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Citation: Lee, K.; Back, K. Escherichia
coli RimI Encodes Serotonin
N-Acetyltransferase Activity and Its
Overexpression Leads to Enhanced
Growth and Melatonin Biosynthesis.
Biomolecules 2023,13, 908. https://
doi.org/10.3390/biom13060908
Academic Editor: Christophe
Ribelayga
Received: 9 May 2023
Revised: 25 May 2023
Accepted: 27 May 2023
Published: 30 May 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
biomolecules
Article
Escherichia coli RimI Encodes Serotonin N-Acetyltransferase
Activity and Its Overexpression Leads to Enhanced Growth
and Melatonin Biosynthesis
Kyungjin Lee and Kyoungwhan Back *
Department of Molecular Biotechnology, College of Agriculture and Life Sciences, Chonnam National University,
Gwangju 61186, Republic of Korea; nicekj7@hanmail.net
*Correspondence: kback@chonnam.ac.kr; Tel.: +82-62-530-2165
Abstract:
Serotonin N-acetyltransferase (SNAT) functions as the penultimate or final enzyme in
melatonin biosynthesis, depending on the substrate. The Escherichia coli orthologue of archaeal SNAT
from Thermoplasma volcanium was identified as RimI (EcRimI), with 42% amino acid similarity to
archaeal SNAT. EcRimI has been reported to be an N-acetyltransferase enzyme. Here, we investigated
whether EcRimI also exhibits SNAT enzyme activity. To achieve this goal, we purified recombinant
EcRimI and examined its SNAT enzyme kinetics. As expected, EcRimI showed SNAT activity toward
various amine substrates including serotonin and 5-methoxytryptamine, with K
m
and V
max
values of
531
µ
M and 528 pmol/min/mg protein toward serotonin and 201
µ
M and 587 pmol/min/mg protein
toward 5-methoxytryptamine, respectively. In contrast to the rimI mutant E. coli strain that showed no
growth defect, the EcRimI overexpression strain exhibited a 2-fold higher growth rate than the control
strain after 24 h incubation in nutrient-rich medium. The EcRimI overexpression strain produced
more melatonin than the control strain in the presence of 5-methoxytryptamine. The enhanced growth
effect of EcRimI overexpression was also observed under cadmium stress. The higher growth rate
associated with EcRimI expression was attributed to increased protein N-acetyltransferase activity,
increased synthesis of melatonin, or the combined effects of both.
Keywords:
archaea; Escherichia coli;N-acetylserotonin; protein acetylation; RimI; melatonin;
5-methoxytryptamine
1. Introduction
Melatonin is a universal molecule present in almost all living organisms, including
bacteria, archaea, plants, and animals [
1
–
3
]. Its primary identified function is associated
with its potent antioxidant activity, as one molecule of melatonin can scavenge free radicals
in a reaction cascade consuming up to 10 free radicals [
4
,
5
]. In addition to its intrinsic antiox-
idant activity, melatonin plays an important biological role in a number of species-specific
functions. For example, it is a well-known pineal hormone that regulates the circadian
rhythm and seasonal reproduction in animals [
6
,
7
]. By contrast, in plants, it acts as a master
regulator of growth and development, affecting seed germination [
8
,
9
], photomorphogene-
sis [
10
], flowering [
11
,
12
], senescence [
13
], and grain yield [
14
,
15
] in concert with numerous
plant hormones [
16
,
17
]. It also plays a pivotal role in alleviating plant damage resulting
from various environment stresses, including abiotic and biotic stresses, either through
enhancement of antioxidant activities and various defense genes
[18,19]
or improvement
of protein quality control [
12
,
20
]. Melatonin is synthesized by bacteria, and appears to
be involved in detoxifying reactive oxygen species to prevent free radical attack [
21
–
23
],
although it has also been reported to inhibit the growth of plant-pathogenic bacteria [24].
In melatonin biosynthesis, the aromatic amino acid tryptophan serves as the initial
substrate and melatonin is synthesized via four enzymatic reactions with tryptophan
5-hydroxylase (TPH), aromatic amino acid decarboxylase (tryptophan decarboxylase),
Biomolecules 2023,13, 908. https://doi.org/10.3390/biom13060908 https://www.mdpi.com/journal/biomolecules
Biomolecules 2023,13, 908 2 of 15
serotonin N-acetyltransferase (SNAT; also named arylakylamine N-acetyltransferase), and
N-acetylserotonin O-methyltransferase (ASMT) in animals. Analogously, plants also em-
ploy four enzymes, but TPH is replaced with tryptamine 5-hydroxylase, which catalyzes
tryptamine into serotonin [
25
]. Among these four enzymes, SNAT plays a key role in
melatonin biosynthesis, functioning as either the penultimate or final enzyme of melatonin
biosynthesis, depending on substrate, in both animals and plants [
2
,
25
]. SNAT acetylates
serotonin and 5-methoxytryptamine into N-acetylserotonin and melatonin, respectively [
26
].
In 1995, the first SNAT gene was cloned from sheep using a cDNA expression library [
27
],
and its orthologues have been cloned and characterized from a wide range of species
including Gram-positive bacteria and fungi but not from higher plants, nematodes, or
arthropods [
28
]. Later, the first plant SNAT gene was cloned from the rice GCN5-related
N-acetyltransferase (GNAT) family [
29
]. As expected, the SNAT protein from rice did not
exhibit sequence homology with that from animals. The possible ancestor of the plant
SNAT gene has been cloned and characterized in cyanobacteria [30].
Despite reports of SNAT genes from a diverse array of species, the presence of SNAT
in archaea has long remained a mystery. Surprisingly, the archaeal SNAT gene from Thermo-
plasma volcanium was recently cloned [
3
] and a human orthologue was discovered [
31
]. This
archaeal SNAT also belongs to the GNAT family, but it is classified as a new clade of SNAT,
distinct from those of animals and plants. In particular, the Gram-negative bacterium
Escherichia coli had long been reported to produce melatonin [
32
], but no SNAT homolog
genes have yet been described. However, based on the discovery of archaeal SNAT, we
identified an orthologous gene in E.coli.
In this study, we selected E.coli RimI (EcRimI), an archaeal SNAT orthologue expressed
in E.coli, and purified recombinant EcRimI. We found that EcRimI possessed SNAT enzyme
activity. Furthermore, its overexpression of EcRimI in E.coli was functionally linked to
enhanced melatonin biosynthesis in the presence of 5-methoxytryptamine. The EcRimI
overexpression strain of E.coli exhibited enhanced growth at 28 and 37
◦
C compared to the
control strain. We concluded that enhanced synthesis of melatonin in the EcRimI overex-
pression strain was in part responsible for the enhanced growth rate due to ameliorating
of starvation and stationary-phase stress, although we cannot rule out a possible role of
protein acetylation.
2. Materials and Methods
2.1. Synthesis of Escherichia coli RimI Gene
Based on the nucleotide sequence information of E. coli RimI (GenBank accession
number WP_137442509), the full-length EcRimI gene with the length of 447 bp was custom-
synthesized at Bioneer (Daejeon, Republic of Korea).
2.2. Escherichia coli Expression and Purification of Recombinant EcRimI Protein
The full-length synthetic EcRimI gene was amplified by PCR using primer set (RimI
forward primer, 5
0
-GCC ATG GGA AAC ACG ATT TCT TCC CTC GAA-3
0
;RimI reverse
primer, 5
0
-CTC GAG CAT ACT GAT TGG CAA CGC-3
0
) with a template plasmid containing
EcRimI DNA such as pBHA-RimI, which was provided by Bioneer. The PCR product was
ligated into the TA vector (RBC Bioscience, New Taipei City, Taiwan) followed by NcoI and
XhoI digestion. The NcoI and XhoI insert of EcRimI was then ligated into the same restriction
sites of the E. coli expression vector pET28b (Invitrogen, Carlsbad, CA, USA), leading to
the generation of pET28b-RimI. The pET28b-RimI plasmid was transformed into E. coli
strain BL21(DE3) (Invitrogen). An overnight culture (10 mL) grown in Lennox broth (LB)
medium (10 g/L pancreatic digest of casein, 5 g/L yeast extract, and 5 g/L NaCl) containing
antibiotic kanamycin (50 mg/L) was inoculated into 100 mL of Terrific broth (TB) medium
[20 g/L Bacto-tryptone, 24 g/L Bacto-yeast extract, glycerol 4 mL/L, and phosphate buffer
(0.017 M KH
2
PO
4
and 0.072 M K
2
HPO
4
)] containing 50 mg/L kanamycin and incubated
at 37
◦
C for 4 h, followed by the addition of 1 mM isopropyl-
β
-D-thiogalactopyranoside
(IPTG; Sigma, St. Louis, MO, USA). The culture was further grown at 24
◦
C and shaken
Biomolecules 2023,13, 908 3 of 15
at 180 rpm for 12 h. The purification procedure using affinity (Ni
2+
) chromatography was
performed according to the manufacturer’s recommendations (Qiagen, Tokyo, Japan).
2.3. Homology Analysis
The analysis of amino acid sequence homology was carried out using the BLASTp tool
in the non-redundant protein sequences databases at the National Center for Biotechnology
Information (http://www.ncbi.nlm.nih.gov/, accessed on 3 September 2019). Phylogenetic
tree analysis was performed using the BLAST-Explorer program [33].
2.4. Measurement of Serotonin N-Acetyltransferase Enzyme Kinetics
The purified recombinant EcRimI protein (3
µ
g or varying concentrations) was incu-
bated in a total volume of 100
µ
L containing 0.5 mM serotonin (or other substrates) and
0.5 mM acetyl-CoA in 100 mM potassium phosphate (pH 8.8 or varying pH) at 55
◦
C (or
other temperatures) for 30 min. Enzymatic reaction products such as N-acetylserotonin,
N-acetyltryptamine, N-acetyltyramine, and melatonin were subjected to high-performance
liquid chromatography (HPLC) analysis as described previously [
11
]. Lineweaver–Burk
plots were employed to calculate substrate affinity (K
m
) and the maximum reaction rate
(V
max
) using two substrates such as serotonin and 5-methoxytryptamine. Protein concen-
trations were determined using the Bradford method and a protein assay dye (Bio-Rad,
Hercules, CA, USA). The analysis was performed in triplicate.
2.5. Growth Measurement of Escherichia coli
A 1 mL seed culture that had been incubated overnight at 37
◦
C in Lennox broth (LB)
medium (10 g/L pancreatic digest of casein, 5 g/L yeast extract, 5 g/L NaCl) containing
50 mg/L kanamycin was inoculated into 10 mL Terrific broth (TB) medium containing
50 mg/L kanamycin in 40 mL polypropylene conical tubes (SPL Life Science, Pocheon-si,
Republic of Korea) and continuously cultured at various temperatures. The absorbance
at 600 nm of the culture was measured using a spectrophotometer (MicroDigital Nabi,
GyungGi, Republic of Korea) periodically until 24 h.
2.6. Melatonin Measurement in Escherichia coli
One hundred microliters of seed culture incubated overnight at 37
◦
C in LB medium
containing 50 mg/L kanamycin was inoculated into 1 mL LB medium containing 50 mg/L
kanamycin and incubated at 37 ◦C until the optical density of the E.coli culture at 600 nm
(OD600) reached 1.0. After the addition of 1 mM isopropyl-
β
-D-thiogalactopyranoside
and 1 mM 5-methoxytryptamine, the culture was grown and shaken at 250 rpm at varying
temperatures such as 28, 37, and 42
◦
C for the indicated time periods. The resulting cul-
tures were centrifuged at 11,500
×
gfor 5 min and separated into cell pellet and medium
(supernatant) fractions. The medium fractions (0.2 mL) were mixed with 0.2 mL of 100%
methanol. The resulting 10
µ
L aliquots were subjected to high-performance liquid chro-
matography (HPLC) using a fluorescence detector system (Waters, Milford, MA, USA) as
described previously [34].
2.7. Cadmium Treatment of Escherichia coli
One hundred microliters of seed culture incubated overnight at 37
◦
C in LB medium
containing 50 mg/L kanamycin was inoculated into 1 mL LB medium containing 50 mg/L
kanamycin and incubated at 37 ◦C until the optical density of the E.coli culture at 600 nm
(OD
600
) reached either 0.5 or 1.0. After adding 1 or 5 mM cadmium chloride (CdCl
2
),
the culture was grown at 37
◦
C for 20 h with shaking at 250 rpm. The absorbance of the
resulting cultures was measured at 600 nm using a spectrophotometer (MicroDigital Nabi).
2.8. Statistical Analysis
The data were analyzed by analysis of variance using IBM SPSS Statistics 23 software
(IBM Corp., Armonk, NY, USA). Means with different letters indicate significantly different
Biomolecules 2023,13, 908 4 of 15
values at p< 0.05 according to Tukey’s post hoc honestly significant difference (HSD) test.
Data are presented as means ±standard deviations.
3. Results
3.1. Gene Selection and Synthesis of the Escherichia coli RimI Gene
The BLASTp program (http://www.ncbi.nih.gov/, accessed on 3 November 2019)
revealed that the archaeal SNAT (TvSNAT) protein [
3
] had ~23% identity and ~42% simi-
larity to the RimI protein of E.coli (EcRimI), which encodes the protein N-acetyltransferase
enzyme (Figure 1A). EcRimI acetylates a number of proteins, including ribosomal protein
S18 [
35
], translation elongation factor Tu [
36
], and other proteins [
37
]. The human protein
N-acetyltransferase Naa50, an orthologue of archaeal SNAT, shows SNAT enzyme activ-
ity [
31
], indicating that E.coli RimI was likely to also exhibit SNAT activity. Phylogenetic
analysis indicated that EcRimI belonged to the SNAT family and was positioned between
the plant and archaeal SNAT subfamilies, but much closer to the archaeal subfamily than
the plant SNAT subfamily (Figure 1B). The full-length EcRimI nucleotide sequence was
synthesized in accordance with sequence information reported in the GenBank database
(WP_137442509).
Biomolecules 2023, 13, x FOR PEER REVIEW 4 of 15
culture was grown at 37 °C for 20 h with shaking at 250 rpm. The absorbance of the re-
sulting cultures was measured at 600 nm using a spectrophotometer (MicroDigital Nabi).
2.8. Statistical Analysis
The data were analyzed by analysis of variance using IBM SPSS Statistics 23 software
(IBM Corp., Armonk, NY, USA). Means with different letters indicate significantly differ-
ent values at p < 0.05 according to Tukey’s post hoc honestly significant difference (HSD)
test. Data are presented as means ± standard deviations.
3. Results
3.1. Gene Selection and Synthesis of the Escherichia coli RimI Gene
The BLASTp program (http://www.ncbi.nih.gov/, accessed on 3 November 2019) re-
vealed that the archaeal SNAT (TvSNAT) protein [3] had ~23% identity and ~42% similar-
ity to the RimI protein of E. coli (EcRimI), which encodes the protein N-acetyltransferase
enzyme (Figure 1A). EcRimI acetylates a number of proteins, including ribosomal protein
S18 [35], translation elongation factor Tu [36], and other proteins [37]. The human protein
N-acetyltransferase Naa50, an orthologue of archaeal SNAT, shows SNAT enzyme activ-
ity [31], indicating that E. coli RimI was likely to also exhibit SNAT activity. Phylogenetic
analysis indicated that EcRimI belonged to the SNAT family and was positioned between
the plant and archaeal SNAT subfamilies, but much closer to the archaeal subfamily than
the plant SNAT subfamily (Figure 1B). The full-length EcRimI nucleotide sequence was
synthesized in accordance with sequence information reported in the GenBank database
(WP_137442509).
Figure 1. (A) Amino acid sequence alignment of TvSNAT and E. coli RimI (SNAT). The conserved
acetyl coenzyme A binding sites are underlined. Plus signs (+) denote similar amino acids. Dashes
denote gaps. (B) Phylogenetic tree showing E. coli SNAT, archaeal orthologues and rice SNAT genes.
The scale bar represents 0.3 substitutions per site. GenBank accession numbers are: TvSNAT
(NC_002689), E. coli RimI (WP_137442509), rice SNAT1 (AK059369), rice SNAT2 (AK068156), hu-
man Naa50 (BAB14397), and Arabidopsis Naa50 (NM_121172).
Figure 1.
(
A
) Amino acid sequence alignment of TvSNAT and E.coli RimI (SNAT). The conserved
acetyl coenzyme A binding sites are underlined. Plus signs (+) denote similar amino acids. Dashes
denote gaps. (
B
) Phylogenetic tree showing E.coli SNAT, archaeal orthologues and rice SNAT
genes. The scale bar represents 0.3 substitutions per site. GenBank accession numbers are: TvSNAT
(NC_002689), E.coli RimI (WP_137442509), rice SNAT1 (AK059369), rice SNAT2 (AK068156), human
Naa50 (BAB14397), and Arabidopsis Naa50 (NM_121172).
3.2. Enzyme Kinetic Analysis of Recombinant EcRimI
The synthetic full-length EcRimI gene was cloned for expression as a fusion protein
with a C-terminal hexa-histidine tag, followed by Ni
2+
affinity purification, as illustrated in
Figure 2A. The purified recombinant EcRimI protein was first investigated for the capacity
to catalyze serotonin into N-acetylserotonin (NAS). As shown in Figure 2B, NAS was
produced
in vitro
by recombinant EcRimI protein in a concentration-dependent manner.
The optimal SNAT activity was observed at a temperature of 55
◦
C and half-peak activity
occurred at 45
◦
C. SNAT activity at 37
◦
C was a quarter of that at 55
◦
C. No NAS was
Biomolecules 2023,13, 908 5 of 15
produced at 70
◦
C, which was consistent with archaeal SNAT [
3
]. EcRimI exhibited peak
activity at pH 8.8, followed by pH 7.8 and pH 6.5, and very low SNAT activity was
observed at pH 5.5. This pH preference of EcRimI SNAT activity was identical to that
of archaeal SNAT, but differed from human Naa50, which has peak SNAT activity at
pH 7.8 [
3
,
31
]. In addition to serotonin, several other amines were accepted as substrates.
The maximum SNAT activity occurred with 5-methoxytryptamine, which was catalyzed
into melatonin by the EcRimI enzyme, followed in rank order by tryptamine, serotonin,
and tyramine. The substrate preference order of EcRimI differed from that of archaeal
SNAT, in which tyramine is the optimal substrate and 5-methoxytryptamine is the least
favorable substrate [
3
]. Tyramine has been reported as the optimal substrate for sheep
SNAT and rice SNAT1 [
38
]. In terms of the possible acceptance of polyamines such as
spermidine and octopamine as RimI substrates, we conducted an SNAT inhibition assay
(0.5 mM serotonin) in the presence of each substrate at various concentrations to determine
whether SNAT activity was affected by the presence of these polyamines. These inhibition
analyses were performed because no standard compounds of N-acetylspermidine and
N-acetyloctopamine are commercially available. As shown in Figure 3, spermidine did not
inhibit SNAT activity at concentrations up to 1 mM under the 45
◦
C temperature condition.
Similarly, octopamine slightly inhibited SNAT activity at 0.2 mM, but enhanced SNAT
activity at 0.5 mM, shifting to 90% inhibition at 1 mM (Figure 3A). Under the 55
◦
C assay
condition, spermidine enhanced SNAT activity, whereas octopamine did not alter SNAT
activity except at 0.5 mM, where it showed a 50% inhibition rate (Figure 3B). Collectively,
E.coli SNAT (EcSNAT or EcRimI) likely did not accept polyamines as substrates, in sharp
contrast to archaeal SNAT, which accepts polyamines as substrates [3].
The K
m
and V
max
values of EcRimI toward serotonin as a substrate were 531
µ
M and
528 pmol/min/mg protein, respectively (Figure 4A). For the substrate 5-methoxytryptamine,
EcRimI exhibited K
m
and V
max
values of 201
µ
M and 587 pmol/min/mg protein, re-
spectively (Figure 4B). The catalytic efficiency (V
max
/K
m
) was 3-fold higher toward 5-
methoxytryptamine than toward serotonin, suggesting that EcRimI preferred 5-
methoxytryptamine to serotonin as a substrate for melatonin biosynthesis. The enzyme
kinetics values of EcRimI were very similar to those of archaeal SNAT, supporting EcRimI
as an archaeal SNAT orthologue. By contrast, human Naa50, a known archaeal SNAT
orthologue, exhibited much higher K
m
and V
max
values for serotonin of 986
µ
M and
1800 pmol/min/mg protein, respectively. Based on the data presented above regarding
the enzymatic properties of SNAT, EcRimI possessed SNAT activity, in accordance with
archaeal SNAT and human Naa50 [
3
,
31
]. However, the exact mechanism through which
EcRimI synthesizes melatonin in E.coli awaits further in-depth analysis, particularly in
view of the dual roles of EcRimI in melatonin synthesis and protein N-acetylation.
Biomolecules 2023,13, 908 6 of 15
Biomolecules 2023, 13, x FOR PEER REVIEW 5 of 15
3.2. Enzyme Kinetic Analysis of Recombinant EcRimI
The synthetic full-length EcRimI gene was cloned for expression as a fusion protein
with a C-terminal hexa-histidine tag, followed by Ni2+ affinity purification, as illustrated
in Figure 2A. The purified recombinant EcRimI protein was first investigated for the ca-
pacity to catalyze serotonin into N-acetylserotonin (NAS). As shown in Figure 2B, NAS
was produced in vitro by recombinant EcRimI protein in a concentration-dependent man-
ner. The optimal SNAT activity was observed at a temperature of 55 °C and half-peak
activity occurred at 45 °C. SNAT activity at 37 °C was a quarter of that at 55 °C. No NAS
was produced at 70 °C, which was consistent with archaeal SNAT [3]. EcRimI exhibited
peak activity at pH 8.8, followed by pH 7.8 and pH 6.5, and very low SNAT activity was
observed at pH 5.5. This pH preference of EcRimI SNAT activity was identical to that of
archaeal SNAT, but differed from human Naa50, which has peak SNAT activity at pH 7.8
[3,31]. In addition to serotonin, several other amines were accepted as substrates. The
maximum SNAT activity occurred with 5-methoxytryptamine, which was catalyzed into
melatonin by the EcRimI enzyme, followed in rank order by tryptamine, serotonin, and
tyramine. The substrate preference order of EcRimI differed from that of archaeal SNAT,
in which tyramine is the optimal substrate and 5-methoxytryptamine is the least favorable
substrate [3]. Tyramine has been reported as the optimal substrate for sheep SNAT and
rice SNAT1 [38]. In terms of the possible acceptance of polyamines such as spermidine
and octopamine as RimI substrates, we conducted an SNAT inhibition assay (0.5 mM ser-
otonin) in the presence of each substrate at various concentrations to determine whether
SNAT activity was affected by the presence of these polyamines. These inhibition analyses
were performed because no standard compounds of N-acetylspermidine and N-acety-
loctopamine are commercially available. As shown in Figure 3, spermidine did not inhibit
SNAT activity at concentrations up to 1 mM under the 45 °C temperature condition. Sim-
ilarly, octopamine slightly inhibited SNAT activity at 0.2 mM, but enhanced SNAT activ-
ity at 0.5 mM, shifting to 90% inhibition at 1 mM (Figure 3A). Under the 55 °C assay con-
dition, spermidine enhanced SNAT activity, whereas octopamine did not alter SNAT ac-
tivity except at 0.5 mM, where it showed a 50% inhibition rate (Figure 3B). Collectively, E.
coli SNAT (EcSNAT or EcRimI) likely did not accept polyamines as substrates, in sharp
contrast to archaeal SNAT, which accepts polyamines as substrates [3].
Figure 2. Affinity purification and enzymatic characteristics of E. coli RimI (EcRimI) protein. (A)
Purification of C-terminal 6× His-tagged EcRimI protein. E. coli BL21 (DE3) cells harboring the
Figure 2.
Affinity purification and enzymatic characteristics of E. coli RimI (EcRimI) protein. (
A
) Pu-
rification of C-terminal 6
×
His-tagged EcRimI protein. E. coli BL21 (DE3) cells harboring the pET28b-
EcRimI plasmid were induced with isopropyl
β
-D-1-thiogalactopyranoside (IPTG) for 24 h at 24
◦
C.
M, molecular mass standards; lane 1, total protein in 30
µ
L bacterial culture without IPTG; lane
2, total proteins in 30
µ
L bacterial culture with IPTG; lane 3, 10
µ
g soluble protein; lane 4, 5
µ
g
protein purified through affinity chromatography. Protein samples were separated using 12% sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie blue.
Serotonin N-acetyltransferase enzyme activity as a function of (
B
) protein concentration, (
C
) tempera-
ture, (
D
) pH, and (
E
) substrate. Recombinant purified EcRimI (3
µ
g) was assayed for 0.5 h at 55
◦
C
(varying temperature or pH) in the presence of 0.5 mM serotonin (or other substrate) and 0.5 mM
acetyl coenzyme A, followed by high-performance liquid chromatography (HPLC) detection. NAS
represents N-acetylserotonin. Values are means ±standard deviation (SD; n= 3).
Biomolecules 2023, 13, x FOR PEER REVIEW 6 of 15
pET28b-EcRimI plasmid were induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) for 24 h
at 24 °C. M, molecular mass standards; lane 1, total protein in 30 μL bacterial culture without IPTG;
lane 2, total proteins in 30 μL bacterial culture with IPTG; lane 3, 10 μg soluble protein; lane 4, 5 μg
protein purified through affinity chromatography. Protein samples were separated using 12% so-
dium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie
blue. Serotonin N-acetyltransferase enzyme activity as a function of (B) protein concentration, (C)
temperature, (D) pH, and (E) substrate. Recombinant purified EcRimI (3 μg) was assayed for 0.5 h
at 55 °C (varying temperature or pH) in the presence of 0.5 mM serotonin (or other substrate) and
0.5 mM acetyl coenzyme A, followed by high-performance liquid chromatography (HPLC) detec-
tion. NAS represents N-acetylserotonin. Values are means ± standard deviation (SD; n = 3).
Figure 3. Dose-dependent inhibition of SNAT enzyme activity of recombinant EcRimI by polyam-
ines. (A) SNAT enzyme inhibition in the presence of either spermidine or octopamine at 45 °C. (B)
SNAT enzyme inhibition in the presence of either spermidine or octopamine at 55 °C. SNAT enzyme
activity was measured in the presence of various levels of polyamines and 0.5 mM serotonin. SNAT
activity is expressed as a percentage relative to that in the absence of polyamines. Values are means
± SD (n = 3).
The Km and Vmax values of EcRimI toward serotonin as a substrate were 531 μM and
528 pmol/min/mg protein, respectively (Figure 4A). For the substrate 5-methoxytrypta-
mine, EcRimI exhibited Km and Vmax values of 201 μM and 587 pmol/min/mg protein, re-
spectively (Figure 4B). The catalytic efficiency (Vmax/Km) was 3-fold higher toward 5-meth-
oxytryptamine than toward serotonin, suggesting that EcRimI preferred 5-methoxytryp-
tamine to serotonin as a substrate for melatonin biosynthesis. The enzyme kinetics values
of EcRimI were very similar to those of archaeal SNAT, supporting EcRimI as an archaeal
SNAT orthologue. By contrast, human Naa50, a known archaeal SNAT orthologue, exhib-
ited much higher Km and Vmax values for serotonin of 986 μM and 1800 pmol/min/mg pro-
tein, respectively. Based on the data presented above regarding the enzymatic properties
of SNAT, EcRimI possessed SNAT activity, in accordance with archaeal SNAT and human
Naa50 [3,31]. However, the exact mechanism through which EcRimI synthesizes melato-
nin in E. coli awaits further in-depth analysis, particularly in view of the dual roles of
EcRimI in melatonin synthesis and protein N-acetylation.
Figure 3.
Dose-dependent inhibition of SNAT enzyme activity of recombinant EcRimI by polyamines.
(
A
) SNAT enzyme inhibition in the presence of either spermidine or octopamine at 45
◦
C. (
B
) SNAT
enzyme inhibition in the presence of either spermidine or octopamine at 55
◦
C. SNAT enzyme activity
was measured in the presence of various levels of polyamines and 0.5 mM serotonin. SNAT activity is
expressed as a percentage relative to that in the absence of polyamines. Values are
means ±SD
(
n= 3
).
Biomolecules 2023,13, 908 7 of 15
Biomolecules 2023, 13, x FOR PEER REVIEW 7 of 15
Figure 4. Determination of Km and Vmax values for recombinant EcRimI toward the substrates (A)
serotonin and (B) 5-methoxytryptamine. Km and Vmax values were determined using Lineweaver–
Burk plots at 55 °C.
3.3. Escherichia coli Growth Curves for the EcRimI Overexpression Strain
The E. coli rimI mutant strain exhibits moderate growth retardation in minimal me-
dium, but this growth difference is nonsignificant in nutrient-rich medium such as LB
medium [36]. Importantly, as temperature strongly affects the solubility of the recombi-
nant protein expressed in E. coli [39], we tested a wide range of temperatures for compar-
ison of E. coli growth between the control (pET28b) and overexpression (pET28b-RimI)
strains. In contrast to the previous study with the rimI mutant strain, we used nutrient-
rich media such as Terrific broth (TB) medium to examine the growth effects of EcRimI
overexpression. A significant growth disadvantage of the EcRimI overexpression strain
relative to the control strain was apparent during the first 8 h of culture, but this growth
retardation of the EcRimI overexpression strain was overcome at 12 h, and 2-fold higher
growth was achieved at 24 h under the 28 °C culture temperature condition (Figure 5A).
The growth recovery effect in the EcRimI overexpression strain was faster and more pro-
nounced at the 37 °C culture temperature than at 28 °C. The EcRimI overexpression strain
grew more slowly than the control during the first 4 h incubation and reached an equal
growth rate comparable to the control strain at 6 h, then grew faster than the control strain
from 8 h, and finally showed more than 2-fold higher growth than the control strain at 24
h (Figure 5B). At the 42 °C culture temperature, a similar growth disadvantage of the
EcRimI overexpression strain was observed until 8 h, and OD600 values equal to those of
the control strain were achieved at 12 h. At the end of 24 h of culturing at 42 °C, the growth
advantage of EcRimI overexpression was marginal compared to that at 28 and 37 °C (Fig-
ure 5C). These data clearly indicated that EcRimI overexpression conferred a strong ability
to enhance E. coli growth at the later stage of bacterial cultivation compared to the control
strain, although whether these positive effects resulted from either protein N-acetylation
or melatonin synthesis remained unclear. This mechanism should be investigated in the
near future.
Figure 4.
Determination of K
m
and V
max
values for recombinant EcRimI toward the substrates
(
A
) serotonin and (
B
) 5-methoxytryptamine. K
m
and V
max
values were determined using Lineweaver–
Burk plots at 55 ◦C.
3.3. Escherichia coli Growth Curves for the EcRimI Overexpression Strain
The E.coli rimI mutant strain exhibits moderate growth retardation in minimal
medium, but this growth difference is nonsignificant in nutrient-rich medium such as
LB medium [
36
]. Importantly, as temperature strongly affects the solubility of the recombi-
nant protein expressed in E.coli [
39
], we tested a wide range of temperatures for comparison
of E.coli growth between the control (pET28b) and overexpression (pET28b-RimI) strains.
In contrast to the previous study with the rimI mutant strain, we used nutrient-rich media
such as Terrific broth (TB) medium to examine the growth effects of EcRimI overexpression.
A significant growth disadvantage of the EcRimI overexpression strain relative to the con-
trol strain was apparent during the first 8 h of culture, but this growth retardation of the
EcRimI overexpression strain was overcome at 12 h, and 2-fold higher growth was achieved
at 24 h under the 28
◦
C culture temperature condition (Figure 5A). The growth recovery
effect in the EcRimI overexpression strain was faster and more pronounced at the 37
◦
C
culture temperature than at 28
◦
C. The EcRimI overexpression strain grew more slowly than
the control during the first 4 h incubation and reached an equal growth rate comparable
to the control strain at 6 h, then grew faster than the control strain from 8 h, and finally
showed more than 2-fold higher growth than the control strain at 24 h (Figure 5B). At the
42
◦
C culture temperature, a similar growth disadvantage of the EcRimI overexpression
strain was observed until 8 h, and OD
600
values equal to those of the control strain were
achieved at 12 h. At the end of 24 h of culturing at 42 ◦C, the growth advantage of EcRimI
overexpression was marginal compared to that at 28 and 37
◦
C (Figure 5C). These data
clearly indicated that EcRimI overexpression conferred a strong ability to enhance E.coli
growth at the later stage of bacterial cultivation compared to the control strain, although
whether these positive effects resulted from either protein N-acetylation or melatonin
synthesis remained unclear. This mechanism should be investigated in the near future.
3.4. Melatonin Production in Escherichia coli
E.coli produces small amounts of melatonin (>1 ng/mL) [
34
]. To identify melatonin
in E.coli, 5-methoxytryptamine, a direct substrate of melatonin production by SNAT,
was added to the culture medium. After 24 h incubation in the presence of 1 mM 5-
methoxytryptamine and 1 mM isopropyl
β
-D-1-thiogalactopyranoside (IPTG) at various
temperatures, melatonin levels were quantified in the medium fraction of the E.coli culture,
as the majority of melatonin was found in the medium fraction rather than in E.coli cells [
34
].
As shown in Figure 6A, the control E.coli strain (pET28b) also produced melatonin at
concentrations of 383, 322, and 301 ng/mL at the incubation temperatures of 28, 37, and
42
◦
C, respectively. By contrast, the EcRimI overexpression E.coli strain (pET28b-RimI)
Biomolecules 2023,13, 908 8 of 15
produced melatonin at 617, 707, and 301 ng/mL at 28, 37, and 42
◦
C, respectively. Thus,
the EcRimI overexpression strain produced melatonin at rates 1.6- and 2.2-fold higher
than the control strain when E.coli was incubated at 28 and 37
◦
C, respectively. However,
no differences in melatonin production between the EcRimI overexpression and control
strains were observed at 42
◦
C culture temperature. These results were consistent with the
growth curves at 42
◦
C. Thus, the optimal temperature for melatonin production in the
EcRimI overexpression E.coli strain was 37
◦
C, followed by 28
◦
C. Time-course analysis
of melatonin production was performed at the optimal temperature of 37
◦
C (Figure 6B).
Melatonin production increased as the incubation time was prolonged. However, in the
absence of 5-methoxytryptamine, the melatonin level was below our HPLC detection limit
of 1 ng/mL. Our data suggested that E.coli could synthesize melatonin in the presence of
5-methoxytryptamine, which is present in the diet [
40
,
41
]. A biosynthetic pathway from
serotonin to N-acetylserotonin and then to melatonin is also possible, but is unlikely due to
the requirement of an additional enzyme, such as ASMT. These possibilities remain to be
investigated in future research.
Biomolecules 2023, 13, x FOR PEER REVIEW 8 of 15
Figure 5. Growth curves of control (pET28b) and EcRimI overexpression (pET28b-RimI) E. coli
strains at (A) 28 °C, (B) 37 °C, and (C) 42 °C. Each point represents three independent replicates. E.
coli was grown in nutrient-rich TB medium.
3.4. Melatonin Production in Escherichia coli
E. coli produces small amounts of melatonin (>1 ng/mL) [34]. To identify melatonin
in E. coli, 5-methoxytryptamine, a direct substrate of melatonin production by SNAT, was
added to the culture medium. After 24 h incubation in the presence of 1 mM 5-methoxy-
tryptamine and 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at various tempera-
tures, melatonin levels were quantified in the medium fraction of the E. coli culture, as the
majority of melatonin was found in the medium fraction rather than in E. coli cells [34].
As shown in Figure 6A, the control E. coli strain (pET28b) also produced melatonin at
concentrations of 383, 322, and 301 ng/mL at the incubation temperatures of 28, 37, and 42
°C, respectively. By contrast, the EcRimI overexpression E. coli strain (pET28b-RimI) pro-
duced melatonin at 617, 707, and 301 ng/mL at 28, 37, and 42 °C, respectively. Thus, the
EcRimI overexpression strain produced melatonin at rates 1.6- and 2.2-fold higher than
the control strain when E. coli was incubated at 28 and 37 °C, respectively. However, no
differences in melatonin production between the EcRimI overexpression and control
strains were observed at 42 °C culture temperature. These results were consistent with the
growth curves at 42 °C. Thus, the optimal temperature for melatonin production in the
EcRimI overexpression E. coli strain was 37 °C, followed by 28 °C. Time-course analysis of
melatonin production was performed at the optimal temperature of 37 °C (Figure 6B).
Melatonin production increased as the incubation time was prolonged. However, in the
absence of 5-methoxytryptamine, the melatonin level was below our HPLC detection limit
of 1 ng/mL. Our data suggested that E. coli could synthesize melatonin in the presence of
5-methoxytryptamine, which is present in the diet [40,41]. A biosynthetic pathway from
serotonin to N-acetylserotonin and then to melatonin is also possible, but is unlikely due
to the requirement of an additional enzyme, such as ASMT. These possibilities remain to
be investigated in future research.
Figure 5.
Growth curves of control (pET28b) and EcRimI overexpression (pET28b-RimI) E.coli strains
at (
A
) 28
◦
C, (
B
) 37
◦
C, and (
C
) 42
◦
C. Each point represents three independent replicates. E.coli was
grown in nutrient-rich TB medium.
Biomolecules 2023, 13, x FOR PEER REVIEW 9 of 15
Figure 6. (A) Melatonin production of the control (pET28b) and EcRimI overexpression (pET28b-
RimI) strains at 24 h under various culture temperature conditions. (B) Time course of melatonin
production by the control (pET28b) and EcRimI overexpression (pET28b-RimI) strains at 37 °C. Me-
dium fractions were subjected to HPLC analysis for melatonin quantification. Different letters indi-
cate significant differences from the wild type (Tukey’s honest significant difference (HSD) test; p <
0.05). Values are presented as means ± SD (n = 3).
3.5. Cadmium Response of the EcRimI Overexpression Strain
Cadmium treatment of E. coli inhibits antioxidant enzymes and lowers the levels of
antioxidants such as glutathione, which decreases the scavenging of reactive oxygen spe-
cies and leads to mechanical damage [42]. Correspondingly, adding glutathione alleviates
damage from oxidative stress and increases the growth rate of E. coli [43]. In contrast to
glutathione, melatonin treatment hampers the growth of Xanthomonas oryzae, a plant-
pathogenic bacterium [24]. To assess the impacts of cadmium stress, the EcRimI overex-
pression strain was challenged with cadmium treatment. As shown in Figure 7, the EcRimI
overexpression strain did not exhibit cadmium stress tolerance compared to the control.
However, even in the presence of cadmium, the EcRimI overexpression strain of E. coli
showed significantly enhanced growth relative to the control strain, regardless of the cell
density. These data suggested that the growth enhancement associated with EcRimI over-
expression was barely affected by heavy metal stress, indicating that EcRimI played a
general role in improving E. coli growth through modulation of protein N-acetylation,
melatonin biosynthesis, or both.
Figure 7. Growth curves of the control (pET28b) and EcRimI overexpression (pET28b-RimI) E. coli
strains in the presence of cadmium. (A) Cadmium was applied to a culture at OD600 = 1.0 and incu-
bated for 20 h at 37 °C. (B) Cadmium was applied to a culture at OD600 = 0.5 and incubated for 20 h
Figure 6.
(
A
) Melatonin production of the control (pET28b) and EcRimI overexpression (pET28b-RimI)
strains at 24 h under various culture temperature conditions. (
B
) Time course of melatonin production
by the control (pET28b) and EcRimI overexpression (pET28b-RimI) strains at 37
◦
C. Medium fractions
were subjected to HPLC analysis for melatonin quantification. Different letters indicate significant
differences from the wild type (Tukey’s honest significant difference (HSD) test; p< 0.05). Values are
presented as means ±SD (n= 3).
Biomolecules 2023,13, 908 9 of 15
3.5. Cadmium Response of the EcRimI Overexpression Strain
Cadmium treatment of E.coli inhibits antioxidant enzymes and lowers the levels
of antioxidants such as glutathione, which decreases the scavenging of reactive oxygen
species and leads to mechanical damage [
42
]. Correspondingly, adding glutathione al-
leviates damage from oxidative stress and increases the growth rate of E.coli [
43
]. In
contrast to glutathione, melatonin treatment hampers the growth of Xanthomonas oryzae,
a plant-pathogenic bacterium [
24
]. To assess the impacts of cadmium stress, the EcRimI
overexpression strain was challenged with cadmium treatment. As shown in Figure 7, the
EcRimI overexpression strain did not exhibit cadmium stress tolerance compared to the
control. However, even in the presence of cadmium, the EcRimI overexpression strain of E.
coli showed significantly enhanced growth relative to the control strain, regardless of the
cell density. These data suggested that the growth enhancement associated with EcRimI
overexpression was barely affected by heavy metal stress, indicating that EcRimI played
a general role in improving E.coli growth through modulation of protein N-acetylation,
melatonin biosynthesis, or both.
Biomolecules 2023, 13, x FOR PEER REVIEW 9 of 15
Figure 6. (A) Melatonin production of the control (pET28b) and EcRimI overexpression (pET28b-
RimI) strains at 24 h under various culture temperature conditions. (B) Time course of melatonin
production by the control (pET28b) and EcRimI overexpression (pET28b-RimI) strains at 37 °C. Me-
dium fractions were subjected to HPLC analysis for melatonin quantification. Different letters indi-
cate significant differences from the wild type (Tukey’s honest significant difference (HSD) test; p <
0.05). Values are presented as means ± SD (n = 3).
3.5. Cadmium Response of the EcRimI Overexpression Strain
Cadmium treatment of E. coli inhibits antioxidant enzymes and lowers the levels of
antioxidants such as glutathione, which decreases the scavenging of reactive oxygen spe-
cies and leads to mechanical damage [42]. Correspondingly, adding glutathione alleviates
damage from oxidative stress and increases the growth rate of E. coli [43]. In contrast to
glutathione, melatonin treatment hampers the growth of Xanthomonas oryzae, a plant-
pathogenic bacterium [24]. To assess the impacts of cadmium stress, the EcRimI overex-
pression strain was challenged with cadmium treatment. As shown in Figure 7, the EcRimI
overexpression strain did not exhibit cadmium stress tolerance compared to the control.
However, even in the presence of cadmium, the EcRimI overexpression strain of E. coli
showed significantly enhanced growth relative to the control strain, regardless of the cell
density. These data suggested that the growth enhancement associated with EcRimI over-
expression was barely affected by heavy metal stress, indicating that EcRimI played a
general role in improving E. coli growth through modulation of protein N-acetylation,
melatonin biosynthesis, or both.
Figure 7. Growth curves of the control (pET28b) and EcRimI overexpression (pET28b-RimI) E. coli
strains in the presence of cadmium. (A) Cadmium was applied to a culture at OD600 = 1.0 and incu-
bated for 20 h at 37 °C. (B) Cadmium was applied to a culture at OD600 = 0.5 and incubated for 20 h
Figure 7.
Growth curves of the control (pET28b) and EcRimI overexpression (pET28b-RimI) E.coli
strains in the presence of cadmium. (
A
) Cadmium was applied to a culture at OD
600
= 1.0 and
incubated for 20 h at 37
◦
C. (
B
) Cadmium was applied to a culture at OD
600
= 0.5 and incubated for
20 h at 37
◦
C. The medium used in this experiment was LB (Lennox broth). Overnight seed cultures of
100
µ
L were inoculated into 1 mL fresh LB medium containing the antibiotic kanamycin (50
µ
g/mL)
and grown at 37
◦
C until reaching OD
600
= 1.0 or 0.5, followed by cadmium treatment for 20 h. Each
point represents three independent replicates. Different letters indicate significant differences from
the wild type (Tukey’s HSD test; p< 0.05).
4. Discussion
The biosynthetic pathways and biological roles of melatonin have been well docu-
mented since its discovery in the pineal gland of cows in 1958 [
44
] and in several plant
species in 1995 [
45
,
46
]. Accordingly, increasing numbers of melatonin biosynthesis-related
genes have been cloned and characterized from prokaryotes and eukaryotes through se-
quence homology-based cloning strategies using sheep SNAT [
27
,
47
]. Among such genes,
SNAT has been most widely studied, as it plays a key role in melatonin biosynthesis in
diverse organisms [
48
]. Consequently, many sheep SNAT orthologues have been cloned
from human [
49
], yeast [
50
], Drosophila melanogaster [
51
], Chlamydomonas reinhardtii [
52
],
and Xanthomonas oryzae [
53
] (Table 1). Similarly, plant SNAT genes have been cloned from
various species [
54
]. In contrast to the single copy of SNAT present in animals [
28
], plant
SNAT exists as a gene family containing at least 3 isogenes with very low amino acid
sequence homology [
25
]. These SNAT genes include rice SNAT1 [
29
,
55
] and SNAT2 [
38
],
Arabidopsis SNAT1 [
56
] and SNAT2 [
11
], tobacco SNAT1 and SNAT2 [
57
], apple SNAT3 [
58
],
red algal SNAT [
59
], and cyanobacterial SNAT [
30
]. All plant SNAT1 and SNAT2 expression
Biomolecules 2023,13, 908 10 of 15
is localized in chloroplasts, except apple SNAT3, which is expressed in mitochondria [
58
].
A mitochondrial apple SNAT3 orthologue has been found in rice, but its function as an
SNAT enzyme in rice remains unknown [
25
]. Collectively, rice likely contains up to four
SNAT isogenes including an archaeal orthologue, which is expressed in the cytoplasm,
in accordance with human Naa50, another archaeal SNAT orthologue [
31
]. The archaeal
SNAT gene was recently cloned and characterized from archaeal GNAT family genes [
3
].
This archaeal SNAT from Thermoplasma volcanium was previously annotated as TvArd1
(arrest-defective-1), encoding a protein with N-terminal acetyltransferase activity that trans-
fers an acetyl group from acetyl-coenzyme A to the N-termini of various proteins [
60
–
62
].
Ard1 is also called Naa10, and it is one of six NAT enzyme complexes containing Nat10
to Nat60 [
63
]. The closest orthologue of archaeal SNAT in humans is Naa50, N-alpha-
acetyltransferase 50, which was recently revealed to possess SNAT enzyme activity [
31
].
These data suggest that archaeal SNAT proteins with N-acetyltransferase activity represent
a novel class of SNAT family genes. In support of this possibility, E.coli RimI, an archaeal
SNAT orthologue that exhibits protein N-acetyltransferase activity, also showed SNAT en-
zyme activity, indicating that E.coli have the genetic capacity to synthesize melatonin from
its penultimate precursor, serotonin, as well as the direct substrate 5-methoxytryptamine
(Figure 2). Phylogenetic analysis indicated that EcRimI belongs to the archaea SNAT family
comprising TvSNAT [
3
] and human Naa50 [
31
] and is distantly related to the animal SNAT
and plant SNAT family, suggesting that EcRimI is a functional E. coli orthologue of archaeal
SNAT. In addition to the SNAT orthologue genes found in animals, plants, and archaea,
enhanced intracellular survival (Eis) proteins containing two NAT domains and one sterol
carrier domain also exhibit SNAT enzyme activity; however, Eis homolog proteins are
present mainly in mycobacteria and certain Gram-positive bacteria, but are not found in
animals and plants [64].
The different activities of SNAT enzymes derived from plants and archaea have trig-
gered fundamental questions about their biological functions, such as whether transgenic
phenotypes showing gain or loss of SNAT gene function can be attributed to changes in
melatonin or protein acetylation. Arabidopsis SNAT1 exhibits N-acetyltransferase activity
toward chloroplast proteins [
65
], and its knockout mutant of SNAT1 (snat1) exhibited
stunted growth depending on light intensity [
12
]. Whether the main reason for altered phe-
notypes in the snat1 mutant of Arabidopsis is decreased biosynthesis of melatonin, altered
levels of chloroplast protein acetylation, or the combined effects of both changes remains
unclear [
12
,
20
,
65
]. Analogously, Naa50 knockdown disrupted centromeric sister chromatid
cohesion in Drosophila [
66
] and centromeric cohesion in HeLa cells [
67
], suggesting that
protein N-acetyltransferase activity plays an important role in cell division. However, these
findings do not eliminate the possible involvement of melatonin in cell division. This
possibility remains to be elucidated in future research. Notably, the phenotypic abnormality
observed in humans and Drosophila was not found in Naa50 knockdown yeast [
68
]. E.coli
RimI is responsible for the N-terminal acetylation of the ribosomal protein S18 [
69
] and
elongation factor Tu [
36
]. Consequently, an E.coli strain devoid of RimI showed slightly
reduced growth on minimal medium, which was not observed in nutrient-rich medium,
possibly driven by reduced efficiency of translation [
36
]. Assessing whether the growth
retardation of the RimI-lacking strain is associated with melatonin biosynthesis remains an
intriguing question.
Biomolecules 2023,13, 908 11 of 15
Table 1. Enzyme kinetics of SNAT proteins from various organism.
Organism Enzyme
Km(µM) Vmax Reference
Serotonin nmol/min/mg Protein
Animal SNAT orthologue proteins
Human SNAT 1235 - [49]
Sheep SNAT 85 0.67 [46]
Yeast SNAT 5100 - [50]
Drosophila
melanogaster SNAT 1620 - [51]
Xanthomonas oryzae SNAT 709 - [53]
Chlamydomonas
reinhardtii SNAT 247 0.325 [52]
Plant SNAT orthologue proteins
Rice SNAT1 270 3.3 [55]
Rice SNAT2 371 4.7 [38]
Arabidopsis SNAT1 309 1.4 [56]
Arabidopsis SNAT2 232 2.1 [11]
Tobacco SNAT1 579 8.1 [57]
Tobacco SNAT2 326 1.5 [57]
Apple SNAT3 55 0.0009 [58]
Red algae SNAT 467 28 [59]
Cyanobacteria SNAT 823 1.6 [30]
Archaea SNAT orthologue proteins
Thermoplasma
volcanium SNAT 621 0.416 [3]
Human SNAT 986 1.8 [31]
Escherichia coli SNAT 531 0.528 This paper
Enhanced intracellular survival (Eis) protein
Saccharopolyspora
erythraea SNAT 13,000 - [64]
All SNAT genes belong to the GCN5-related N-acetyltransferase (GNAT) superfam-
ily of enzymes and share a common acetyl coenzyme A binding domain [
70
]. Although
whether animal SNAT proteins such as sheep SNAT have protein N-acetyltransferase
activity remains unclear, SNAT proteins derived from plants and archaea exhibit such ac-
tivity [
36
,
65
]. The dual activity of SNAT proteins gives rise to the production of melatonin,
N-acetylated proteins, or both simultaneously. Both products play diverse biological roles
in organisms. Melatonin is a universal and pleiotropic molecule orchestrating the day–
night waking cycle, seasonal reproduction, antitumor functions, and immune responses in
animals [
2
,
71
] while acting as a master regulator of plant growth and development [
72
].
N-acetylation is a universal protein modification process, with 80–90% of soluble pro-
teins being N-terminally acetylated [
62
], and regulates protein degradation, subcellular
translocation, and protein complex formation [63].
As reported in animals and plants, melatonin may have certain biological func-
tions, such as antioxidation, in E.coli [
4
,
41
,
73
,
74
]. The EcRimI mutant strain showed
no growth inhibition in nutrient-rich LB medium [
37
]. In addition, the EcRimI overexpres-
sion strain exhibited far less N-acetylation activity toward E.coli proteins
in vivo
than other
N-acetyltransferases such as YfiQ, Yjab, and YiaC, suggesting that the role of EcRimI as a
protein N-acetyltransferase is minimal [
37
]. Although no direct evidence connects mela-
tonin with E.coli growth, a beneficial effect of melatonin on E.coli growth cannot be ruled
out, as the EcRimI overexpression E.coli strain showed enhanced growth in conjunction
with increased melatonin production in this study. Melatonin has long been proposed
to be synthesized in bacteria, especially in E. coli, but no biosynthesis pathway has been
found. Due to the discovery SNAT gene in E. coli, we open a new window to studying the
Biomolecules 2023,13, 908 12 of 15
function of melatonin in bacteria by way of gene manipulation and to engineer melatonin
biosynthesis for its overproduction in E. coli. This is the first report of successful cloning of
SNAT from E.coli. Further studies will shed light on the physiological roles of melatonin in
E.coli during growth and under oxidative stress.
5. Conclusions
With the help of the first successful cloning of archaeal SNAT, we revealed that E.coli
RimI, encoding a protein N-acetyltransferase, exhibited SNAT enzyme activity
in vitro
and
that EcRimI overexpression
in vivo
was associated with enhanced melatonin production in
E.coli. The EcRimI overexpression E.coli strain exhibited enhanced growth compared to
the control strain. The EcRimI overexpression E.coli strain showed strong tolerance against
stationary-phase stress based on its growth curves. This enhanced growth effect of EcRimI
overexpression compared to that of the control remained in the presence of cadmium
stress. In summary, E.coli clearly has the capacity to synthesize melatonin through the
enzymatic activity of EcRimI using 5-methoxytryptamine present in the diet, and EcRimI
overexpression plays an important role in E.coli growth, possibly associated with melatonin
synthesis, protein acetylation, or both.
Author Contributions:
Conceptualization, K.B.; formal analysis, K.B. and K.L.; investigation, K.L.;
writing—original draft preparation, K.B.; writing—review and editing, K.B.; funding acquisition, K.B.
All authors have read and agreed to the published version of the manuscript.
Funding:
This research was supported from grants by the Basic Science Research Program of the Na-
tional Research Foundation of Korea (NRF-2021R1I1A2042237) funded by the Ministry of Education.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
The data that support the finding of this study are available from the
corresponding author upon reasonable request.
Conflicts of Interest: The authors declare no conflict of interest.
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