Biosynthesis of trigonelline from nicotinate mononucleotide in mungbean seedlings

Article abstract of DOI:10.1016/j.phytochem.2007.08.008

To determine the biosynthetic pathway to trigonelline, the metabolism of [carboxyl-¹⁴C]nicotinate mononucleotide (NaMN) and [carboxyl-¹⁴C]nicotinate riboside (NaR) in protein extracts and tissues of embryonic axes from germinating mungbeans (Phaseolus aureus) was investigated. In crude cell-free protein extracts, in the presence of S-adenosyl-l-methionine, radioactivity from [¹⁴C]NaMN was incorporated into NaR, nicotinate and trigonelline. Activities of NaMN nucleotidase, NaR nucleosidase and trigonelline synthase were also observed in the extracts. Exogenously supplied [¹⁴C]NaR, taken up by embryonic axes segments, was readily converted to nicotinate and trigonelline. It is concluded that the NaMN → NaR → nicotinate → trigonelline pathway is operative in the embryonic axes of mungbean seedlings. This result suggests that trigonelline is synthesised not only from NAD but also via the de novo biosynthetic pathway of pyridine nucleotides.

Full text of DOI:10.1016/j.phytochem.2007.08.008

Available online at www.sciencedirect.com
PHYTOCHEMISTRY
Phytochemistry 69 (2008) 390–395
www.elsevier.com/locate/phytochem
Biosynthesis of trigonelline from nicotinate mononucleotide
in mungbean seedlings
a,b
a
a,
*
Xin-Qiang Zheng , Ayu Matsui , Hiroshi Ashihara
a
Department of Biological Sciences, Graduate School of Humanities and Sciences, Ochanomizu University, Bunkyo-ku, Tokyo 112-8610, Japan
b
Tea Research Institute, Zhejiang University, Kaixuan Road, Hangzhou 310029, China
Received 27 May 2007; received in revised form 19 July 2007
Available online 20 September 2007
Abstract
To determine the biosynthetic pathway to trigonelline, the metabolism of [carboxyl- C]nicotinate mononucleotide (NaMN) and [car-
1
4
1
4
boxyl- C]nicotinate riboside (NaR) in protein extracts and tissues of embryonic axes from germinating mungbeans (Phaseolus aureus)
1
4
was investigated. In crude cell-free protein extracts, in the presence of S-adenosyl-L-methionine, radioactivity from [ C]NaMN was
incorporated into NaR, nicotinate and trigonelline. Activities of NaMN nucleotidase, NaR nucleosidase and trigonelline synthase were
1
4
also observed in the extracts. Exogenously supplied [ C]NaR, taken up by embryonic axes segments, was readily converted to nicotinate
and trigonelline. It is concluded that the NaMN ! NaR ! nicotinate ! trigonelline pathway is operative in the embryonic axes of
mungbean seedlings. This result suggests that trigonelline is synthesised not only from NAD but also via the de novo biosynthetic path-
way of pyridine nucleotides.
Ó 2007 Elsevier Ltd. All rights reserved.
Keywords: Mungbean; Phaseolus aureus; Leguminosae; Biosynthesis; Pyridine alkaloid; Trigonelline
1
. Introduction
has not been fully elucidated. Since nicotinate (4) is derived
from the degradation of NAD (7), it has been proposed
that trigonelline (5) is a product of the pyridine nucleotide
cycle originating from the pyridine nucleotides NAD and
NADP (Zheng et al., 2004; Ashihara et al., 2005; Ashihara,
2006; Matsui et al., 2007). However, it is not clear that trig-
onelline (5) is synthesised exclusively by this pathway in
plants. Direct nicotinate (4) formation from NaMN (2) ini-
tiated by de novo nucleotide biosynthesis is also plausible.
The direct route from NaMN (2) to nicotinate (4) in nico-
tine-producing tobacco roots has been proposed by Wag-
ner et al. (1986), who suggested that NaMN
glycohydrolase contributes to this conversion. However,
the Km value for NaMN (2) of NaMN glycohydrolase
was extremely high (4 mM). The cellular concentration of
NaMN (2) in mungbean seedlings is so low that it is diffi-
cult to detect using HPLC (X.-Q. Zheng, unpublished
observation). Even if the pathway from NaMN (2) to
nicotinate (4) is operative, other enzymes may therefore
participate in this process in trigonelline (5)-forming
Trigonelline (N-methylnicotinate) (5) (see Fig. 1) is a
secondary metabolite formed from nicotinate (4) (Joshi
and Handler, 1960). In many legumes and some species
of other plant families, such as coffee (Coffea spp.), large
amounts of trigonelline (5) (1–60 lmol/g fresh weight)
accumulate in seeds (Koshiro et al., 2006; Matsui et al.,
2
007). Several physiological functions of trigonelline (5)
have been proposed (Tramontano et al., 1982; Lynn
et al., 1984; Ueda et al., 1995; Tramontano and Jouve,
1
2
997; Wood, 1999; Minorsky, 2002; Zheng and Ashihara,
004), but the biosynthesis and degradation of this alkaloid
0
Abbreviations: IMP, inosine-5 -mononucleotide; NaAD, nicotinate
adenine nucleotide; NaMN, nicotinate mononucleotide; NaR, nicotinate
riboside; NMN, nicotinamide mononucleotide; NR, nicotinamide ribo-
side; PPi, pyrophosphate; PRPP, 5-phosphoribosyl-1-pyrophosphate.
*
Corresponding author. Tel./fax: +81 3 5978 5358.
E-mail address: ashihara.hiroshi@ocha.ac.jp (H. Ashihara).
0
031-9422/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2007.08.008

X.-Q. Zheng et al. / Phytochemistry 69 (2008) 390–395
391
Fig. 1. Proposed pathway of trigonelline (5) synthesis from NaMN (2). Steps (1)–(3) are respectively, catalysed by NaMN nucleotidase, NaR nucleosidase
and trigonelline synthase. The alternative enzymes, nicotinate phosphoribosyltransferase or NaMN glycohydrolase (reaction is not shown), might
participate in the step (1a), and nucleoside phosphorylase in step (2a), although no evidence was obtained here. Trigonelline (5) synthesis via the pyridine
nucleotide cycle is also shown.
4
0
mungbean plants. Here, we report experimental results that
support formation of trigonelline (5) from NaMN (2) via
NaR (3) in mungbean embryonic axes. The classic pathway
of trigonelline (5) synthesis via pyridine nucleotide cycle,
and the new proposed route are both illustrated in Fig. 1.
NaR 3
Nicotinate 4
Trigonelline 5
30
2
. Results and discussion
2
1
0
0
0
2
.1. Conversion of NaMN (2) to trigonelline (5) in vitro
It has been reported that trigonelline (5) is produced
from the degradation of NAD (7) via the pyridine nucleo-
tide cycle (Zheng et al., 2004, 2005; see Fig. 1). However,
there is no evidence that trigonelline (5) is formed exclu-
sively by this route. It is also speculated that nicotinate
(
4), which is a direct precursor of trigonelline (5), is also
derived from NaMN (2) itself newly synthesised by the de
novo pathway. To demonstrate this hypothesis, we first
examined the conversion of NaMN (2) to trigonelline (5)
in the enzyme extracts in vitro.
0
30
60
90
120
Time (min)
1
4
Fig. 2. Metabolic fate of 18 lM [ C]NaMN (2) (3.7 kBq) in the presence
of 1 mM SAM and 10 mM MgCl in soluble protein extracts (320 lg)
from embryonic axes taken from 3-day-old mungbean seedlings. Values
are expressed as a percentage of total radioactivity (3.7 kBq) and SD
n = 3) in the reaction mixture. The rest of radioactivity was recovered in
unmetabolised NaMN (2).
2
Fig. 2 shows the time-course of the in vitro metabolism
1
4
of [carboxyl- C]NaMN (2) in the presence of 1 mM
SAM and dialysed protein extracts from embryonic axes
of 3-day-old mungbean seedlings. Two min after adminis-
(

392
X.-Q. Zheng et al. / Phytochemistry 69 (2008) 390–395
1
4
tration of [ C]NaMN (2), radioactivity was incorporated
30
25
NaAD 6
into NaR (3) (ca. 4.8% of radioactivity administered). After
0 min, radioactivity was also with associated nicotinate (4)
NaR 3
1
Nicotinate 4
and trigonelline (5). This finding suggests that NaR (3) is
the initial metabolite of NaMN (2). The radioactivity in
NaR (3) increased transiently up to 30 min and then
decreased. After 30 min, the most heavily labelled com-
pound was nicotinate (4) (14.9–35.5% of radioactivity
2
1
1
0
5
0
5
0
1
4
administered). As expected, radioactivity from [ C]NaMN
2) was detected in trigonelline (5), and it increased linearly
(
with incubation time. The direct pathway from NaMN (2)
to trigonelline (5) via NaR (3) and nicotinate (4) therefore
functions at least in the crude protein extracts from mung-
bean embryonic axes. Conversion of NaMN (2) to NaR (3)
is catalysed by nucleotidase(s), and conversion of NaR (3)
to nicotinate (4) is hydrolysed by nucleosidase(s), although
we do not know if NaMN (2) specific-nucleotidase and
NaR (3) specific-nucleosidase are present in mungbean
extracts.
0
10
20
30
Time (min)
1
.2. Metabolic fate of [ C]NaMN (2) in vitro
4
2
14
Fig. 3. Metabolic fate of 18 lM [ C]NaMN (2) (3.7 kBq) in the presence
of 1 mM ATP and 10 mM MgCl in soluble protein extracts (320 lg) from
2
NaMN (2) has a crucial role in pyridine metabolism
Fig. 1). In addition to the conversion to NaR (3), NaMN
2) is converted to NaAD (6) by NaMN adenylyltransfer-
embryonic axes taken from 3-day-old mungbean seedlings. Values are
expressed as a percentage of total radioactivity (3.7 kBq) and SD (n = 3) in
the reaction mixture. The rest of radioactivity was recovered in unmetab-
olised NaMN (2).
(
(
ase (EC 2.7.7.18) and utilised for NAD (7) synthesis in
many organisms including plants (Katoh and Hashimoto,
2
004; Noctor et al., 2006; Hashida et al., 2007). Therefore,
Table 1
1
4
Activities of enzymes involved in NaMN (2) metabolism
we examined the in vitro metabolic fate of [ C]NaMN (2)
in the presence of 1 mM ATP, another substrate of NaMN
adenylyltransferase (Fig. 3). 2 min after addition of
Enzyme name
EC number
Activity
NaMN adenylyltransferase
NaMN nucleotidase
NaR nucleosidase
2.7.7.18
40.6
11.6
16.7
3.2
1
4
[
C]NaMN (2), 2.7%, 2.5% and 0.7% of administered
Not registered
Not registered
2.1.1.7
NaMN (2) was converted to NaAD (6), NaR (3) and nic-
otinate (4), respectively. However, after longer incubation,
NaAD (6) was the predominant metabolite and it com-
prised ca. 27% of total activity after 30 min. The rest of
the radioactivity was retained in NaMN (2). The data sug-
gest that NaMN (2) is preferentially used for NaAD (6)
synthesis if sufficient ATP is provided, with a smaller
amount of NaMN (2) being converted to NaR (3) and nic-
otinate (4).
Trigonelline synthase
Enzyme activities are expressed as pkat per mg protein.
The values show average of the activity obtained from two separate
experiments.
nucleoside phosphorylase (Fig. 1, reaction 2a) in the con-
version of NaR (3) to nicotinate (4) is not feasible, because
Pi inhibits the conversion of NaR (3) to nicotinate (4) (data
not shown). These reactions therefore appear not to be
involved in the conversion of NaMN (2) to nicotinate (4).
Involvement of NaMN glycohydrolase in this conversion,
which has been suggested to occur in tobacco roots (Wag-
ner et al., 1986), cannot be wholly excluded, because radio-
activity in NaMN (2) was incorporated into nicotinate (4);
however, the rapid incorporation of radioactivity into NaR
(3) was always observed so that the two-step conversion
catalysed by nucleotidase and nucleosidase appears to be
the major route.
2
.3. Activity of enzymes involved in NaMN (2) metabolism
The activities of NaMN adenylyltransferase, NaMN
nucleotidase, NaR nucleosidase and trigonelline synthase
were measured individually using [ C]NaMN (2), [ C]
NaR (3) and [ C]nicotinate (4) as substrates, as shown in
Table 1. NaMN adenylyltransferase had the highest activ-
ity and NaR nucleosidase activity was about 1.5 times that
of the NaMN nucleotidase activity.
1
4
14
1
4
In addition to direct hydrolytic cleavage, the conversion
of NaMN (2) to nicotinate (4) can, in theory, be catalysed
by the reverse reaction of nicotinate phosphoribosyltrans-
ferase (Fig. 1, reaction 1a). However, this pathway appears
to be negligible in mungbean extracts, as up to 1 mM PPi
did not stimulate the conversion. Participation of
1
2.4. Uptake of [carboxyl- C]NaMN (2)
4
The in vitro enzyme activity strongly suggests the con-
version of NaMN (2) to trigonelline (5) occurs in a way
which bypasses the pyridine nucleotide cycle. However,

X.-Q. Zheng et al. / Phytochemistry 69 (2008) 390–395
393
results obtained from in vitro experiments do not always
reflect metabolism in vivo because enzymes are sometimes
located in different intracellular compartments. We there-
fore examined in situ metabolism of 9 lM [car-
(Zheng et al., 2004). The endogenous concentration of
NaMN (2) in mungbean seedlings may be too low to detect
using ordinary HPLC methods.
1
4
boxyl- C]NaMN (2) (37 kBq) in segments of 3-day-old
mungbean embryonic axes. Almost no uptake of
2.6. Concluding remarks
1
4
[
C]NaMN (2) by the embryonic axes was observed, and
Our results using crude extracts of mungbean seedlings
1
4
no radioactive metabolites were detected in methanol-solu-
ble materials (data not shown). This is probably because
the transport system of NaMN (2) is not present in mung-
bean cell membranes.
and in situ tracer experiments with [ C] NaR (3) suggest
that part of NaMN (2) originating from de novo pyridine
nucleotide synthesis is converted to nicotinate (4) via
NaR (3) and used for trigonelline (5) synthesis. This is
the first report that trigonelline (5) is synthesised directly
from the de novo synthesis of NaMN (2) by three enzymatic
systems, namely NaMN nucleotidase, NaR nucleosidase
and trigonelline synthase. NaMN (2) produced by de novo
biosynthesis appears to be utilised preferentially for NAD
(7) synthesis, because NaMN adenylyltransferase activity
is greater than the activity of NaMN nucleotidase. Over-
flow of NaMN (2) produced by the de novo pathway may
be directed to trigonelline (5) synthesis.
1
.5. In situ metabolism of [carboxyl- C] NaR (3)
4
2
1
4
In contrast to [ C]NaMN (2), approximately 2% and
1
4
4
% of the administered 10 lM [carboxyl- C] NaR (3)
(
37 kBq) had been taken up by segments of 3-day-old
mungbean embryonic axes at 2 h and 4 h after incubation,
respectively. The radioactivity was distributed in nucleo-
tides (mainly NaMN (2), NAD (7) and NADP), trigonel-
line (5), nicotinate (4) and unmetabolised NaR (3)
(
(
Fig. 4). The radioactivity in nucleotides and trigonelline
5) increased with time, but radioactivity in nicotinate (4)
3. Experimental
and NaR (3) was almost constant. These results suggest
that NaR (3) was not only utilised for nucleotide synthesis,
but also for trigonelline (5) synthesis. Although NaR (3)
has not yet been isolated from any organism, incorporation
of the radioactivity from [carbonyl- C]nicotinamide (10)
into NaR (3) in leaves of Coffea arabica has been observed
3.1. Chemicals
1
4
[Carboxyl- C]nicotinate mononucleotide (sp. act
1
4
ꢀ1
14
2.0 GBq mmol ), [carboxyl- C]nicotinate riboside (sp.
act 1.9 GBq mmol ) and [carboxyl- C]nicotinate (sp.
act 1.9 GBq mmol ) were obtained from Moravek Bio-
chemicals Inc, Brea, CA, USA. Biochemicals were pur-
chased from Sigma Chemical Co., St. Louis, MO, USA.
ꢀ
1
14
ꢀ
1
1.00
0.75
0.50
0.25
0.00
3.2. Plant materials
2
4
hr
hr
Mungbean seeds (Phaseolus aureus Roxb) were pur-
chased from the Carolina Biological Supply Company,
Burlington, NC, USA. Seeds sterilised with saturated NaO-
Cl solution were germinated on 0.55% agar gel in Erlen-
meyer flasks at 26 °C in the dark, as detailed in previous
papers (Ashihara et al., 1974; Zheng et al., 2005). Embry-
onic axes were removed from cotyledons, and were
1
4
promptly used for enzyme extraction and in situ C-tracer
experiments.
3.3. Extraction of soluble protein extracts
Embryonic axes (ca. 4 g fresh weight) were homogenised
in extraction medium, consisting of 50 mM HEPES-NaOH
buffer (pH 7.6), 2 mM sodium EDTA, 2 mM dithiothreitol
and 5 mM MgCl , using a mortar and pestle. The homog-
2
enate was centrifuged at 20,000g for 20 min at 2 °C. The
supernatant was treated with 80% satd. (NH ) SO , and
4
2
4
1
4
Fig. 4. In situ metabolism of 10 lM [ C] NaR (3) (37 kBq) in embryonic
axes of 3-day-old mungbean seedlings. Values are expressed as a
percentage of total uptake of radioactivity and SD (n = 3). Total uptake
the precipitate was collected by centrifugation at 20,000g
for 15 min. The resulting pellet was dissolved in 50 mM
HEPES-NaOH buffer (pH 7.6) and desalted on a pre-
packed column of Sephadex G-25.
1
4
of [ C] NaR (3) by the embryonic axes (100 mg fresh weight) was
.83 ± 0.15 kBq (2h) and 1.42 ± 0.03 kBq (4h).
0

394
X.-Q. Zheng et al. / Phytochemistry 69 (2008) 390–395
The protein content was determined by the method of
Bradford (1976).
ous paper (Ashihara et al., 2000, 2005). A plant sample
(100 mg fresh weight) and 2.0 ml of 20 mM sodium phos-
phate buffer (pH 5.6) containing 10 mM sucrose were
placed in the main compartment of a 30 ml Erlenmeyer
flask. The flask was fitted with a small glass tube containing
3.4. In vitro metabolism of NaMN (2) in soluble protein
extracts
a piece of filter paper impregnated with 0.1 ml of 20%
1
4
14
In vitro conversion of [ C]NaMN (2) was examined in
the presence of SAM or ATP in the soluble mungbean pro-
tein fraction that was precipitated with 80% saturated
ammonium sulphate. The total volume of the reaction mix-
ture was 100 ll, and incubation took place at 30 °C. The
reaction mixture contained 30 mM HEPES-NaOH buffer
KOH in the centre well, to collect CO . Each reaction
2
was started by adding a solution of radioactive compound
(37 kBq) to the main compartment of the flask. The flasks
were incubated in an oscillating water bath at 27 °C. After
incubation, the plant materials were harvested, washed
with distilled H O, kept in an extraction medium consisting
2
1
4
(
pH 7.6), 18 lM [ C]NaMN (2) (3.7 kBq), 10 mM MgCl ,
of MeOH–H O (4:1, v/v) methanol with 20 mM sodium
2
2
desalted enzyme extracts and 1 mM SAM (or 1 mM ATP).
Degradation of NaMN (2) was also examined using a reac-
tion mixture consisting of 30 mM HEPES-NaOH buffer
diethyldithiocarbamate, and stored at ꢀ30 °C prior to
extraction.
Labelled metabolites were extracted and analysed as
described previously (Zheng and Ashihara, 2004). In sum-
mary, segments of the embryonic axes were homogenised
in a pestle and mortar with the extraction medium. The
resulting MeOH-soluble fraction was concentrated using
a rotary evaporator and was separated by TLC using
microcrystalline cellulose TLC plates (Merck, Darmstadt,
Germany). Solvent system I of the previous paper (Zheng
and Ashihara, 2004) was used to identify radio-labelled
metabolites.
1
4
(
pH 7.6), 18 lM [ C]NaMN (2) (3.7 kBq), 10 mM MgCl
2
and desalted enzyme extracts. The reactions were termi-
nated by transferring the test tubes to a boiling water bath
for 2 min. After brief centrifuging, an aliquot of each
supernatant was loaded onto the cellulose TLC plate.
Labelled compounds were separated by TLC using the sol-
vent system I (n-BuOH–HOAc–H O, 4:1:2 v/v) as
2
described by Zheng and Ashihara (2004).
3
.5. Determination of enzyme activity
3.7. Determination of radioactivity
The same crude protein fraction was used for individual
enzyme assays. The total volume of the reaction mixture
was 100 ll, and incubation took place at 30 °C. The mixture
for NaMN adenylyltransferase contained 30 mM HEPES–
Radioactivity was measured using a multi-purpose scin-
tillation counter (type LS 6500, Beckman, Fullerton, CA,
USA). The distribution of radioactivity on the TLC sheet
was analysed using a Bio-Imaging Analyser (Type, FLA-
2000, Fuji Photo Film Co., Ltd. Tokyo, Japan).
1
4
NaOH buffer (pH 7.6), 18 lM [ C]NaMN (2) (3.7 kBq),
mM ATP, 10 mM MgCl and desalted enzyme extracts.
1
2
The reaction mixtures for NaMN nucleotidase contained
1
4
3
0 mM HEPES–NaOH buffer (pH 7.6), 18 lM [ C]NaMN
Acknowledgement
(
2) (3.7 kBq), 10 mM MgCl and desalted enzyme extracts.
2
The reaction mixture for NaR (3) nucleosidase was the
1
4
This work was partly supported by a Grant-in-Aid for
Scientific Research from the Japanese Society for the Pro-
motion of Science (No. 16570031).
same, except that NaMN (2) was replaced by 20 lM [ C]
NaR (3) (3.7 kBq). The reaction mixture for trigonelline
synthase contained 30 mM HEPES–NaOH buffer (pH
1
4
7
.6), 18 lM [ C]nicotinate (3.7 kBq), 1 mM SAM, 10 mM
MgCl and desalted enzyme extracts. The reaction was per-
2
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