Biosci. Biotechnol. Biochem., 72 (9), 2456–2458, 2008
Note
Conversion of 5-Hydroxytryptophan into Serotonin
by Tryptophan Decarboxylase in Plants,
Escherichia coli, and Yeast
y
Munyoung PARK, Kiyoon KANG, Sangkyu PARK, and Kyoungwhan BACK
Department of Molecular Biotechnology, Agricultural Plant Stress Research Center,
Biotechnology Research Institute, Chonnam National University,
Gwangju, 500-757, South Korea
Received April 4, 2008; Accepted May 19, 2008; Online Publication, September 7, 2008
The L-tryptophan decarboxylase (TDC) gene of rice
was heterologously expressed in various organisms.
Transgenic rice overexpressing TDC showed accumu-
lation of serotonin upon 5-hydroxytryptophan treat-
ment, which was consistent with the in vitro 5-hydroxy-
tryptophan decarboxylase enzyme activity of purified
recombinant rice TDC in a pyridoxal phosphate-de-
pendent manner. Recombinant yeast harboring TDC
produced serotonin at the expense of the endogenous 5-
hydroxytryptophan levels.
Recombinant TDC was purified from E. coli harbor-
ing pET28b-TDC, in which a full-length rice TDC gene
(GenBank accession no. AK069031) was constructed
in-frame with codons for six additional histidine
residues at the carboxy terminus, as described previ-
ously.4) The purified TDC protein was used to determine
whether rice TDC would catalyze 5-OH Trp into
serotonin and would prove to be dependent on pyridoxal
phosphate. As shown in Fig. 1A, TDC activity was
8 nkat mgꢀ1 protein even in the absence of cofactor
pyridoxal phosphate, whereas the addition of pyridoxal
phosphate to the reaction medium increased TDC
activity by 9-fold, suggesting that rice TDC requires
pyridoxal phosphate as a cofactor. In contrast, its 5-
hydroxytryptophan decarboxylase (5-OHTDC) activity
was 8 times lower than TDC activity in the absence
of pyridoxal phosphate, but the addition of pyridoxal
phosphate caused an 18-fold increase in 5-OHTDC
activity. The optimum pyridoxal phosphate concentra-
tion for the activity of both TDC and 5-OHTDC was
about 0.1 mM.
The in vivo activity of 5-OHTDC was further
confirmed in transgenic rice constitutively expressing a
rice TDC gene.4) Three-week-old rice leaves were
employed. One-cm leaf squares were excised with a
razor blade and placed them in 6-cm diameter polysty-
rene Petri dishes containing various concentrations of
5-OH Trp. The tissues were then incubated in a growth
chamber at 25 ꢁC for 12 h. The levels of serotonin were
quantified by HPLC and compared.
Key words: tryptophan decarboxylase; 5-hydroxytrypto-
phan decarboxylase; 5-hydroxytryptophan;
serotonin
L-Tryptophan decarboxylase (TDC) catalyzes trypto-
phan into tryptamine. In plants, TDC is involved in the
biosynthesis of several types of secondary metabolites,
including terpenoid indole alkaloids, serotonin deriva-
tives, and serotonin.1,2) Since the first isolation of a TDC
gene was reported, from Catharanthus roseus,3) only
three other TDC genes have been characterized at the
biochemical and molecular levels.1,4) In addition, the
substrate specificities of TDC proteins as purified
enzymes and in heterologous expression systems have
not been characterized, except for a study in which a
partially purified TDC from tomato was also found to
catalyze 5-hydroxytryptophan (5-OH Trp) into serotonin,
as shown by a simple paper chromatography analysis.5)
Subsequently, there have been no further reports as to
whether TDC can catalyze 5-OH Trp into serotonin or
synthesize serotonin via 5-OH Trp via its 5-hydroxy-
tryptophan decarboxylase activity (5-OHTDC) in heter-
ologous expression systems. Here, in the present study,
we carried out functional analyses of TDC, focusing on
the availability of 5-OH Trp as a substrate and using rice
plants, E. coli, and yeast as heterologous systems.
The leaves of the transgenic rice plants accumulated
28 and 46 mg of serotonin per g fresh weight (fw) when
treated with 100 and 500 mM 5-OH Trp respectively
(Fig. 1B). In contrast, the wild-type produced 2.7 and
17 mg of serotonin per g fw in response to 100 and
500 mM 5-OH Trp respectively. The transgenic leaves
produced 10-fold higher serotonin than the wild-type
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To whom correspondence should be addressed. Fax: +82-62-530-2169; E-mail: kback@chonnam.ac.kr
Abbreviations: 5-OH Trp, 5-hydroxytryptophan; 5-OHTDC, 5-hydroxytryptophan decarboxylase; TDC, tryptophan decarboxylase