Conversion of nicotinic acid to trigonelline is catalyzed by N-methyltransferase belonged to motif B′ methyltransferase family in Coffea arabica

Article abstract of DOI:10.1016/j.bbrc.2014.09.043

Trigonelline (N-methylnicotinate), a member of the pyridine alkaloids, accumulates in coffee beans along with caffeine. The biosynthetic pathway of trigonelline is not fully elucidated. While it is quite likely that the production of trigonelline from nicotinate is catalyzed by N-methyltransferase, as is caffeine synthase (CS), the enzyme(s) and gene(s) involved in N-methylation have not yet been characterized. It should be noted that, similar to caffeine, trigonelline accumulation is initiated during the development of coffee fruits. Interestingly, the expression profiles for two genes homologous to caffeine synthases were similar to the accumulation profile of trigonelline. We presumed that these two CS-homologous genes encoded trigonelline synthases. These genes were then expressed in Escherichia coli, and the resulting recombinant enzymes that were obtained were characterized. Consequently, using the N-methyltransferase assay with S-adenosyl[methyl-¹⁴C]methionine, it was confirmed that these recombinant enzymes catalyzed the conversion of nicotinate to trigonelline, coffee trigonelline synthases (termed CTgS1 and CTgS2) were highly identical (over 95% identity) to each other. The sequence homology between the CTgSs and coffee CCS1 was 82%. The pH-dependent activity curve of CTgS1 and CTgS2 revealed optimum activity at pH 7.5. Nicotinate was the specific methyl acceptor for CTgSs, and no activity was detected with any other nicotinate derivatives, or with any of the typical substrates of B′-MTs. It was concluded that CTgSs have strict substrate specificity. The K_m values of CTgS1 and CTgS2 were 121 and 184 μM with nicotinic acid as a substrate, and 68 and 120 μM with S-adenosyl-l-methionine as a substrate, respectively.

Full text of DOI:10.1016/j.bbrc.2014.09.043

Biochemical and Biophysical Research Communications 452 (2014) 1060–1066
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Conversion of nicotinic acid to trigonelline is catalyzed
by N-methyltransferase belonged to motif B0
methyltransferase family in Coffea arabica
a
b
a
a
Kouichi Mizuno a, , Masahiro Matsuzaki , Shiho Kanazawa , Tetsuo Tokiwano , Yuko Yoshizawa ,
⇑
Misako Kato b
a Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita 010-0195, Japan
b Graduate School of Humanities and Sciences, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 August 2014
Available online 19 September 2014
Trigonelline (N-methylnicotinate), a member of the pyridine alkaloids, accumulates in coffee beans along
with caffeine. The biosynthetic pathway of trigonelline is not fully elucidated. While it is quite likely that
the production of trigonelline from nicotinate is catalyzed by N-methyltransferase, as is caffeine synthase
(CS), the enzyme(s) and gene(s) involved in N-methylation have not yet been characterized. It should be
noted that, similar to caffeine, trigonelline accumulation is initiated during the development of coffee
fruits. Interestingly, the expression profiles for two genes homologous to caffeine synthases were similar
to the accumulation profile of trigonelline. We presumed that these two CS-homologous genes encoded
trigonelline synthases. These genes were then expressed in Escherichia coli, and the resulting recombinant
enzymes that were obtained were characterized. Consequently, using the N-methyltransferase assay with
S-adenosyl[methyl-14C]methionine, it was confirmed that these recombinant enzymes catalyzed the con-
version of nicotinate to trigonelline, coffee trigonelline synthases (termed CTgS1 and CTgS2) were highly
identical (over 95% identity) to each other. The sequence homology between the CTgSs and coffee CCS1
was 82%. The pH-dependent activity curve of CTgS1 and CTgS2 revealed optimum activity at pH 7.5. Nic-
otinate was the specific methyl acceptor for CTgSs, and no activity was detected with any other nicotinate
derivatives, or with any of the typical substrates of B0-MTs. It was concluded that CTgSs have strict
Keywords:
Coffee
Nicotinic acid
Trigonelline
N-methyltransferase
S-adenosylmethionine
substrate specificity. The Km values of CTgS1 and CTgS2 were 121 and 184
substrate, and 68 and 120 M with S-adenosyl- -methionine as a substrate, respectively.
Ó 2014 Elsevier Inc. All rights reserved.
lM with nicotinic acid as a
l
L
1. Introduction
of replicons during the S-phase of the cell cycle [6]. Additionally,
because it is reported that trigonelline contributes to the elonga-
tion of nerve fibers, it is expected that this reagent will be a useful
compound in the treatment of Alzheimer’s disease [7]. Therefore,
there is now great interest in the use of this classic compound,
trigonelline [8].
Trigonelline, N-methylnicotinic acid, was first isolated from the
seeds of Trigonella foenum-graceum and has been found in many
plant species [1,2]. It is known that coffee plants contain trigonel-
line and, in particular, similar to caffeine, there is a large amount of
trigonelline (approximately 1–2% DW) in the coffee seed [3]. Fol-
lowing salt-stress, alfalfa plants have demonstrated a 5-fold
increase in trigonelline, suggesting that trigonelline may play a
role as an osmoregulator in salt-stressed plants [4]. Evans and Tra-
montano previously reported that trigonelline in pea cotyledons
promoted G2 arrest in root and shoot meristems, with an effective
concentration of trigonelline as low as 10ꢀ7 M [5]. Trigonelline
may also act as a cell cycle regulator by preventing the ligation
Trigonelline is the N-methyl conjugate of pyridine alkaloid,
synthesized from nicotinic acid by S-adenosylL-methionine
(SAM)-dependent nicotinic acid N-methyltransferase (trigonelline
synthase, EC 2.1.1.7) (Fig. 1). The enzyme activity was first detected
in crude extracts from peas [9] and has been partially purified from
cell suspension cultures and leaves of Glycine max [10,11]. Ashihara
and collaborators elucidated the biosynthesis of trigonelline and
the pathways of pyridine salvage [12]. However, little is known
about the molecular studied of N-methyltransferases involved in
trigonelline biosynthesis has not been demonstrated yet. Several
studies have been made on methyltransferases involved in the
⇑
Corresponding author. Fax: +81 18 872 1676.
E-mail address: koumno@akita-pu.ac.jp (K. Mizuno).
http://dx.doi.org/10.1016/j.bbrc.2014.09.043
0006-291X/Ó 2014 Elsevier Inc. All rights reserved.

K. Mizuno et al. / Biochemical and Biophysical Research Communications 452 (2014) 1060–1066
1061
broth containing 0.2 lg/ml ampicillin (LA) with vigorous shaking.
A portion (2 ml) of the bacterial culture was added to 100 ml of
fresh LA, and incubated at 37 °C for 2 h with constant shaking
(100 rpm/min). To produce the recombinant protein, we added
300
ll of isopropyl-b-D-thio-galactopyranoside (final concentra-
tion is 0.3
l
M), and maintained the cells at 25 °C for 8 h. E. coli cells
Fig. 1. Possible pathway for biosynthesis of trigonelline. (1) NAD nucleosidase (EC
3.2.2.5); (2) nicotinamidase (EC 3.5.1.19); nicotinate N-methyltransferase (trigo-
nelline synthase) (EC 2.1.1.7). NAD, Nicotine adenine dinucleotide; SAH, S-adeno-
were harvested by centrifugation at 500g for 5 min and then
washed with 20 mM phosphate buffer pH 7.0 containing 0.1 M
NaCl (PBS). Before the sonication, the cell paste was suspended
in 1 ml of PBS and frozen at ꢀ80 °C. This cell suspension was son-
icated and then centrifuged at 10,000g for 10 min at 4 °C. The
supernatant was applied to a Ni–NTA column (1 ml) previously
equilibrated with PBS containing 10 mM imidazole. After washing
with PBS, proteins were eluted by PBS containing 200 mM imidaz-
ole. This purification procedure was carried out using ÄKTAprime
(GE Healthcare). To remove imidazole, the eluted fractions were
combined and subsequently applied to a NAP™-10 gel-filtration
column (GE Healthcare) that was equilibrated with 20 mM Tris/
HCl pH 7.5, containing 150 mM NaCl (TBS). The purified protein
fractions were then used in the enzyme assay for trigonelline
biosynthesis.
syl-L-homocysteine; SAM, S-adenosyl-L-methionine.
methylation of low molecular weight compounds in planta. Joshi
and Chiang [13] reported the characterization of plant O-methyl-
transferase family which catalyzed the methylation of caffeic acid
and their derivatives. Then, salicylic methyltransferase from Clar-
kia breweri which was the first characterized member of motif B0
methyltransferase family and many motif B0 methyltransferases
were subsequently identified [14–20]. Motif B0 methyltransferase
family have the motif B0 and YFFF region in the amino acid
sequence and were also known as SABATH family [21]. In the past
fifteen years, majority of motif B0 methyltransferases catalyze the
methylation of carboxylic acids, on the other hand, the occurrence
of N-methyltransferase and S-methyltransferase were only demon-
strated in purine alkaloid biosynthesis and sulfur-containing
compounds, respectively [20,22]. The atoms of methyl acceptor
were not seemed to be essential for the methylation reaction by
motif B0 methyltransferases.
2.4. Determination of enzyme activity
The reaction mixture for standard assays contained 100 mM
Tris/HCl pH 7.5, 0.2 mM MgCl2, 0.2 mM SAM, 1.5 mM nicotinic
acid, and 100
ll of enzyme preparation in a total volume of
Here we show the molecular information about N-methyltrans-
ferase specific to trigonelline biosynthesis belonged to motif B0
methyltransferase family in Coffea arabica. The previously cloned
CtCS3 and CtCS4 by our group were identified as trigonelline syn-
thase 1 (CTgS1) and 2 (CTgS2), respectively, by the studies on the
substrate specificity of the recombinant enzymes. This is the first
report to describe the cloning of N-methyltransferase, the key
enzyme for the biosynthesis of pyridine alkaloids from nicotinic
acid metabolism, for trigonelline biosynthesis in plants.
300 l. We analyzed reactants of trigonelline synthase by HPLC
l
that was carried out using an L-2130 pump, an L-2200 autosam-
pler, an L-2300 column oven, and an L-2450 diode array detector,
all from Hitachi (Tokyo, Japan). Separation was carried out on an
Inertsil ODS-3 column (/ 4.6 ꢁ 250 mm, 5
was coupled to cartridge guard column (Inertsil ODS-3,
m particle size) from GL Science Inc. (Tokyo,
lm particle size) that
a
4.0 ꢁ 10 mm, 5
l
Japan), with CH3CN: CH3COOH: H2O = 1: 1: 98 as a solvent at a
total flow rate of 0.5 ml/min. The column temperature was 40 °C
and the injection volume was 20 ll for all analyses performed.
Quantification was carried out using calibration curves obtained
using standard solutions of pyridine derivatives, including trigo-
nelline, at 264 nm. The fractions suspected to contain trigonelline
were collected and concentrated, and the condensed product was
then analyzed with ESI mass spectrometry (Exactive, Thermo
Fisher Scientific, Waltham, MA, USA). Determination of trigonelline
synthase activity was based on the transfer of a 14C-labeled methyl
group from [methyl-14C]SAM to an unlabeled substrate, the methyl
acceptor. To determine the kinetic parameters, the reaction condi-
2. Materials and methods
2.1. Materials
Oligonucleotide primers for PCR mutagenesis were purchased
from Hokkaido System Science Co., Ltd. (http://www.hssnet.co.jp/
). [Methyl-14C]SAM (55 mCi/mmol) was purchased from GE
Healthcare UK Ltd. Taq-polymerase and ExTaq were used for all
PCR-based experiments (TaKaRa, Otsu, Japan). All other reagents
were of the highest purity available.
tions were as described above, except that 50
l
M [methyl-14C]SAM
l. The enzymatic
(1.8 kBq) was used and the total volume was 100
l
activity of trigonelline synthase was determined by thin layer
chromatography (TLC)-based densitometric analysis using ImageJ
software (http://rsb.info.nih.gov/ij/). TLC was performed as previ-
ously described [26], except we used n-butanol/acetic acid/water
(4:1:2, v/v) as the solvent. The Km values were derived from
Lineweaver–Burk plots analyzed with ‘Enzyme Kinetics’ software
(Trinity Software, Campton, NH, USA).
2.2. Construction of expression plasmid
Plasmids for expressing CtCS3 and CtCS4 in Escherichia coli were
constructed in the pET23d vector (Merck KGaA, Darmstadt, Ger-
many) [23–25]. As the pET23d vector carries an optimal C-terminal
His-Tag sequence, the 30-termination sites of CtCS3- and CtCS4-
cDNA were replaced by an XhoI restriction site using polymerase
chain reaction (PCR)-directed mutagenesis. The expression plas-
mids for CtCS3 and CtCS4 carrying the C-terminal His-Tag (named
pET23d-CtCS3 and pET23d-CtCS4, respectively) were then
constructed.
2.5. Analysis of gene expression by semi-quantitative RT-PCR
Total cellular RNAs from C. arabica were extracted using a cetyl-
trimethylammonium bromide (CTAB) solution according to the
method described by Chang et al. [27], with the exception of the
use of 5% 2-mercaptoethanol. For semi-quantitative RT-PCR, total
RNAs extracted from developing fruits, flower buds, and leaves of
coffee trees at several stages were treated with RNase-free DNase
I (TaKaRa). First strand cDNA was synthesized using a 30-RACE core
set and an oligo-dT 3 sites adaptor primer (oligo-dT 3SAP)
2.3. Production of recombinant enzymes
The pET23d-CtCS3 and pET23d-CtCS4 expression plasmids
were introduced into E. coli BL21 (DE3). A single colony of the
transformants was cultured at 37 °C overnight in 3 ml of Luria

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K. Mizuno et al. / Biochemical and Biophysical Research Communications 452 (2014) 1060–1066
(TaKaRa). DNA-free total RNA (5
used for first strand cDNA synthesis with a 20
l
g) from each tissue sample was
L reaction volume.
l) contained 0.1 l of first strand
derivatives (nicotinic acid, methylnicotinate, nicotinamide, N-
methylnicotinamide, nicotinic acid hydrazide and 6-methylnicoti-
nicacid), and three purine/pyrimidine derivatives (uracil,
guanosine and adenine). The recombinant enzyme then only had
methyltransferase activity that produced trigonelline from nico-
tinic acid used as a substrate. As shown in the HPLC profile in
Fig. 3A, the product had the same retention time as trigonelline.
The fractions were collected and concentrated, and then the con-
densed product was applied to mass spectrometer. Since the prod-
uct had the same molecular weight and mass spectrum as
trigonelline, it was determined that the product was trigonelline
(Fig. 3B). It was concluded that, CTgS1 and CTgS2 are trigonelline
synthases that can specifically catalyze N-methylation of nicotinic
acid. The optimum temperature for both CTgS1 and CTgS2 was
25 °C. At this temperature, the optimum pH was 7.5. CTgS1 and
CTgS2 displayed Michaelis–Menten kinetics toward nicotinic acid
l
The PCR reaction mixture (20
l
l
cDNA from the above reaction mixture, 2.5 units of Ex Taq
(TaKaRa), 2 mM MgCl2, 0.25 mM each of deoxyribonucleotide tri-
phosphate and 5 lM of gene-specific primers. The gene-specific
primers used were: 50-CCCATTCCCCAGAATACA-30 and 50-GCTGCAT
TCGTCGCAAA-30 for CTgS1, and 50-ATTCCACAGGTTTGCGAC-30 and
50-ATCCTTTCCCCGTATCAG-30 for CTgS2. A set of specific primers
(50-GCTTTCAACACCTTCTTCAG-30 and 50-GCTGCTCAGGGTGGAA-
GAG-30) for
a-tubulin (accession number AF363630) was used for
the control reaction. For PCR, we used a PTC-200 thermal cycler
(Bio-Rad Laboratories, Inc., CA, USA) with the following program:
94 °C for 1 min, 50 °C for 30 s, and 72 °C for 15 s. The amplification
was done for 25 cycles. The amplicons corresponding to CTgS1,
CTgS2, and tubulin were 139, 167, and 133 bp in length, respec-
tively. The reaction products were visualized by UV light on 1.2%
agarose gels stained with ethidium bromide. The intensity of fluo-
rescence was quantitated with a Macintosh computer using ImageJ
software (http://rsb.info.nih.gov/ij/).
with Km values of 121 and 184
23.4 nmol/min/mg of protein, respectively. The Km values of CTgS1
and CTgS2, with SAM as a substrate were 68 and 120 M, respec-
lM, and Vmax values of 5.20 and
l
tively. The specific activity of recombinant CTgS1 and CTgS2 were
0.43 and 1.3 nkat/mg proteins with nicotinic acid, respectively.
2.6. Analytical procedure
3.3. Gene expression of CTgS1 and CTgS2
Protein concentrations were measured using methods previ-
ously described by Bradford [28]. Nucleotide sequencing was
carried out using an ABI PRISMÒ 3100 genetic analyzer (Life Tech-
nologies Corporation, CA, USA) and was conducted with assistance
from the Life Research Support Center in Akita Prefectural Univer-
sity. Nucleotide and protein sequences were analyzed by computer
using MacVector (MacVector, Inc., NC, USA).
As CTgS1 and CTgS2 are highly homologous with other coffee
caffeine synthases (CCS1, CTS1, CTS2 and CmXRS1) at the nucleotide
level (over 80% identical), the amount of transcripts from these
genes cannot be detected by Northern blot analysis. The expression
of CTgS1 and CTgS2 was evaluated by semi-quantitative RT-PCR
designed for specific amplification and detection of these tran-
scripts (Fig. 4). Although the transcripts of CTgS1 and CTgS2 were
detected in all organs (flower buds, closed leaves, young leaves,
and mature leaves) (Fig. 4A), and in the developing stages of fruits
(DAF 0 to DAF 30) used in these experiments, the gene expression
in DAF 30 and the mature leaves were the strongest for those genes
in the evaluated organs (Fig. 4B). The transcript accumulation of
CTgS1 was maximum in DAF 5 fruits, and then tended to decrease
slowly. On the other hand, CTgS1 in leaves was expressed in mature
leaves rather than in young leaves. The signals for CTgS2 transcripts
were extremely weak in leaves.
3. Results
3.1. Comparison of coffee trigonelline synthases (CTgS1 and CTgS2)
with other motif B0-methyltransferases
CTgS1 (Accession Number AB054842) and CTgS2 (Accession
Number AB054843) consisted of 1373-bp and 1434-bp sequences,
respectively, and encoded 386 amino acid residues. Their amino
acid sequences were highly identical (over 95% identity) to each
other. CTgS1 shared a high degree of sequence identity (82.3%,
80.8%, and 82.9%) with coffee caffeine synthase (CCS1), coffee theo-
bromine synthase (CTS1), and coffee 7-methylxanthosine synthase
(CmXRS1), respectively (Fig. 2A). CTgS2 had the same values of
sequence identity for the specified caffeine synthetic enzymes.
These values indicated that CTgS1 and CTgS2 are highly homolo-
gous to caffeine synthetic enzymes from coffee. On the other hand,
CTgS1 and CTgS2 also shared approximately 40% identity with tea
caffeine synthase, cacao theobromine synthase, C. breweri salicy-
late methyltransferase [14], and Arabidopsis thaliana jasmonate
methyltransferase [17], and they have three conserved regions as
SAM binding motifs. (Fig. 2A). The phylogenetic tree among the
CCS family and those related enzymes, such as B0-MTs, is indicated
in Fig. 2B. Thus, CTgS1 and CTgS2 are highly homologous with caf-
feine synthetic enzymes from coffee, and enzymes of the motif B0
methyltransferase family [22,23] (SABATH family).
4. Discussion
In this study, we successfully isolated and characterized the
trigonelline synthase genes from coffee. The amino acid sequence
for CTgS1 and CTgS2 are similar to the coffee caffeine synthases,
with the sequence identity between CCS1 and CTgS1 being more
than 82%. It was then assumed that the enzymes could accept xan-
thine derivatives as substrates, so we attempted to measure the
enzymatic activity with those compounds. However, no enzymatic
activity could be detected when the xanthine derivatives were
used as substrates.
It was previously speculated that trigonelline is transported to
seeds after it is produced in other tissues [29]. The levels of trigo-
nelline synthases gene expression clearly increased in fruits from 1
to 2 weeks after flowering (Fig. 4B). In the process of coffee bean
maturation, the pulp and peel develop first, and at this time, the
endosperm and embryo that form the bean are not yet sufficiently
developed. This observation suggested that the trigonelline was
transported into the developing endosperm after being produced
in the pulp. However, to prove that trigonelline was transported,
the trigonelline synthase expression levels in the seed, fruit pulp,
and peel 30 days after flowering needed to be examined.
3.2. Catalytic properties of CTgS1 and CTgS2
The molecular mass for both the recombinant CTgS1 and CTgS2
was 43.2 kDa. The recombinant enzymes were assayed with typical
substrates for B0-MTs (salicylic acid, benzoic acid, jasmonic acid,
indoleacetic acid and gibberellic acid), xanthine derivatives for
substrates of caffeine synthases (xanthosine, xanthine, 7-methyl-
xanthine, theobromine, theophylline and paraxanthine), pyridine
Previous studies have reported trigonelline biosynthesis based
on biochemical analysis and characterization of enzymes purified
from plant tissue. Here, we identified and characterized the gene

K. Mizuno et al. / Biochemical and Biophysical Research Communications 452 (2014) 1060–1066
1063
Fig. 2. Amino acid sequence comparison of coffee trigonelline synthases and related methyltransferases. (A) The following amino acid sequences (accession numbers in
parentheses) were subjected to sequence alignment: CTgS1(AB054842), CTgS2(AB054843), CmXRS1(AB034699), CTS1(AB034700), CTS2(AB054841), CCS1(AB086414),
TCS1(AB031280), BTS1(AB096699), and CbSAMT(AF133053). Shaded boxes represent conserved amino acid residues, and dashes represent gaps that have been inserted for
optimal alignment. The proposed SAM-binding motifs (A, B0 and C) and the conserved region, designated as the ‘‘YFFF-region’’, are shown by open boxes. Conserved amino
acids for SAM binding are indicated by black-and white inverted letters. The amino acids involved in substrate binding are indicated by arrowheads. (B) Phylogenetic analysis
of the Motif B0 methyltransferase family. The unrooted tree was created with ClustalW using the neighbour joining method (http://www.genome.jp/tools/clustalw/).
Substrates of the enzymes are indicated in parentheses. Abbreviations of substrates are as follows: XR, xanthosine; 7mX, 7-methylxantine; Tb, theobromine; NA, nicotinic
acid. Sources of the sequences are as follows: AtJAMT(AY008434), Arabidopsis thaliana jasmonic acid methyltransferase; ZmAAMT(NM001195208), Zea mays anthranilic acid
methyltransferase; OsBSMT1(XM467504), Oryza sativa benzoate salicylate methyltransferase; AtBSMT1(BT022049), Arabidopsis thaliana BSMT1; AlBSMT1(AY224596),
Arabidopsis lyrata BSMT1; AmBAMT(AF198492), Antirrhinum majus benzoic acid methyltransferase; OsIAMT1(EU375746), Oryza sativa indole-3-acetic acid methyltrans-
ferase; AtIAMT(AK175586), Arabidopsis thaliana IAMT; AtGAMT1(NM118775), A. thaliana gibberellic acid methyltransferase1; AtGAMT2(NM125013), A. thaliana GAMT2;
NsBSMT(AJ628349), Nicotiana suaveolens BSMT; NgNAMT(GU169286), Nicotiana gossei nicotinic acid carboxyl methyltransferase; AbSAMT(AB049752), Atropa belladonna
salicylic acid methyltransferase; AmSAMT(AF515284), Antirrhinum majus SAMT; PCS1(AB207817), Camellia ptilophylla theobromine synthase1; ICS1(AB056108), Camellia
irrawadiensis theobromine synthase1 and those of the other sequences are indicated in Fig. 2A.

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K. Mizuno et al. / Biochemical and Biophysical Research Communications 452 (2014) 1060–1066
Fig. 3. HPLC and MS analysis of in vitro biosynthesis of trigonelline. (A) Standard samples consisted of a mixture of nicotinic acid (indicated by arrowhead) and trigonelline
(indicated by asterisk). The reaction mixtures contained nicotinic acid, SAM, and the purified recombinant CTgS1 or CTgS2 and were incubated for 16 h at 25 °C. (B) Mass
spectra of recombinant CTgS2 reaction product (upper) and authentic trigonelline (lower). Positive-ion ESI-MS analysis of the product corresponding to trigonelline and
authentic trigonelline (m/z 138).
for trigonelline synthases, and demonstrated that they belong to
the family of coffee caffeine synthases (CCS family). The substrate
specificities of a member of the caffeine synthases in coffee are
shown in Table 1.
Although the amino acid sequences of the CCS family enzymes
were very similar to each other and belong to the same clade in the
phylogenetic tree (Fig. 2B), those CCS family enzymes have respec-
tively different substrate specificity. Thus, it was determined that
Table 1
Substrate specificities of the coffee caffeine synthase family.
CTgS1/CTgS2a
CmXRS1b
CTS1/CTS2b
CCS1b
CCS1b
Substrate
Product
Nicotinic acid
Trigonelline
Xanthosine
7-Methylxanthosine
7-Mehtylxanthine
Theobromine
7-Methylxanthine
Theobromine
Theobromine
Caffeine
a
This study.
Mizuno et al. [23,24].
b

K. Mizuno et al. / Biochemical and Biophysical Research Communications 452 (2014) 1060–1066
1065
acid in C. breweri SAMT (CbSAMT) [18]. Homology models of those
enzymes were built using the experimentally-determined struc-
ture of CbSAMT as the template. Since the Q161-F162 residues in
CTgSs were comparable as the structural equivalent of the CbSAMT
M150-W151 salicylate binding site, it is believed that this region
was involved in the binding with the acceptor molecule of methyl
groups. The Q161-F162 sequence of CTgS1 and CTgS2 is presum-
ably involved in binding with nicotinic acid as a methyl group
acceptor. Subsequently, CTgS1 and CTgS2 have a F354-A355-
T356 sequence, the corresponding amino acid residues of CmXRS1
are FAK, and the corresponding amino acid residues of CTS1 and
CCS1 are I/LAK. The variation of the amino acid residues corre-
sponded to the substrate specificities respectively.
On the other hand, the amino acid sequence identity was very
high (over 95%) between CTgS1 and CTgS2, with only 11 amino
acid residues different from each other. It is assumed that the dif-
ference of the 11 amino acid residues is related to the intensity of
the substrate affinity.
The results of semi-quantitative RT-PCR showed that the
expression levels of CTgS1 and CTgS2 in immature fruits were
maximum approximately 2 weeks after flowering (Fig. 4B). The
expression levels were then maintained in the 30 days after flow-
ering. Because the amounts of transcripts for CTgS1 and CTgS2
were maximized later than that of the genes for caffeine synthase
[23,24], it appears that the synthesis of and the accumulation of
trigonelline developed later than those for caffeine. Since organelle
transportation signal sequences were not shown in the sequences
of CTgSs as were other caffeine synthetic enzymes in coffee, these
enzymes were determined to behave in the cytosol, and the pro-
duced trigonelline and caffeine are presumably transported to
the vacuole with another transportation system. It is known that
ATP-binding cassette (ABC) transporters are involved in the trans-
port of endogenous secondary metabolites in plants. Since it is sus-
pected that the transport of trigonelline and caffeine is conducted
by a similar system, this idea is therefore the proposed focus for
future research.
Fig. 4. Relative abundance of CTgS1 and CTgS2 transcripts in developing fruits. (A)
Semi-quantitative RT-PCR of total RNAs from flower buds (FB), closed leaves (CL),
young leaves (YL) and mature leaves (ML) was performed with 18–30 cycles to
determine the linear range of PCR amplification. The results obtained here with 25
cycles were for all samples. (B) Semi-quantitative RT-PCR of total RNAs from
developing fruits at various time periods, designated as days after flowering (DAF)
was performed with 18–30 cycles to determine the linear range of PCR amplifi-
cation. The results obtained here with 26 cycles were for all samples. Relative
transcript levels were calculated, and the highest values (DAF15) were set as 100%.
References
[1] E. Johns, Ueber die alkaloide des bockshornsamens, Ber. Deut. Chem. Ges. 18
(1885) 2518–2523.
[2] W. Barz, Metabolism and Degradation of Nicotinic acid in Plant Cell Cultures,
Primary and Secondary Metabolism of Plant Cell Cultures, Springer-Verlag,
Berlin, 1985. pp. 186–195.
[3] P. Mazzafera, Trigonelline in coffee, Phytochemistry 30 (1991) 2309–2310.
[4] W.A. Tramontano, D. Jouve, Trigonelline accumulation in salt-stressed legumes
and the role of other osmoregulators as cell cycle control agents,
Phytochemistry 44 (1997) 1037–1040.
[5] L.S. Evans, W.A. Tramontano, Trigonelline and promotion of cell arrest in G2 of
various legumes, Phytochemistry 23 (1984) 1837–1840.
[6] S. Mazzuca, M.B. Bitonti, A.M. Innocenti, D. Francis, Inactivation of DNA
replication origins by the cell cycle regulator, trigonelline, in root meristems of
Lactuca sativa, Planta 211 (2000) 127–132.
[7] C. Tohda, N. Nakamura, K. Komatsu, M. Hattori, Trigonelline-induced neurite
outgrowth in human neuroblastoma SK-N-SH cells, Biol. Pharm. Bull. 22
(1999) 679–682.
some specific amino acid residues of those CCS family enzymes
contribute directly to those substrate specificities. We revealed
that the activity of 3-N methylation for the first step of caffeine
biosynthesis needs a histidine residue at the corresponding posi-
tion 161 in the CmXRS1 sequence [25]. The corresponding amino
acid residues in the sequences of CTgS1 and CTgS2 were glutamine,
the same as those for CCS1 and CTS1. Although the neighboring
amino acid residue is tryptophan in the coffee caffeine synthase
(CmXRS1, CTS1, CTS2, and CCS1) sequences, the two trigonelline
synthases (CTgS1 and CTgS2) were shown to have phenylalanine
residues at the corresponding position (Fig. 2A). We focused our
attention on the correlation between the methylation acceptor
molecule and the two amino acid residues at the corresponding
position of those enzymes. Trigonelline synthases (CTgS1 and
CTgS2) have Q161-F162 residues at the corresponding position,
7-methylxanthosine synthase (CmXRS1) corresponds to Q161-
W162 residues, and theobromine synthases (CTS1 and CTS2) and
caffeine synthase (CCS1) correspond to H160-W161. The
substrates for those enzymes are nicotinate, xanthosine, and 7-
methylxanthine or theobromine, respectively. Meanwhile, it was
determined that the region consisting of 7 amino acid residues
containing those two residues was a binding region for salicylic
[8] P.V. Minorsky, The hot and the classic, Plant Physiol. 128 (2002) 7–8.
[9] J.G. Joshi, P. Handler, Biosynthesis of trigonelline, J. Biol. Chem. 235 (1960)
2981–2983.
[10] B. Upmeier, W. Gross, S. Köster, W. Barz, Purification and properties of S-
adenosyl-
L
-methionine:nicotinic
acid-N-methyltransferase
from
cell
suspension cultures of Glycine max L., Arch. Biochem. Biophys. 262 (1988)
445–454.
[11] X. Chen, A.J. Wood, Purification and characterization of S-adenosyl-L-
methionine nicotinic acid-N-methyltransferase from leaves of Glycine max,
Biol. Plant 48 (2004) 531–535.
[12] H. Ashihara, T. Yokota, A. Crozier, Biosynthesis and catabolism of purine
alkaloids, in: G.-G’h. Nathalie (Ed.), Advances in Botanical Research, vol. 68,
Academic Press, Burlington, 2013, pp. 111–138.
[13] C.P. Joshi, V.L. Chiang, Conserved sequence motifs in plant S-adenosyl-L-
methionine-dependent methyltransferases, Plant Mol. Biol. 37 (1998) 663–
674.
[14] J.R. Ross, K.H. Nam, J.C. D’Auria, E. Pichersky, S-Adenosyl-
L-methionine:
salicylic acid carboxyl methyltransferase, an enzyme involved in floral scent

1066
K. Mizuno et al. / Biochemical and Biophysical Research Communications 452 (2014) 1060–1066
production and plant defense, represents
methyltransferases, Arch. Biochem. Biophys. 367 (1999) 9–16.
a
new class of plant
[22] M. Kato, K. Mizuno, Caffeine synthase and related methyltransferases in
plants, Front. Biosci. 9 (2004) 1833–1842.
[15] M. Kato, K. Mizuno, A. Crozier, T. Fujimura, H. Ashihara, Caffeine synthase gene
from tea leaves, Nature 406 (2000) 956–957.
[16] N. Dudareva, L.M. Murfitt, C.J. Mann, N. Gorenstein, N. Kolosova, C.M. Kish, C.
Bonham, K. Wood, Developmental regulation of methyl benzoate biosynthesis
and emission in snapdragon flowers, Plant Cell 12 (2000) 949–961.
[17] H.S. Seo, J.T. Song, J.J. Cheong, Y.H. Lee, Y.W. Lee, I. Hwang, J.S. Lee, Y.D. Choi,
[23] K. Mizuno, A. Okuda, M. Kato, N. Yoneyama, H. Tanaka, H. Ashihara, T.
Fujimura, Isolation of a new dual-functional caffeine synthase gene encoding
an enzyme for the conversion of 7-methylxanthine to caffeine from coffee
(Coffea arabica L.), FEBS Lett. 534 (2003) 75–81.
[24] K. Mizuno, M. Kato, F. Irino, N. Yoneyama, T. Fujimura, H. Ashihara, The first
committed step reaction of caffeine biosynthesis: 7-methylxanthosine
synthase is closely homologous to caffeine synthases in coffee (Coffea
arabica L.), FEBS Lett. 547 (2003) 56–60.
Jasmonic acid carboxyl methyltransferase:
a key enzyme for jasmonate-
regulated plant responses, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 4788–4793.
[18] C. Zubieta, J.R. Ross, P. Koscheski, Y. Yang, E. Pichersky, J.P. Noel, Structural
basis for substrate recognition in the salicylic acid carboxyl methyltransferase
family, Plant Cell 15 (2003) 1704–1716.
[25] K. Mizuno, S. Kurosawa, Y. Yoshizawa, M. Kato, Essential region for 3-N
methylation in N-methyltransferases involved in caffeine biosynthesis, Z.
Naturforsch. 65c (2010) 257–265.
[19] T.G. Köllner, C. Lenk, N. Zhao, I. Seidl-Adams, J. Gershenzon, F. Chen, J.
Degenhardt, Herbivore-induced SABATH methyltransferases of maize that
[26] M. Kato, T. Kanehara, H. Shimizu, T. Suzuki, F. Gillies, A. Crozier, H. Ashihara,
Caffeine biosynthesis in young leaves of Camellia sinensis: in vitro studies on N-
methyltransferase activity involved in conversion of xanthosine to caffeine,
Physiol. Plant 98 (1996) 629–636.
methylate anthranilic acid using S-adenosyl-L-methionine, Plant Physiol. 153
(2010) 1795–1807.
[20] N. Zhao, J.L. Ferrer, H.S. Moon, J. Kapteyn, X. Zhuang, M. Hasebe, C.N. Stewart
Jr., D.R. Gang, F. Chen, SABATH Methyltransferase from the moss
[27] S. Chang, J. Puryear, J. Cairney, Simple and efficient method for isolating RNA
from pine trees, Plant Mol. Biol. Rep. 11 (1993) 113–116.
A
Physcomitrella patens catalyzes S-methylation of thiols and has
detoxification, Phytochemistry 81 (2012) 31–41.
[21] J.C. D’Auria, F. Chen, E. Pichersky, The SABATH family of MTs in Arabidopsis
thaliana and other plant species, in: JT Romeo (Ed.), Recent Advances in
Phytochemistry, vol. 37, Elsevier Science, Oxford, UK, 2003, pp. 253–283.
a
role in
[28] M. Bradford, A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein–dye binding, Anal.
Biochem. 7 (1976) 248–254.
[29] H. Ashihara, Trigonelline (N-methylnicotinic acid) biosynthesis and its
biological role in plants, Nat. Prod. Commun. 3 (2008) 1423–1428.