Melatonin and Phytomelatonin: Chemistry, Biosynthesis, Metabolism, Distribution and Bioactivity in Plants and Animals-An Overview - MDPI
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International Journal of
Molecular Sciences
Review
Melatonin and Phytomelatonin: Chemistry, Biosynthesis,
Metabolism, Distribution and Bioactivity in Plants and
Animals—An Overview
Giuseppe Mannino 1 , Carlo Pernici 1 , Graziella Serio 2 , Carla Gentile 2, * and Cinzia M. Bertea 1, *
1 Department of Life Sciences and Systems Biology, Plant Physiology Unit, University of Turin, Via Quarello
15/A, 10135 Turin, Italy; giuseppe.mannino@unito.it (G.M.); carlo.pernici@edu.unito.it (C.P.)
2 Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of
Palermo, Viale delle Scienze, 90128 Palermo, Italy; graziella.serio01@unipa.it
* Correspondence: carla.gentile@unipa.it (C.G.); cinzia.bertea@unito.it (C.M.B.); Tel.: +39-091-2389-7423 (C.G.);
+39-011-670-6361 (C.M.B.)
Abstract: Melatonin is a ubiquitous indolamine, largely investigated for its key role in the regulation
of several physiological processes in both animals and plants. In the last century, it was reported
that this molecule may be produced in high concentrations by several species belonging to the
plant kingdom and stored in specialized tissues. In this review, the main information related to the
chemistry of melatonin and its metabolism has been summarized. Furthermore, the biosynthetic
pathway characteristics of animal and plant cells have been compared, and the main differences
between the two systems highlighted. Additionally, in order to investigate the distribution of this
indolamine in the plant kingdom, distribution cluster analysis was performed using a database
Citation: Mannino, G.; Pernici, C.; composed by 47 previously published articles reporting the content of melatonin in different plant
Serio, G.; Gentile, C.; Bertea, C.M. families, species and tissues. Finally, the potential pharmacological and biostimulant benefits derived
Melatonin and Phytomelatonin: from the administration of exogenous melatonin on animals or plants via the intake of dietary
Chemistry, Biosynthesis, Metabolism, supplements or the application of biostimulant formulation have been largely discussed.
Distribution and Bioactivity in Plants
and Animals—An Overview. Int. J. Keywords: indolamine; biostimulant; dietary supplements; cluster analysis; N-acetyl-5- methoxytrip-
Mol. Sci. 2021, 22, 9996. https://
tamine
doi.org/10.3390/ijms22189996
Academic Editor: Małgorzata
M. Posmyk
1. Introduction
Received: 30 August 2021
Melatonin (N-acetyl-5-methoxytriptamine) is an indolamine originally discovered
Accepted: 15 September 2021 in 1958 in extracts from bovine pineal gland, but this compound was first isolated and
Published: 16 September 2021 identified as a small molecule with a molecular weight of 232 Daltons in 1960 by Lerner [1].
The name was initially related to its ability to aggregate pigment granules (melanin)
Publisher’s Note: MDPI stays neutral in the chromatophores of frog and fish skin. For more than 30 years, it was assumed
with regard to jurisdictional claims in that melatonin was exclusively produced in the pineal gland of animals, in which the
published maps and institutional affil- indolamine acts as a neurohormone; however, nowadays it is known that melatonin is also
iations. produced by several organisms belonging the Eukarya and Bacteria domains, whereas
no information has been found for Archea. Its extensive distribution has supported the
theory that this indolamine is an ancient molecule retained throughout the evolution of
all organisms [2,3]. Organisms such as Rhodospirillum rubrum [4,5], Arthrospira platensis
Copyright: © 2021 by the authors.
(syn. Spirulina platensis) [6,7], Lingulodinium polyedrum (syn. Gonyaulax polyedra) [8–10]
Licensee MDPI, Basel, Switzerland. and Pterygophora californica [11] have acquired the ability to produce melatonin more than
This article is an open access article 2.5–3.5 billion years ago with the aim to mitigate the oxidative stress of reactive oxygen
distributed under the terms and species (ROS) produced as a consequence of their aerobic metabolism [12]. Plants produce
conditions of the Creative Commons melatonin in different anatomical districts and in order to discriminate plant melatonin
Attribution (CC BY) license (https:// from melatonin produced by all other organisms, in 2004 the term ‘phytomelatonin’ was
creativecommons.org/licenses/by/ proposed [13].
4.0/).
Int. J. Mol. Sci. 2021, 22, 9996. https://doi.org/10.3390/ijms22189996 https://www.mdpi.com/journal/ijms
In vertebrates, melatonin is rhythmically secreted by the pineal gland after photo
stimulation caused by dark or light-suppression [14] and it regulates the sleep–wake cycl
Int. J. Mol. Sci. 2021, 22, 9996
and other seasonal rhythms. In these animals, the nocturnal melatonin peak2also of 39
control
the reproductive capability [15]. The role of melatonin as circadian regulator appears to
have evolved polyphyletically, since it was observed also in invertebrate animals, includ
In vertebrates,
ing the marine annelid melatonin is rhythmically
Platynereis dumerilii secreted by the[16,17].
(Polychaeta) pineal gland
Thanks after photo-
to its antioxidan
stimulation caused by dark or light-suppression [14] and it regulates the
and radical scavenging properties [18], melatonin interacts also with the immune system sleep–wake cycle
and other seasonal rhythms. In these animals, the nocturnal melatonin peak also controls
of mammals acting as an immunostimulatory [19] and anti-inflammatory molecule [14]
the reproductive capability [15]. The role of melatonin as circadian regulator appears to
On the contrary, despite some reports of a circadian melatonin production rhythm in both
have evolved polyphyletically, since it was observed also in invertebrate animals, including
seaweed
the marineLingulodinium
annelid Platynereis polyedrum
dumerilii[20] and dicotyledon
(Polychaeta) Chenopodium
[16,17]. Thanks rubrum and
to its antioxidant [21], it doe
not seem
radical that melatonin
scavenging propertiesmay plays
[18], a role in
melatonin the control
interacts of seaweed
also with the immune andsystem
plant photoperi
of
odism [22].
mammals It isasmore
acting likely that melatonin
an immunostimulatory might
[19] and be involved in
anti-inflammatory other plant
molecule [14]. Onfunctions
the contrary,
including despiteand
growth some reports of a circadian
development, acting asmelatonin production
an auxin-like moleculerhythm in For
[23]. bothinstance
scientificLingulodinium
seaweed evidence haspolyedrum
suggested [20] andmelatonin
that dicotyledon Chenopodium
could modify rootrubrum [21], it doesand mor
architecture
not seem that[24–26],
phogenesis melatonin may plays
flowering a role in[27],
processes the control of seaweed
leaf senescence andand
[28], plant photope-
fruit ripening [29]
riodism [22]. It is more likely that melatonin might be involved in other plant functions,
chlorophyll, proline and carbohydrate content in leaves and fruits [30]. Recent finding
including growth and development, acting as an auxin-like molecule [23]. For instance,
also revealed its contribution as signalling molecule during biotic and abiotic stres
scientific evidence has suggested that melatonin could modify root architecture and mor-
[22,31–34],[24–26],
phogenesis influencing plantprocesses
flowering defence[27],
responses against [28],
leaf senescence several
and pathogen
fruit ripeningattacks
[29], and en
hancing stress
chlorophyll, prolinetolerance to cold, content
and carbohydrate drought, heavyand
in leaves metals, ultraRecent
fruits [30]. violetfindings
radiations,
also or sal
[22,35].
revealed its contribution as signalling molecule during biotic and abiotic stress [22,31–34],
influencing
In thisplant
work,defence responses against
the chemistry several pathogen
and biosynthetic attacks involved
pathways and enhancing stress
in melatonin pro
tolerance to cold, drought, heavy metals, ultra violet radiations, or salt
duction in both animal and plant cells will be discussed. The two biosynthetic pathway [22,35].
will In thisbework,
then the chemistry
compared. and biosynthetic
Consequently, pathways
a meta-analytic involvedwill
approach in melatonin
be employed pro- in orde
duction in both animal and plant cells will be discussed. The two biosynthetic pathways
to investigate the distribution of melatonin in the plant kingdom, highlighting the main
will then be compared. Consequently, a meta-analytic approach will be employed in order
sources of phytomelatonin. Finally, since melatonin has been shown to have importan
to investigate the distribution of melatonin in the plant kingdom, highlighting the main
physiological
sources roles in bothFinally,
of phytomelatonin. plantssince
and melatonin
animals, the haspotential
been shown effects derived
to have from the ap
important
plication of exogenous melatonin as a plant biostimulant or supplement
physiological roles in both plants and animals, the potential effects derived from the appli- for human use
will beofinvestigated.
cation exogenous melatonin as a plant biostimulant or supplement for human use will
be investigated.
2. Chemistry of Melatonin
2. Chemistry of Melatonin
From a physio-chemical point of view, pure melatonin resembles an off-white pow
From a physio-chemical point of view, pure melatonin resembles an off-white powder,
der, having 232.28 g/mol as molecular weight and a density of 1.175 g/cm3. The melting
having 232.28 g/mol as molecular weight and a density of 1.175 g/cm3 . The melting point
point ranges
ranges between between
116.5 ◦ C116.5 °C and
and 118 118boiling
◦ C; the °C; thepoint
boiling point
is 512.8 ◦ Cis[14].
512.8 °C [14].
From From a chem
a chemical
ical point of view, melatonin is identified by the chemical formula C
point of view, melatonin is identified by the chemical formula C13 H16 N2 O2 . The indole13 H 16 N 2 O 2. The indol
chemicalscaffold
chemical scaffoldis is functionalized
functionalized withwith a 3-amide
a 3-amide groupgroup
and a and a 5-alkoxygroup
5-alkoxygroup (Figure 1).(Figure 1)
Moreover,
Moreover,since it is
since it originated starting
is originated fromfrom
starting a molecule of tryptophan,
a molecule is classified
of tryptophan, as an
is classified as an
indolamine compound [36]. This particular chemical structure confers great
indolamine compound [36]. This particular chemical structure confers great stability by stability by
high
highresonance
resonance mesomerism.
mesomerism.
Figure1.1.Chemical
Figure Chemical structure
structure of melatonin.
of melatonin.
Moreover,
Moreover, thethe
3-amide
3-amidegroup and and
group 5-alkoxygroup are also
5-alkoxygroup arethe main
also theresponsible of the of the
main responsible
amphiphilicity of this molecule. This property makes melatonin able to cross biological
amphiphilicity of this molecule. This property makes melatonin able to cross biologica
membranes and enter any cellular and subcellular compartments [36,37], allowing not
membranes and enter any cellular and subcellular compartments [36,37], allowing no
only its easy distribution but also a high protection against oxidative stress in various
onlycompartments
cell its easy distribution
[36,38]. but
The also a high protection
antioxidant protection against oxidative
of melatonin stress inboth
is correlated various cel
compartments [36,38]. The antioxidant protection of melatonin is correlated both to it
Int. J. Mol. Sci. 2021, 22, 9996 3 of 39
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 39
to its own redox active properties and to metabolites originated during its metabolism.
Indeed, a series of new compounds having noteworthy antioxidant properties may be
own redox
further active by
generated properties andoxidation
melatonin to metabolites
in a originated duringknown
set of reactions its metabolism. Indeed,
as melatonin antiox-
a series of new compounds having noteworthy antioxidant properties may be further gen-
idant cascade [39,40]. Among these metabolites, cyclic 3-hydroxymelatonin (C3-OHM),
erated by melatonin oxidation in a set of reactions known as melatonin antioxidant cas-
N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), N1-acetyl-5-methoxykynuramine
cade [39,40]. Among these metabolites, cyclic 3-hydroxymelatonin (C3-OHM), N1-acetyl-
(AMK), 6-hydroxymelatonin (6-OHM), 2-hydroxymelatonin (2-OHM) are the most known
N2-formyl-5-methoxykynuramine (AFMK), N1-acetyl-5-methoxykynuramine (AMK), 6-
(Figure 2).
hydroxymelatonin (6-OHM), 2-hydroxymelatonin (2-OHM) are the most known (Figure 2).
Figure
Figure 2. Melatonin
2. Melatonin metabolismand
metabolism and its
its related
related metabolites.
metabolites. ONOO
ONOO
− − = peroxynitrite;
= peroxynitrite; ROS = reactive oxygen species;
ROS = reactive oxygenRNS
species;
= reactive nitrogen species; O2-: superoxide
− anion; OH-: hydroxyl radical;
− AMFK: N1-acetyl-N2-formyl-5-methoxykynura-
RNS = reactive nitrogen species; O2 : superoxide anion; OH : hydroxyl radical; AMFK: N1-acetyl-N2-formyl-5-
mine; AMK: N1-acetyl-5-methoxykynuramine; AMMC: 3-acetamidomethyl-6-methoxycinnolinone; AMNK: N1-acetyl-5-
methoxykynuramine; AMK: N1-acetyl-5-methoxykynuramine; AMMC: 3-acetamidomethyl-6-methoxycinnolinone; AMNK:
methoxy-3 nitrokynura-mine.
N1-acetyl-5-methoxy-3 nitrokynura-mine.
2.1. N1-Acetyl-N2-Formyl-5-Methoxykynuramine (AFMK)
2.1. N1-Acetyl-N2-Formyl-5-Methoxykynuramine (AFMK)
Kynuramine compounds, such as AFMK and its de-formylated form (AMK), are mol-
Kynuramine
ecules compounds,
produced during suchdegradation.
tryptamine as AFMK and Theits de-formylated
redox activity and form (AMK), are
antioxidative
molecules
propertiesproduced
of AFMK haveduring
beentryptamine
evaluated indegradation. The redox
several experimental models.activity
Unlike and antiox-
antiox-
idative
idants,properties of AFMK
such as vitamin C andhave been
vitamin evaluated
E, AMFK in several
can donate moreexperimental models.
than one electron [41]. Un-
like
In antioxidants,
particular, Rosensuch
andas vitamin showed
colleagues C and vitamin
that AFMK E, can
AMFK can
donate donate
four more
electrons than one
leading
to the production
electron of indolinone
[41]. In particular, derivatives,
Rosen such as Z-,showed
and colleagues E- isomers
thatof AFMK
N-(1-formyl-5-meth-
can donate four
oxy-3oxo-2,3-dihydro-1H-indol-2-ylidenemethyl)-acetamide
electrons leading to the production of indolinone derivatives, andsuchN-(1-formyl-2-hy-
as Z-, E- isomers of
droxy-5-methoxy-3-oxo-2,3-dihydro-1H-indol-2-ylmethyl)-acetamide
N-(1-formyl-5-methoxy-3oxo-2,3-dihydro-1H-indol-2-ylidenemethyl)-acetamide [42]. However, and N-
AFMK was reported to be a less effective free radical scavenger than AMK and melatonin [42].
(1-formyl-2-hydroxy-5-methoxy-3-oxo-2,3-dihydro-1H-indol-2-ylmethyl)-acetamide
[43–46]. The
However, AFMKantioxidant properties
was reported to of
beAFMK
a lesswere demonstrated
effective also scavenger
free radical in biologicalthan
mod-AMK
els. In particular, Tan and colleagues showed that the addition of AFMK to calf thymus
and melatonin [43–46]. The antioxidant properties of AFMK were demonstrated also in
DNA in presence of a mixture of prooxidant agents strongly reduced in a dose-dependent
biological models. In particular, Tan and colleagues showed that the addition of AFMK
way the levels of 8-OH-dG (an indicator of DNA damage) [41]. Moreover, in rat liver ho-
to calf thymus DNA in presence of a mixture of prooxidant agents strongly reduced in
mogenates incubated with H2O2 and Fe2+, 100 µM AFMK inhibited lipid peroxidation
a dose-dependent
(LPO) and improved waycell
the levels of
viability, 8-OH-dG
although Fe2+(an indicator
chelation was of
not DNA damage)
observed [41]. [41]. More-
2+
over, in rat liver homogenates incubated with H2 O2 and Fe , 100 µM AFMK inhibited
lipid peroxidation (LPO) and improved cell viability, although Fe2+ chelation was not
observed [41].
Int. J. Mol. Sci. 2021, 22, 9996 4 of 39
2.2. N1-Acetyl-5-Methoxykynuramine (AMK)
AFMK can be both enzymatically and non-enzymatically de-formylated resulting in
the formation of AMK [47,48]. This compound showed a higher efficiency for scavenging
ROS and preventing protein oxidation with respect to AFMK [49]. Radical scavenging
action of AMK leads to the production of AMK oligomers, such as 3-acetamidomethyl-6-
methoxycinnolinone and N1-acetyl-5-methoxy-3 nitrokynuramine. This property of AMK
strongly depends on the environmental conditions [46]. Indeed, it has been observed that
in aqueous solution, AMK is a radical scavenger stronger than melatonin, although it is a
good scavenger also in nonpolar environment. In particular, AMK is a better OH•− and
NO scavenger than both melatonin [50] and AFMK [51].
2.3. 3-Hydroxymelatonin (C3-OHM)
Melatonin oxidation by reactive oxygen species (ROS) and reactive nitrogen species
(RNS) scavenging may produce also C3-OHM. Experimental data showed an antioxidant
protection by radicals. In particular, as with AFMK, also C3-OHM prevented DNA oxi-
dation induced by Fenton reaction [52]. The presence of C3-OHM was always coupled
to AFMK formation, both in vitro and in vivo experimentations [53]. The ratio between
oxidants and melatonin affected the amount of melatonin oxidation products. In particular,
higher were the ROS levels, more AFMK was produced [54]. Indeed, in this condition,
C3-OHM can also be oxidised to AFMK.
2.4. 6-Hydroxymelatonin (6-OHM)
6-Hydroxymelatonin (6-OHM), for the first time discovered in animal urine in the
form of 6-hydroxymelatonin sulfate, is one of the major melatonin catabolites in animals.
Experimental data showed that 6-OHM prevented lipid peroxidation [55] and DNA dam-
age induced by environmental pollutants, chromium [56], and OH•− generated by Fenton
reaction [57]. Duan et al. also showed neuronal protection by 6-OHM in a model of
ischemia/reperfusion-mediated injury. In this model, the anti-apoptotic action involved
the inhibition of cytochrome C, inhibition of caspase 3 activity, and stabilization of the
mitochondrial membrane potential [58]. Although the known protective effect of 6-OHM,
a slight prooxidant activity was also shown. In particular, it was reported that 6-OHM
caused oxidative DNA damage with double-strand breaks via redox cycling [59].
2.5. 2-Hydroxymelatonin (2-OHM)
Melatonin oxidation also leads to the production of 2-OHM, especially after scav-
enging of HClO [60], oxoferryl haemoglobin [61] and OH•− [62]. Conversely to 3-OHM,
2-OHM is one the prevalent products of the hydroxylation of melatonin in plants. 2-
OHM production is coupled to the formation of the keto tautomer melatonin 2-indolinone.
In addition, in cytochrome C in vitro models the oxidation 2-OHM into AFMK was ob-
served [63].
3. Biosynthesis of Melatonin
3.1. Biosynthetic Route in Plants
It has been shown that the cellular compartments with the highest melatonin levels
in plants are mitochondria and chloroplasts [31]. This observation, together with the
demonstrated localization of serotonin N-acetyltransferase (SNAT), one of the rate-limiting
enzymes involved in melatonin biosynthesis, in chloroplasts [64,65] and in mitochon-
dria [66], leads to hypothesize that these organelles are the major sites involved in the
biosynthesis of this indolamine. The genes encoding for all the enzymes catalysing the
whole melatonin biosynthetic pathway in plants have been discovered in several plant
species, with the exception of one putative gene encoding for a tryptophan hydroxylase
(TPH), which catalyses the conversion of tryptophan into 5-hydroxytryptophan [67]. In
particular, this enzyme, already known in vertebrates, was only recently proposed in plants.
Int. J.Sci.
Int. J. Mol. Mol. Sci. 2021,
2021, 22, x FOR PEER REVIEW
22, 9996 5 of 39 5 of 39
particular, this enzyme, already known in vertebrates, was only recently proposed in
Melatonin biosynthesis begins with the amino acid tryptophan, a compound that
plants.
plants Melatonin
are able tobiosynthesis de novowith
synthesize begins via the shikimate
the amino acid pathway
tryptophan, (Figure 3). Thisthat
a compound pathway
plants of
consists areseven
able todifferent
synthesize de novo
steps thatvia the shikimate
allow pathway (Figure
to the biosynthesis of all 3). This pathway
aromatic amino acids
in consists of seven different
plants, including steps [68].
tryptophan that allow to the
Briefly, biosynthesis of all aromatic amino7-phosphate
3-Deoxy-D-arabinoheptulosonate acids
in plants,
(DAHP) including
synthase (ECtryptophan [68]. Briefly,phosphoenol
2.5.1.54) transforms 3-Deoxy-D-arabinoheptulosonate
pyruvate (PEP) and7-phos- erythrose-4-
phate (DAHP)
phosphate in DAHP,synthase (EC
that is then2.5.1.54)
cyclizedtransforms phosphoenol pyruvate
into 3-dehydroquinate (DHQ) (PEP)by theandaction of
erythrose-4-phosphate in DAHP, that is then cyclized into 3-dehydroquinate (DHQ) by
the DHQ synthase (EC 4.2.3.4). Finally, shikimate is synthetized through the dehydration
the action of the DHQ synthase (EC 4.2.3.4). Finally, shikimate is synthetized through the
and dehydrogenation catalyzed by DHQ dehydratase (EC 4.2.1.10) and shikimate dehy-
dehydration and dehydrogenation catalyzed by DHQ dehydratase (EC 4.2.1.10) and shi-
drogenase (EC 1.1.1.25).(EC
kimate dehydrogenase Therefore, shikimateshikimate
1.1.1.25). Therefore, is phosphorylated by theby
is phosphorylated shikimate
the shiki-kinase
(EC 2.7.1.71) and converted in 5-enolpyruvylshikimate-3-phosphate
mate kinase (EC 2.7.1.71) and converted in 5-enolpyruvylshikimate-3-phosphate (EPSP) (EPSP) by the EPSP
synthase (EC 2.5.1.19). Finally, chorismate is formed through the
by the EPSP synthase (EC 2.5.1.19). Finally, chorismate is formed through the activity activity of chorismate
of
synthase
chorismate synthase (EC 4.2.3.5) that converts EPSP in chorismate, the essential interme-in tryp-
(EC 4.2.3.5) that converts EPSP in chorismate, the essential intermediate
tophan biosynthesis
diate in (Figure 3). Chorismate
tryptophan biosynthesis is converted
(Figure 3). Chorismate in anthranilate
is converted via anthranilate
in anthranilate via
anthranilate
synthase synthasethat
(EC 4.1.3.27) (EC is4.1.3.27) that is consequently
consequently condensed with condensed with phosphoribo-
phosphoribosylpyrophosphate
sylpyrophosphate
(PRPP), generating(PRPP), generatinganthranilate
phosphoribosyl phosphoribosyl anthranilate
(PRA). (PRA).
The ribose Theadded
ring ribose in
ring
this last
added in this last reaction is then opened by PRA isomerase (PRAI; EC 5.3.1.24),
reaction is then opened by PRA isomerase (PRAI; EC 5.3.1.24), subjected to reductive decar- subjected
to reductive
boxylation in decarboxylation in order to form indole-3-glycerol
order to form indole-3-glycerol phosphate thatphosphate that is sponta-
is spontaneously converted
neously converted into the indole scaffold. Finally, tryptophan is produced via the reac-
into the indole scaffold. Finally, tryptophan is produced via the reaction of the indole with
tion of the indole with serine through the action of tryptophan synthase (TPS; EC 4.2.1.20)
serine through the action of tryptophan synthase (TPS; EC 4.2.1.20) (Figure 3).
(Figure 3).
Figure 3. Biosynthetic pathway involved in the synthesis of tryptophan, the key compound for the formation of melatonin
Figure 3. Biosynthetic pathway involved in the synthesis of tryptophan, the key compound for the formation of melatonin
in plants. PEP: 2-phosphoenolpyruvate; DAHP: 3-deoxy-D-arabinoheptulosonate 7-phosphate; DHQ: 3-dehydroquinic
in plants.
acid;PEP:
DHS:2-phosphoenolpyruvate;
3-dehydroshikimate; PEP:DAHP: 3-deoxy-D-arabinoheptulosonate
2-phosphoenolpyruvate; 7-phosphate; DHQ: 3-dehydroquinic
EPSP: 5-enolpyruvylshikimate-3-phosphate; PRA: Phos- acid;
DHS: poribosyl
3-dehydroshikimate; PEP:PRA
antranilate; PRAI: 2-phosphoenolpyruvate; EPSP: 5-enolpyruvylshikimate-3-phosphate;
isomerase; PRPP: phosphoribosylpyrophosphate; PRA: Phosporibosyl
IGP: indole-3-glycerol phosphate; EC:
enzymePRAI:
antranilate; commission number). PRPP: phosphoribosylpyrophosphate; IGP: indole-3-glycerol phosphate; EC: enzyme
PRA isomerase;
commission number).
At least six enzymes are known to be involved in melatonin biosynthesis from tryp-
tophan, indicating
At least that multiple
six enzymes biosynthetic
are known to bepathways
involved mayin be present inbiosynthesis
melatonin this process. from
The six enzymes known to be involved in the synthesis of melatonin are: (i) L-tryptophan
tryptophan, indicating that multiple biosynthetic pathways may be present in this pro-
cess. The six enzymes known to be involved in the synthesis of melatonin are: (i) L-
tryptophan decarboxylase (TDC), (ii) tryptamine 5-hydroxylase (T5H), (iii) serotonin N-
acetyltransferase (SNAT), (iv) acetylserotonin O-methyltransferase (ASMT), (v) caffeic acid
3-O-methyltransferase (COMT), and (vi) a putative tryptophan hydroxylase (TPH) not
yet identified.
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 of 39
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 of 39
Int. J. Mol. Sci. 2021, 22, 9996 decarboxylase (TDC), (ii) tryptamine 5-hydroxylase (T5H), (iii) serotonin N-acetyltrans- 6 of 39
decarboxylase (TDC), (ii) tryptamine 5-hydroxylase (T5H), (iii) serotonin N-acetyltrans-
ferase (SNAT), (iv) acetylserotonin O-methyltransferase (ASMT), (v) caffeic acid 3-O-me-
ferase (SNAT), (iv) acetylserotonin O-methyltransferase (ASMT), (v) caffeic acid 3-O-me-
thyltransferase (COMT), and (vi) a putative tryptophan hydroxylase (TPH) not yet iden-
thyltransferase (COMT), and (vi) a putative tryptophan hydroxylase (TPH) not yet iden-
tified.
tified.
The
Thefirst
firststep
stepofofthe
themelatonin
melatoninbiosynthetic
biosyntheticprocess
processininplants
plantsis isrelated
related to
to the
the production
produc-
oftion The firstfrom
serotonin step tryptophan.
of the melatonin
Twobiosynthetic
different process inmay
pathways plants beisbe
related to(Figure
involved the produc-
4).4).The
of serotonin from tryptophan. Two different pathways may involved (Figure
tionway
first of serotoninwithfrom tryptophan. Two different pathways may be involved (Figure 4).
The firstbegins
way begins withthe decarboxylation
the decarboxylationof tryptophan
of tryptophan intointo
tryptamine
tryptamine by byTPH,
TPH, andandthen
The first
tryptamine way begins with
is hydroxylated the decarboxylation
to serotonin of
by TDC. tryptophan
On Onthe theinto
other tryptamine
hand, by
another TPH, and
possibility
then tryptamine is hydroxylated to serotonin by TDC. other hand, another possi-
theninvolves
first tryptamine is hydroxylatedoftotryptophan
the hydroxylation serotonin byintoTDC. On the other hand,by
5-hydroxytryptophan another
TPH, possi-
bility first involves the hydroxylation of tryptophan into 5-hydroxytryptophan byand
TPH, then
bility first involves the hydroxylation of tryptophan into 5-hydroxytryptophan by TPH,
the
and decarboxylation of 5-hydrotryptophan
then the decarboxylation into serotonin
of 5-hydrotryptophan by TDC.
into serotonin byThese
TDC. routes are both
These routes
and then the decarboxylation of 5-hydrotryptophan into serotonin by TDC. These routes
are bothbecause
possible, possible,TDC because
shows TDC
a good shows a for
affinity good affinity
both for both
tryptophan andtryptophan and 5-
5-hydroxytriptophan
are both possible, because TDC shows a good affinity for both tryptophan and 5-
inhydroxytriptophan
vitro [69]. However, in vitro [69]. been
it has However, it has been demonstrated
demonstrated that in plantsthat in plants
is more is morethe
frequent
hydroxytriptophan in vitro [69]. However, it has been demonstrated that in plants is more
frequent the decarboxylation
decarboxylation than the hydroxylation
than the hydroxylation as first step as first step [69].
[69].
frequent the decarboxylation than the hydroxylation as first step [69].
Figure 4. First
4. First twotwo reactions of themelatonin
reactions melatonin biosynthetic pathway
pathway leading to the formation
formationofofthe essential intermediate
Figure
Figure 4. First
serotonin. TDC: two reactionsofofthe
L-tryptophan the melatoninbiosynthetic
decarboxylase; biosynthetic pathway
TPH: tryptophan
leadingto
leading
hydroxylase;
tothe
theformation
T5H:
the essential
tryptamineof5-hydroxylase;
the essential intermediate
intermediate
EC: enzyme
serotonin. TDC:
serotonin. TDC:L-tryptophan
L-tryptophan decarboxylase; TPH: tryptophan hydroxylase; T5H: tryptamine 5-hydroxylase; EC:EC:
decarboxylase; TPH: tryptophan hydroxylase; T5H: tryptamine 5-hydroxylase; enzyme
enzyme
commission number).
commission
commission number).
number).
Melatonin
Melatonin synthesis
synthesis from serotoninisisaatwo-step
from serotonin two-stepreaction
reactioninvolving
involving three
three different
different
Melatonin synthesis from serotonin is a two-step reaction involving three different
enzymes (SNATs, ASMTs, and COMT) that may have various isoforms.
enzymes (SNATs, ASMTs, and COMT) that may have various isoforms. The first en- The first enzyme
enzymes (SNATs, ASMTs, and COMT) that may have various isoforms. The first enzyme
catalyzes
zyme an acetylation,
catalyzes whereas
an acetylation, the other
whereas thetwo enzymes
other are methyltransferases
two enzymes [69] (Fig-[69]
are methyltransferases
catalyzes an acetylation, whereas the other two enzymes are methyltransferases [69] (Fig-
ure 5). 5).
(Figure AsAsthethe
tree enzymes
tree enzymesexhibit a substrate
exhibit a substrateaffinity forfor
affinity serotonin, N-acetylserotonin,
serotonin, N-acetylserotonin,
ure 5). As the tree enzymes exhibit a substrate affinity for serotonin, N-acetylserotonin,
and5-methoxytryptamine,
and 5-methoxytryptamine, also also in
in this
thiscase
casethe
theorder
orderby bywhich
whichthe
thedifferent enzymes
different enzymes actact
and 5-methoxytryptamine, also in this case the order by which the different enzymes act
canvary
can vary[70–72].
[70–72].
can vary [70–72].
Figure 5. The last two potential reactions leading to the formation of melatonin. SNATs: serotonin N-acetyltransferase;
Figure
Figure
ASMTs: 5.acetylserotonin
5. TheThe last
last two
two potential
potential reactionsleading
reactions leading
O-methyltransferase; to the
to
COMT: the formation
formation
caffeic of
of melatonin.
melatonin.SNATs:
SNATs:
acid 3-O-methyltransferase; serotonin N-acetyltransferase;
serotonin
EC: enzyme N-acetyltransferase;
commission num-
ASMTs: acetylserotonin O-methyltransferase; COMT: caffeic acid 3-O-methyltransferase; EC: enzyme
ber. acetylserotonin O-methyltransferase; COMT: caffeic acid 3-O-methyltransferase; EC: enzyme commission commission num-
ASMTs: number.
ber.
The enzymes involved in melatonin biosynthesis from tryptophan have a different
distribution in plant cells. TDC is localized in the cytoplasm [73], T5H in the endoplasmic
reticulum [39], SNAT is expressed in chloroplasts [39], whereas ASMT and COMT are in
the cytoplasm [74]. Among the four possible melatonin biosynthetic pathways reported in
Figure 6, the first and the second pathways result in serotonin synthesis in the endoplasmic
reticulum, whereas the third and fourth in cytoplasmic environment [69]. ASMTs/COMT
are exclusively located in the cytoplasm and SNATs in the chloroplast, the final subcellular
The enzymes involved in melatonin biosynthesis from tryptophan have a different
distribution in plant cells. TDC is localized in the cytoplasm [73], T5H in the endoplasmic
reticulum [39], SNAT is expressed in chloroplasts [39], whereas ASMT and COMT are in
Int. J. Mol. Sci. 2021, 22, 9996 7 of 39
the cytoplasm [74]. Among the four possible melatonin biosynthetic pathways reported
in Figure 6, the first and the second pathways result in serotonin synthesis in the endo-
plasmic reticulum, whereas the third and fourth in cytoplasmic environment [69]. AS-
MTs/COMT
sites are exclusively
of melatonin synthesis located in the cytoplasm
and accumulation and SNATs
may vary. in the chloroplast,
For example, the
in the cytoplasm
final subcellular sites of melatonin synthesis and accumulation may vary. For example,
serotonin is rapidly metabolized into phenylpropanoid amides, such as feruloylserotonin, in
the cytoplasm serotonin is rapidly metabolized into phenylpropanoid amides,
by the serotonin N-hydroxycinnamoyl transferase (SHT) [75] and melatonin is rapidly such as fer-
uloylserotonin,
converted by the
into cyclic serotonin N-hydroxycinnamoyl
3-hydroxymelatonin (3-OHM) by transferase (SHT)
the melatonin [75] and melato-
3-hydroxylase (M3H),
nin is rapidly converted into cyclic 3-hydroxymelatonin (3-OHM) by
whereas in chloroplasts melatonin can be metabolized into 2-hydroxymelatoninthe melatonin(2-OHM)
3-hy-
droxylase
by (M3H),
the melatonin whereas in chloroplasts
2-hydroxylase (M2H) [76,77]. melatonin can be metabolized into 2-hy-
droxymelatonin (2-OHM) by the melatonin 2-hydroxylase (M2H) [76,77].
Figure
Figure 6. Subcellular
6. Subcellular localization
localization of of melatonin
melatonin intermediates
intermediates and
and enzymes
enzymes involved
involved inin the
the transformationofoftryptophan
transformation tryptophaninto
into melatonin in the different melatonin biosynthetic routes. TDC: L-tryptophan decarboxylase; TPH: tryptophan hy-
melatonin in the different melatonin biosynthetic routes. TDC: L-tryptophan decarboxylase; TPH: tryptophan hydroxylase;
droxylase; T5H: tryptamine 5-hydroxylase; SNATs: serotonin N-acetyltransferase; ASMTs: acetylserotonin O-methyl-
T5H: tryptamine
transferase; 5-hydroxylase;
COMT: SNATs:
caffeic acid serotonin N-acetyltransferase; ASMTs: acetylserotonin O-methyltransferase; COMT:
3-O-methyltransferase.
caffeic acid 3-O-methyltransferase.
The selection of the pathway for melatonin biosynthesis depends on plant growth
The selection
conditions. Indeed,ofunder
the pathway
standard for melatonin
or stress biosynthesis
conditions depends
that do not cause aon highplant growth
accumu-
conditions. Indeed, under standard or stress conditions that do not cause
lation of serotonin, the melatonin biosynthetic pathway proceeds from tryptophan via the a high accu-
mulation of serotonin,
intermediate the melatonin biosynthetic pathway
tryptamine/serotonin/N-acetylserotonin proceeds from
up to melatonin tryptophan
[78] (Figure 6). Invia
the intermediate
this tryptamine/serotonin/N-acetylserotonin
pathway, serotonin levels are relatively low and this molecule up to melatonin [78] (Figure
is preferentially acety- 6).
Inlated
thistopathway, serotoninbylevels
N-acetylserotonin SNATare duerelatively lowaffinity
to its higher and this
(Kmmolecule is preferentially
= 0.385 mmol/L) for ser-
acetylated to N-acetylserotonin
otonin in comparison to ASMT by (KmSNAT
= 1.035due to its higher
mmol/L) and COMTaffinity
(Km(K=m3.396
= 0.385 mmol/L)
mmol/L). The for
serotonin in comparison to ASMT
produced N-acetylserotonin is rapidly (K = 1.035 mmol/L) and COMT (K =
m O-methylated into melatonin bymeither ASMT or 3.396 mmol/L).
The
COMTproduced
with a N-acetylserotonin is rapidly
30-fold higher catalytic O-methylated
efficiency than SNAT,into melatonin
leading by either
to low levels ASMT
of N-ac-
oretylserotonin.
COMT withBased a 30-fold
on previously published data, it was observed that COMT exhibits of
higher catalytic efficiency than SNAT, leading to low levels
N-acetylserotonin. Based on
higher catalytic efficiency thanpreviously
ASMT at published
37 °C but in data,
vivoitexperiments,
was observed andthat COMT
it was ob- ex-
hibits
servedhigher catalytic
that the efficiency
activity of COMT thanto ASMT
methylate ◦ C but in vivo experiments,
at 37N-acetylserotonin into melatonin andwasit was
markedlythat
observed inhibited due to of
the activity theCOMT
fact that
toCOMT
methylatepreferred methylate other
N-acetylserotonin substrates
into melatonin suchwas
markedly inhibited due to the fact that COMT preferred methylate other substrates such
as caffeic acid and 5-hydroxyconiferaldehyde [79,80]. These phenomena resulted in the
functional loss of COMT activity for melatonin synthesis and a dominant role of ASMT in
methylating N-acetylserotonin into melatonin (Figure 6).
On the other hand, during plant senescence or under abiotic stress conditions, plants
tend to accumulate large amounts of melatonin intermediates (such as tryptophan, tryptamine
and serotonin) [35]. Consequently, the biosynthetic route preferably proceeds from tryp-
tophan via the intermediates tryptamine/serotonin/5-MT up to melatonin [69], where
Int. J. Mol. Sci. 2021, 22, 9996 8 of 39
serotonin is O-methylated into 5-MT by COMT, and then it is acylated by SNAT leading
to the formation of melatonin (Figure 6). However, it was observed that a serotonin boost
was not proportionally correlated to a significant increase in melatonin level. Indeed, in
several experimental conditions, despite the content of tryptophan and serotonin was slightly
enhanced during the senescence, an equal increment of melatonin level was not observed.
For example, data from senescent detached rice leaves have shown a difference of more
than threefold in metabolic capacity between serotonin and melatonin synthesis [81,82].
This huge difference between the two compounds could be explained by the relatively low
catalytic efficiencies of COMT and SNAT in senescence compared to those under normal
growth conditions. However, despite SNATs auto-inhibition by serotonin was not currently
observed and other regulatory roles of serotonin on the melatonin synthetic pathway are still
unknown, low levels of melatonin and relatively high levels of 5-MT are obtained compared
to N-acetylserotonin [83,84].
Despite the melatonin biosynthetic pathway under normal conditions producing more
melatonin that in senescence and serotonin boost conditions, the melatonin levels are not
related anyway to the levels of tryptophan and serotonin present in the cells. Consequently,
the limiting step could be attributed to the production of N-acetylserotonin by SNAT [84].
Indeed, N-acetylserotonin must first cross the chloroplast membrane into the cytoplasm
where ASMT or COMT can now transform it into melatonin [72,84].
Another potential route based on the studies conducted on T5H-deficient and T5H-
suppression rice plants seems to be related to 5-hydroxytryptophan-mediated serotonin
synthesis. In the investigated plants, the serotonin levels were much lower compared to
control plants, but melatonin levels were higher. This interesting result was incompatible
with the previously described melatonin biosynthetic pathways [85,86]. In this case, it was
supposed that 5-HT is produced from tryptophan by the action of a putative TPH and then
converted into serotonin by TDC. Given the low levels of serotonin and the high levels of
melatonin found in T5H-deficient plants, the 5-HT pathway does not result in a serotonin
boost but plays a key role in inducing melatonin levels.
Finally, other melatonin biosynthetic routes may exist [87,88], including those inde-
pendent from the formation of serotonin. However, the involved enzymes are not yet
identified and those already known seem not to be involved in this process [69].
3.2. Biosynthetic Route in Animals
A clear difference in melatonin synthesis between animals and plants concerns the
availability of the precursor tryptophan. Unlike plants, animals cannot synthetize tryp-
tophan de novo (Figure 3), but it must be taken in through the diet. Similarly to plants,
also animal mitochondria are the main biosynthetic sites and the compartments with the
highest concentration of melatonin [89]. The melatonin biosynthetic pathway in mammals
was first discovered by Axelrod’s group in 1960, and now it is well defined [90]. However,
the classic melatonin biosynthetic pathway has been expanded in all vertebrates and can
be applied also to other animals, including insects [91].
The pathway involves five enzymatic steps (Figure 7). In the first step, tryptophan
is hydroxylated to 5-hydroxytryptophan by TPH, that is subsequently decarboxylated
to serotonin (5-hydroxytryptamine) by the aromatic amino acid decarboxylase (AADC).
The two final steps were cryptic for several years. Indeed, it was not clear neither the
biosynthetic location of melatonin nor the enzymes involved in the synthesis. In 1960,
when the melatonin biosynthetic pathway was discovered in animals, it was proposed
that serotonin is acetylated by AANAT to N-acetylserotonin exclusively in pineal gland
and liver [92]. However, since ASMT was originally detected in the pineal gland, it was
wrongly suggested that melatonin production was not localized in the liver. For this
reason, melatonin was initially classified as a pineal-related neurohormone [93]. However,
to date it is well known that melatonin is also produced by many peripheral tissues
and organs, such as retina, Harderian gland, ovary, testis, bone marrow, lymphocytes,
hepatic cholangiocytes, gut, and skin [93]. In particular, it was observed that skin and
melatonin biosynthetic pathway was discovered in animals, it was proposed that seroto-
nin is acetylated by AANAT to N-acetylserotonin exclusively in pineal gland and liver
[92]. However, since ASMT was originally detected in the pineal gland, it was wrongly
suggested that melatonin production was not localized in the liver. For this reason, mela-
Int. J. Mol. Sci. 2021, 22, 9996 tonin was initially classified as a pineal-related neurohormone [93]. However, to date9itofis39
well known that melatonin is also produced by many peripheral tissues and organs, such
as retina, Harderian gland, ovary, testis, bone marrow, lymphocytes, hepatic cholangio-
cytes, gut, and skin [93]. In particular, it was observed that skin and gut produce more
gut produce more melatonin than the pineal gland [94]. Thus, the concept of melatonin
melatonin than the pineal gland [94]. Thus, the concept of melatonin as a neurohormone
as a neurohormone was modified and the observed ubiquitous presence of extra-pineal
was modified and the observed ubiquitous presence of extra-pineal melatonin in mam-
melatonin in mammals was explained [95,96].
mals was explained [95,96].
Figure7.7.The
Figure Theclassic
classicmelatonin
melatoninsynthetic
syntheticpathway
pathway in
in animals.
animals. TPH:
TPH: tryptophan
tryptophan hydroxylase;
hydroxylase;AADC:
AADC:aromatic
aromaticamino
aminoacid
acid
decarboxylase; AANAT: aralkylamine N-acetyltransferase; ASMTs: acetylserotonin O-methyltransferase.
decarboxylase; AANAT: aralkylamine N-acetyltransferase; ASMTs: acetylserotonin O-methyltransferase.
Later, it
Later, it was
was observed
observedthat thatthe enzyme
the enzyme activity of ASMT
activity of ASMT for N-acetylserotonin
for N-acetylserotoninwas
approximately 14-fold higher in presence of serotonin, thus it was
was approximately 14-fold higher in presence of serotonin, thus it was supposed that supposed that N-ace-
tylserotonin was the
N-acetylserotonin waspreferable substratesubstrate
the preferable of ASMTofrather ASMT than serotonin
rather than [97]. Based [97].
serotonin on
these observations, it was assumed that serotonin is first acetylated to form
Based on these observations, it was assumed that serotonin is first acetylated to form N- N-acetylsero-
tonin by AANAT
acetylserotonin byand
AANATthe resulting
and the N-acetylserotonin methylatedmethylated
resulting N-acetylserotonin to melatoninto by ASMT
melatonin
(Figure 7). AANAT is widely accepted as the limiting factor for the production
by ASMT (Figure 7). AANAT is widely accepted as the limiting factor for the production of melato-
nin.
of Indeed, inIndeed,
melatonin. mammals the main melatonin
in mammals biosynthetic
the main melatonin regulatory regulatory
biosynthetic factor is light, par-is
factor
ticularly blue light (~420–480 nm) [98]. In order to achieve a relatively long-term
light, particularly blue light (~420–480 nm) [98]. In order to achieve a relatively long-term effect
[95], this
effect kind
[95], thisofkind
irradiation during the
of irradiation day immediately
during suppressessuppresses
the day immediately melatonin biosynthe-
melatonin
sis by inhibiting
biosynthesis the activity
by inhibiting theofactivity
AANAT ofboth
AANAT via protein
both via dephosphorylation and gene
protein dephosphorylation
down-regulation [99]. Other factors that may impact on animal
and gene down-regulation [99]. Other factors that may impact on animal melatonin melatonin biosynthesis
include food include
biosynthesis intake, temperature
food intake,alterations,
temperature andalterations,
diseases [100].and diseases [100].
3.3. Focus
3.3. Focus onon the
the Enzymes Involved in the Biosynthetic
Biosynthetic Routes
Routes
Aspreviously
As previously described,
described, thethe biosynthetic
biosynthetic pathways involved in the synthesis of mel- mela-
atoninininboth
tonin bothplants
plantsand
andanimals
animalshavehave some
some similarities
similarities and differences,
differences, mainly
mainlyrelated
related
tothe
to theenzymes,
enzymes, their
their substrate
substrate affinity and cellular
cellular localization.
localization.Moreover,
Moreover,differently
differentlytoto
animals, plants have an additional melatonin biosynthetic pathway that proceeds fromfrom
animals, plants have an additional melatonin biosynthetic pathway that proceeds sero-
serotonin
tonin to 5-MT
to 5-MT with with the consequent
the consequent acetylation
acetylation of intermediate
of this this intermediate to melatonin
to melatonin (Fig-8).
(Figure
ure 8).
This This evidence
evidence is basedis on
based
someon recent
some recent
studies studies
in whichin which
it wasit was reported
reported howhow the the
two
two melatonin-generating
melatonin-generating sitescrosstalk
sites can can crosstalk to maintain
to maintain a stablea stable
supply supply of melatonin
of melatonin in
in plants
plantsthe
when when the chloroplast
chloroplast pathwaypathway is blocked.
is blocked. In this particular
In this particular context,
context, plants canplants
switchcanthe
switch the chloroplast-based
chloroplast-based productionproduction into mitochondria,
into mitochondria, using a biosynthetic
using a biosynthetic pathway pathway
similar to
similar
that foundto that found in However,
in animals. animals. However, some differences
some differences related to related to the and
the origin origin and ac-of
activity
tivity of enzymes have been found. In this section, the main differences
enzymes have been found. In this section, the main differences in the activity observed in the activity
observed
for plant andfor plant
animalandenzymes
animal enzymes
involvedinvolved in melatonin
in melatonin biosynthesis
biosynthesis will bewill be inves-
investigated
tigated and
and discussed. discussed.
3.3.1. L-Tryptophan Decarboxylase (TDC) (EC 4.1.1.105)
TDC is the enzyme catalyzing the conversion of 5-hidroxytryptophan into sero-
tonin [73]. It was originally identified in Catharanthus roseus as a soluble cytosolic and ho-
modimeric protein, composed by monomers having molecular weight equal to 54,000 u.m.a.
TDC of C. roseus showed higher substrate affinity to tryptophan (Km = 0.075 mmol/L)
compared to 5-hydroxytryptophan (Km = 1.3 mmol/L). On the contrary, TDC could not
accept L-DOPA (dioxyphenylalanine) or phenylalanine as substrates [101]. TDC proteins
purified from Ophiorrhiza pumila, Oryza sativa and Rauvolfia verticillata showed Km values
for tryptophan (0.72, 0.69 and 2.89 mmol/L, respectively), 10-fold higher than the values
showed for TDC purified from C. roseus. However, it is also known that other species,
including Oryza sativa, expressed multiple TDC proteins in contrast to C. roseus that has
only one isoform [102,103].
Int. J. Mol. Sci. 2021, 22, 9996 10 of 39
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 10 of 39
Figure
Figure 8. The
8. The melatonin
melatonin biosyntheticpathway
biosynthetic pathwayininmitochondria
mitochondria (orange
(orange broken
brokenarrows)
arrows)andandininchloroplasts
chloroplasts(solid green
(solid green
arrows). In plants both pathways are probably present: when the main chloroplast pathway is interrupted (like in Sekigu-
arrows). In plants both pathways are probably present: when the main chloroplast pathway is interrupted (like in
chi mutant rice) the mitochondrial pathway takes over to compensate for the lack [85,86]. TDC: L-tryptophan decarbox-
Sekiguchi mutant rice) the mitochondrial pathway takes over to compensate for the lack [85,86]. TDC: L-tryptophan
ylase; TPH: tryptophan hydroxylase; T5H: tryptamine 5-hydroxylase; SNATs: serotonin N-acetyltransferase; ASMTs: ac-
decarboxylase;
etylserotonin TPH: tryptophan hydroxylase;
O-methyltransferase; T5H:
COMT: caffeic tryptamine
acid 5-hydroxylase; SNATs: serotonin N-acetyltransferase;
3-O-methyltransferase.
ASMTs: acetylserotonin O-methyltransferase; COMT: caffeic acid 3-O-methyltransferase.
3.3.1. L-Tryptophan Decarboxylase (TDC) (EC 4.1.1.105)
3.3.2. Tryptamine 5-Hydroxylase (T5H) (EC 1.14.-.-)
TDC is the enzyme catalyzing the conversion of 5-hidroxytryptophan into serotonin
[73].Regarding the hydroxylation
It was originally reaction occurring
identified in Catharanthus roseus asduring
a soluble melatonin
cytosolic biosynthesis,
and homodi- it
ismeric
predominantly mediated in plants by two cytochrome
protein, composed by monomers having molecular weight equal to 54,000 P450-dependent monooxyge-
u.m.a.
nases
TDC(T5H and the
of C. roseus putative
showed TPH).
higher Likelyaffinity
substrate aromatic amino acid(Khydroxylase,
to tryptophan m = 0.075 mmol/L) both com-
require
tetrahydrobiopterin as a co-substrate
pared to 5-hydroxytryptophan (Km = 1.3 [104]. Tryptamine
mmol/L). 5-hydroxylase
On the contrary, TDC could(T5H) notbelongs
accept to
the cytochrome
L-DOPA P450 monooxygenase
(dioxyphenylalanine) family [105],
or phenylalanine asand it is responsible
substrates [101]. TDC forproteins
the addition
puri- of
one
fiedhydroxyl group to pumila,
from Ophiorrhiza the 5 position of tryptamine.
Oryza sativa and RauvolfiaThisverticillata
reaction leads
showed to the
Km formation
values for of
5-hydroxytryptamine
tryptophan (0.72, 0.69[67]. andT5H2.89displays
mmol/L,not only a high10-fold
respectively), substrate affinity
higher thanforthetryptamine
values
(Kshowed
m = 0.0073
for mmol/L),
TDC purifiedbut also
from a
C.high turnover
roseus. However, number
it is (K
alsocat = 45/min)
known that [105].
other However,
species,
including
T5H enzymeOryza
does sativa, expressed
not catalyze multiple TDC
the conversion proteins in into
of tryptophan contrast to C. roseus that has
5-hydroxytryptophan [105].
only
The one isoform
catalytic [102,103].
efficiency (Kcat /Km ) measured for rice T5H was 6164/min. This value was
25-fold higher than the value measured for rice TDC (Kcat /Km = 247/min) suggesting
a 3.3.2.
rapidTryptamine
conversion5-Hydroxylase
of tryptamine(T5H) (EC 1.14.-.-)
produced by TDC to serotonin (Figure 9). This data
could explain
Regarding thethe
high content of melatonin
hydroxylation in close-related
reaction occurring duringspecies
melatonin [106,107]. A suggestion
biosynthesis, it is
ofpredominantly
the existence mediated
of one or in plants
more by two cytochrome
enzymes involved inP450-dependent
the synthesis ofmonooxygenases
melatonin comes
(T5HSekiguchi
from and the putative
mutantTPH). Likely aromatic
rice, which completely amino acid
lacks in hydroxylase,
T5H activity,both but require tetra- it
nevertheless
ishydrobiopterin
able to synthetize as a melatonin
co-substrateat[104].
lowerTryptamine 5-hydroxylase
levels compared to the wild (T5H)typebelongs
[86]. to the
Further
cytochrome
analysis P450that
showed monooxygenase
Sekiguchi ricefamily [105],the
switched and it is responsible
melatonin synthetic forpathway
the additionfromofthe
one hydroxyl
classic group
plant type usingto the 5 position
a route similarof to
tryptamine.
that usedThis reaction leads
by animals, to thetryptophan
in which formation is
hydroxylated to 5-hydroxytryptophan by a tryptophan hydroxylase (TPH). On trypta-
of 5-hydroxytryptamine [67]. T5H displays not only a high substrate affinity for the other
minealthough
hand, (Km = 0.0073 mmol/L),
no animal TPHbuthomologs
also a high turnover
were number
detected (Kcatgenome
in plant = 45/min) [105].
[85], How-
other scien-
ever, T5H enzyme does not catalyze the conversion of tryptophan
tific evidences support the presence of TPH-like enzymes in plants. However, this route into 5-hydroxytrypto-
phan [105].
exhibits Thelow
a very catalytic
serotoninefficiency (Kcat/Km)flux
biosynthetic measured
rate if for rice T5Htowas
compared the6164/min.
main melatoninThis
value was 25-fold higher than the value measured for rice TDC
biosynthetic plant pathway. For example, (i) Griffonia simplicifolia seeds are notoriously(K cat/Km = 247/min) sug-
gesting
rich a rapid conversion of with
in 5-hydroxytryptophan, tryptamine
amounts produced by TDC
justifiable to serotonin
by assuming the (Figure
presence 9).of
This
TPH-
data could explain the high content of melatonin in close-related species
like enzymes [67]; (ii) in in vitro cultures of St John’s wort (Hypericum perforatum L.) was [106,107]. A sug-
gestion of the existence of one or more enzymes involved in the synthesis of melatonin
observed that the synthesis of serotonin mainly occurs via 5-hydroxytryptophan when
14C-tryptophan was added in culture medium, suggesting that it was produced from tryp-
tophan by a TPH-like enzyme [108]; (iii) the soluble fraction of rice root extracts exhibited a
tetrahydropterin-dependent amino acid hydroxylase activity, similar to TPH [109]; (iv) Parkscientific evidences support the presence of TPH-like enzymes in plants. However, this
route exhibits a very low serotonin biosynthetic flux rate if compared to the main melato-
nin biosynthetic plant pathway. For example, (i) Griffonia simplicifolia seeds are notori-
ously rich in 5-hydroxytryptophan, with amounts justifiable by assuming the presence of
TPH-like enzymes [67]; (ii) in in vitro cultures of St John’s wort (Hypericum perforatum L.)
Int. J. Mol. Sci. 2021, 22, 9996 11 of 39
was observed that the synthesis of serotonin mainly occurs via 5-hydroxytryptophan
when 14C-tryptophan was added in culture medium, suggesting that it was produced
from tryptophan by a TPH-like enzyme [108]; (iii) the soluble fraction of rice root extracts
exhibited
and a tetrahydropterin-dependent
colleagues observed a concomitant amino
increaseacid
of hydroxylase activity,
the content of similar
melatonin, to TPH
tryptamine,
[109]; (iv) Park and colleagues observed a concomitant increase of the content of
tryptophan and 5-HT in transgenic rice plants (cv. Dongjin) with transcriptionally suppres-melato-
nin,oftryptamine,
sion tryptophan
T5H with respect and
to the 5-HT
wild in [85].
type transgenic rice plants (cv. Dongjin) with tran-
scriptionally suppression of T5H with respect to the wild type [85].
Figure 9. Synthetic capacity of serotonin from tryptophan.
Figure 9. Synthetic capacity of serotonin from tryptophan.
3.3.3. Serotonin N-Acetyltransferase (SNAT) (EC 2.3.1.87)
3.3.3. Serotonin N-Acetyltransferase (SNAT) (EC 2.3.1.87)
SNAT, also known in animals with the name of aralkylamine N-acetyltransferase
SNAT, also known in animals with the name of aralkylamine N-acetyltransferase
(AANAT) [110], catalyzes the transfer of the acetyl group of Acetyl-CoA to the primary
(AANAT) [110], catalyzes the transfer of the acetyl group of Acetyl-CoA to the primary
amine of serotonin, producing CoA and N-acetylserotonin. The comparison of plant
amine of serotonin, producing CoA and N-acetylserotonin. The comparison of plant
SNATs and animal AANAT revealed clear differences in enzyme kinetics. Concerning the
SNATs and animal AANAT revealed clear differences in enzyme kinetics. Concerning
catalytic activity (Vmax/Km), sheep AANAT activity is 10-fold higher than rice SNAT. More-
the catalytic
over, it was activity
shown that (Vmax
SNATs/Km ),can sheep AANAT
accept variousactivity is 10-fold higher
amine substrates, includingthan rice SNAT.
serotonin,
Moreover, it was shown that SNATs can accept various amine
tryptamine, and 5-MT, with different affinity. In particular, the preferred substrate ofsubstrates, including sero-
tonin,
AANAT is serotonin, whereas for SNAT is generally 5-MT. In rice SNAT has comparable of
tryptamine, and 5-MT, with different affinity. In particular, the preferred substrate
AANAT
affinity is forserotonin,
serotonin whereas
and 5-MT for(KSNAT is generally 5-MT. In rice SNAT has comparable
m = 0.385 mmol/L, 0.375mmol/L), while in A. thaliana
affinity
SNAT affinity for 5-MT is sixfold higher thanmmol/L,
for serotonin and 5-MT (K m = 0.385 0.375mmol/L),
that of serotonin (Km = 0.051 while in A. 0.309
mmol/L, thaliana
SNAT affinity for 5-MT is sixfold higher than that of serotonin
mmol/L) [70]. Moreover, considering the turnover number of SNAT in A. thaliana, the cat- (K m = 0.051 mmol/L,
0.309
alyticmmol/L)
efficiency [70].
(KcatMoreover,
/Km) for the considering
conversionthe ofturnover
5-MT to number
serotoninofisSNAT23-fold A. thaliana,
in higher thanthe
catalytic efficiency
that recorded for the (K /K ) for
catconversion
m the conversion of 5-MT to serotonin is 23-fold
of serotonin into melatonin. The genes encoding for higher than
that recorded for the conversion of serotonin into melatonin. The
SNATs have been isolated and identified in many vertebrates, yeasts and bacteria. Re- genes encoding for SNATs
have been
cently, theyisolated andfound
were also identified in many
in different vertebrates,
algae and plants, yeasts and bacteria.
including Recently,
Chlamydomonas they
rein-
were also
hardtii found
[111], in different
Pyropia yezoensisalgae
[112],and plants,
Oryza sativaincluding Chlamydomonas
[113], Arabidopsis reinhardtii
thaliana [84] and Pinus [111],
Pyropia yezoensis
taeda [114]. In plants, Oryza sativa
[112], SNATs were [113],
mainly Arabidopsis
identified thaliana [84] and
in chloroplast Pinus taedaOn
[84,111,113]. [114].
the In
plants, SNATs
other hand, thewere mainly
enzyme AANATidentified in chloroplast
from vertebrates shows[84,111,113].
homology On withthean other hand, the
alphaproteo-
enzyme
bacterial AANAT
enzymefrom and itvertebrates
is inheritedshows homology with
via mitochondria [115].anBased
alphaproteobacterial
on the theory of endo-enzyme
symbiosis
and proposed
it is inherited viaby Sagan, α-proteobacteria
mitochondria [115]. Basedare onthe
theprecursors of mitochondria,proposed
theory of endosymbiosis while
bycyanobacteria of chloroplastsare
Sagan, α-proteobacteria [116].
theThis theory led
precursors of to the hypothesiswhile
mitochondria, that these organelles of
cyanobacteria
inherited the[116].
chloroplasts melatonin
This synthetic
theory led machinery from their that
to the hypothesis prokaryotic ancestors. inherited the
these organelles
melatonin synthetic machinery from their prokaryotic ancestors.
Despite the similar enzymatic activity of SNAT and AANAT, the major difference is
related to the stabilization mechanism of these enzymes. Indeed, AANAT contains regula-
tory flanking regions which have not been identified in plant SNATs. In several mammals,
especially primates and ungulates, the stabilization of AANAT prevails over transcriptional
up-regulation of the gene and it is decisive for post-transcriptional regulation of circadian
AANAT rhythmicity [117].
3.3.4. Acetylserotonin O-Methyltransferase (ASMT) (EC 2.1.1.4)
ASMT is an enzyme catalyzing the final reaction in melatonin biosynthesis, converting
N-acetylserotonin to melatonin. It is also known as hydroxyindole-O-methyltransferase
(HIOMT) [118]. Plant ASMTs lack in homology with the animal isoforms, causing the
impossibility to identify and clone them until 2011 [119]. In the same year, the gene se-
quence of the first ASMT was identified in rice, and the protein was finally purified. The
purified recombinant rice ASMT displayed low enzyme activities for N-acetylserotonin
at 30 ◦ C (Km = 0.864 mmol/L; Vmax = 0.21 pkat/mg protein) [120], showing instead 2800-
fold higher catalytic efficiency at the optimal temperature of 55 ◦ C (Km = 0.222 mmol/L;You can also read
