, C.M.,

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

17

Bonham, C., and Wood, K. (2000) Plant Cell 12: 949-961.

(23) Cheng, X., and Blumenthal, R.M. (1999) S-Adenosylmethionine-Dependent

Methyltransferases: Structure and Functions. World Scientific, Singapore.

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

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

18

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

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

19

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).

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

7

-

m

X 3-

m

X 1-

m

X T

b T

p P

x X X

R X

M

P

S

u

b

s

t

r

a

t

eM

a

t

e

r

i

a

l R

e

f

e

r

e

n

c

e

s

N

a

t

i

v

e

T

C

S

1

(

t

e

a

)1

0

0 1

7

.

6 4

.

2 2

6

.

8 t r 2

1

0 t

r n

d n

d (

14

)

C

r

u

d

e

e

n

z

y

m

e

(

c

o

f

f

e

e

f

r

u

i

t

s

)1

0

0 1

2

75

.

7 4

.

6 1

7

5 n

d n

d (

8

)

R

e

c

o

m

b

i

n

a

n

t

C

a

M

X

M

T

(

c

o

f

f

e

e

)1

0

0 n

d n

d 1

5 n

d T

h

i

s

s

t

u

d

yn

d n

d n

d n

d

T

a

b

l

e

1

S

u

b

s

t

r

a

t

e

s

p

e

c

i

f

i

c

i

t

y

o

f

C

a

M

X

M

T

R

e

c

o

m

b

i

n

a

n

t

T

C

S

1

(

t

e

a

)1

0

0 1

.

0 1

2

.

3 1

8

.

5 <

0

.

1 2

3

0 n

d ( 15)

R

e

l

a

t

i

v

e

e

n

z

y

m

e

a

c

t

i

v

i

t

i

e

s

o

f

C

a

M

X

M

T

,

a

c

r

u

d

e

e

x

t

r

a

c

t

s

f

r

o

m

c

o

f

f

e

e

f

r

u

i

t

s

,

a

n

d

n

a

t

i

v

e

a

n

d

r

e

c

o

m

b

i

n

a

n

t

c

a

f

f

e

i

n

e

s

y

n

t

h

a

s

e

(

T

C

S

1

)

f

r

o

m

t

e

a

w

e

r

e

c

o

m

p

a

r

e

d

.

A

c

t

i

v

i

t

y

o

f

e

a

c

h

t

o

w

a

r

d

s

7

m

X

w

a

s

s

e

t

a

s

1

0

0

a

n

d

t

h

e

i

r

r

e

l

a

t

i

v

e

a

c

t

i

v

i

t

i

e

s

a

r

e

s

h

o

w

n

.

F

o

r

a

b

b

r

e

v

i

a

t

i

o

n

s

,

s

e

e

F

i

g

.

3

l

e

g

e

n

d

;

X

R

,

x

a

n

t

h

i

n

e

r

i

b

o

s

i

d

e

;

X

M

P

,

x

a

n

t

h

o

s

i

n

e

m

o

n

o

p

h

o

s

p

h

a

t

e

;

n

d

,

n

o

t

d

e

t

e

c

t

e

d

;

t

r

,

t

r

a

c

e

.

by guest on April 7, 2018 http://www.jbc.org/ Downloaded from

3

,

7

-

D

i

m

e

t

h

y

l

x

a

n

t

h

i

n

e(

T

h

e

o

b

r

o

m

i

n

e

)1

,

3

,

7

-

T

r

i

m

e

t

h

y

l

x

a

n

t

h

i

n

e(

C

a

f

f

e

i

n

e

)

7

-

M

e

t

h

y

l

x

a

n

t

h

i

n

e7

-

M

e

t

h

y

l

x

a

n

t

h

o

s

i

n

e

X

M

T

X

a

n

t

h

o

s

i

n

e

N

NH

N

NH

O

O

R

i

b

o

s

e

N

N+H

N

NH

O

O

R

i

b

o

s

e

C

H3

N

NH

N

NH

O

O

C

H3

N

NH

N

N

C

H3

O

O

C

H3

N

NN

N

C

H3

O

O

C

H3H3C

N

NN

N

O

O

C

H3H3C

H

P

a

r

a

x

a

n

t

h

i

n

eA

M

P

,

G

M

P

F

i

g

.

1

O

g

a

w

a

e

t

a

l

.

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

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.

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

A B

6

6

9

7

4

5

3

0

1

4

( k

D

a

)

c l o

n

e

6

c l o

n

e

3

5

c l o

n

e

4

5

c

l

o

n

e

6 c

l

o

n

e

3

5 c

l

o

n

e

4

5

T

b

C

f

T

pX 7

m

X

T

b

P

x

T

pX 7

m

X

T

b

P

x

T

pX 7

m

X

T

b

P

x

R e

t

e

n

i

o

n

t

i

m

e

(

m

i

n

)

A

b

s

o

r

b

a

n

c

e

C

0

5

1 0

1 5

2 0

2 5

3 0

C

f

T

b7 m

X

P

x

S

t

a

n

d

a

r

d R

e

a

c

t

i

o

n C

o

n

t

r

o

l

*

F

i

g

.

3

O

g

a

w

a

e

t

a

l

.

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

M - - - - - - E L Q E V L H M N E G E G D T S Y A K N A S Y NM E L A T A G K V N E V L F M N R G E G E S S Y A Q N S S F TM - - - - - - D V R Q V L H M K G G A G E N S Y A M N S F I QM - - - - K V M K K L L C M N I A G D G E T S Y A N N S G L Q

L A L A K V K P F L E Q C I R E L L R A N L P N I N K C I K VQ Q V A S M A Q P A L E N A V E T L F S R D F - H L Q A L N AR Q V I S I T K P I T E A A I T A L Y S G D T - V T T R L A IK V M M S K S L H V L D E T L K D I I G D H V G F P K C F K M

C

a

M

X

M

TT

C

S

1S

A

M

TB

A

M

T

2 5

3 1

2 5

2 7

5 6

6 1

5 5

5 8

C

a

M

X

M

TT

C

S

1S

A

M

TB

A

M

T

A D L G C A S G P N T L L T V R D I V Q S I D K V G Q E E K NA D L G C A A G P N T F A V I S T I K R M M E K K C R E L N -A D L G C S S G P N A L F A V T E L I K T V E E L R K K M G RM D M G C S S G P N A L L V M S G I I N T I E D L Y T E K N I

E L E R P T I Q I F L N D L F Q N D F N S V F K L L P S F Y R- C Q T L E L Q V Y L N D L F G N D F N T L F K G L S S E V I- E N S P E Y Q I F L N D L P G N D F N A I F R S L P I E N D- N E L P E F E V F L N D L P D N D F N N L F K L L S H E - -

K L E K E N G R K I G S C L I S A M P G S F Y G R L F P E E SG N K C - - - - E E V P C Y V M G V P G S F H G R L F P R N S- - - - - - - - V D G V C F I N G V P G S F Y G R L F P R N T- - - - - - - - - N G N C F V Y G L P G S F Y G R L L P K K S

M H F L H S C Y S V H W L S Q V P S G L V I E L G I G A N K GL H L V H S S Y S V H W L T Q A P K G L T S R E G L A L N K GL H F I H S S Y S L MW L S Q V P I G I E - - - - - - S N K GL H F A Y S S Y S I H W L S Q V P E G L E D - - - - - N N R Q

S I Y S S K G C R P P V Q K A Y L D Q F T K D F T T F L R I HK I Y I S K T S P P V V R E A Y L S Q F H E D F T M F L N A RN I Y M A N T C P Q S V L N A Y Y K Q F Q E D H A L F L R C RN I Y M A T E S P P E V Y K A Y A K Q Y E R D F S T F L K L R

8 7

9 1

8 6

8 9

1 1

8

1 2

1

1 1

6

1 1

7

1 4

9

1 4

8

1 3

9

1 3

9

1 8

0

1 7

9

1 6

4

1 6

5

2 1

1

2 1

0

1 9

5

1 9

6

C

a

M

X

M

TT

C

S

1S

A

M

TB

A

M

T

C

a

M

X

M

TT

C

S

1S

A

M

TB

A

M

T

C

a

M

X

M

TT

C

S

1S

A

M

TB

A

M

T

C

a

M

X

M

TT

C

S

1S

A

M

TB

A

M

T

C

a

M

X

M

TT

C

S

1S

A

M

TB

A

M

T

S K E L F S R G R M L L T C I C K V D E F D E P N P - - - - LS Q E V V P N G C M V L I L R G R Q C S D P S D M Q S C F T WA Q E V V P G G R M V L T I L G R R S E D R A S T E C C L I WG E E I V P G G R M V L T F N G R S V E D P S S K D D L A I F

D L L D M A I N D L I V E G L L E E E K L D S F N I P F F T PE L L A M A I A E L V S Q G L I D E D K L D T F N I P S Y F AQ L L A M A L N Q M V S E G L I E E E K M D K F N I P Q Y T PT L L A K T L V D M V A E G L V K M D D L Y S F N I P I Y S P

S A E E V K C I V E E E G S C E I L Y L E T F K A H Y D A A FS L E E V K D I V E R D G S F T I D H I E G F D L D S V E M QS P T E V E A E I L K E G S F L I D H I E A S E I YW S S C TC T R E V E A A I L S E G S F T L D R L E V F R V C W D A S D

S I D D D Y P V R - - - S H E Q I K A E Y V A S L I R S V Y EE N D K W V R G - - - - - - - - - - - E K F T K V V R A F T EK D G D G G G S - - - - - - V E E E G Y N V A R C M R A V A EY T D D D D Q Q D P S I F G K Q R S G K F V A D C V R A I T E

P I L A S H F G E A I M P D L F H R L A K H A A K V L H M G KP I I S N Q F G P E I M D K L Y D K F T H I V V S D L E A K LP L L L D H F G E A I I E D V F H R Y K L L I I E R M S K E KP M L A S H F G S T I M D L L F G K Y A K K I V E H L S V E N

G C Y N N L I I S L A K K P E K S D VP K T T S I I L V L S K I D G - - - -T K F I N V I V S L I R K S D - - - -S S Y F S I V V S L S R R - - - - - -

2 3

8

2 4

1

2 2

6

2 2

7

2 6

9

2 7

2

2 5

7

2 5

8

3 0

0

3 0

3

2 8

8

2 8

9

3 2

8

3 2

3

3 1

3

3 2

0

3 5

9

3 5

4

3 4

4

3 5

1

3 7

8

3 6

9

3 5

9

3 6

4

C

a

M

X

M

TT

C

S

1S

A

M

TB

A

M

T

C

a

M

X

M

TT

C

S

1S

A

M

TB

A

M

T

C

a

M

X

M

TT

C

S

1S

A

M

TB

A

M

T

C

a

M

X

M

TT

C

S

1S

A

M

TB

A

M

T

C

a

M

X

M

TT

C

S

1S

A

M

TB

A

M

T

C

a

M

X

M

TT

C

S

1S

A

M

TB

A

M

T

C

a

M

T

L

1

S

A

M

T

B

A

M

T

C

a

M

T

L

2 C

a

M

T

L

3C

a

M

X

M

T

T

C

S

1

0

.

1

B

A

M

o

t

i

f

I

M

o

t

i

f

I

II

F

i

g

.

4

O

g

a

w

a

e

t

a

l

.

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

p H

R

e

l

a

t

i

v

e

a

c

t

i

v

i

t

y

(

%

)

0

5 0

1 0

0A

v v

B

C

4 5 6 7 8 9 1 0 1

1

0

0 .

2

0 .

4

0 .

6

0 .

8

1 .

0

1 .

2

0 5 0 1

0

0 1

5

0 2

0

0 2

5

0

[ 7

-

m

e

t

h

y

l

x

a

n

t

h

i

n

e

]

(µM

)

00 .

2

0 .

4

0 .

6

0 .

8

1 .

0

1 .

2

1 .

4

1 .

6

1 .

8

0 1 0 2

0 3

0 4

0 5

0

[

A

d

o

M

e

t

]

(µM

)

F

i

g

.

5

O

g

a

w

a

e

t

a

l

.

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Ro

o

t

St

e

m

Ol

d

l

e

a

f

Yo

u

n

g

l

e

a

f

C

a

M

T

L

1

,

2

C

a

M

T

L

3

C

a

M

X

M

T

X

M

T

A

a

b D

c

d

B

F

i

g

.

6

O

g

a

w

a

e

t

a

l

.

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

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:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from