Discovery of overlooked enzyme in onion and its application

Article abstract of DOI:10.1080/10426507.2019.1603704

When onions (Allium cepa) are chopped, cells are broken, propanthial S-oxide (lachrymatory factor; LF) is released and it makes our eyes water. LF had long been believed to be formed non-enzymatically from 1-propenesulfenic acid (PSA), a putative reaction product of alliinase acting on trans-(+)-S-(1-propenyl)-L-cysteine sulfoxide (1-PRENCSO). During the course of our study for the discoloration of Allium plants, however, we got a clue that some unknown enzyme responsible for the LF formation should be present in the crude onion alliinase preparation. In this study, we report the discovery of this new enzyme called lachrymatory factor synthase (LFS), and its application such as non-lachrymatory onions.

Full text of DOI:10.1080/10426507.2019.1603704

Phosphorus, Sulfur, and Silicon and the Related Elements
ISSN: 1042-6507 (Print) 1563-5325 (Online) Journal homepage: https://www.tandfonline.com/loi/gpss20
Discovery of overlooked enzyme in onion and its
application
Takahiro Kamoi
To cite this article: Takahiro Kamoi (2019) Discovery of overlooked enzyme in onion and its
application, Phosphorus, Sulfur, and Silicon and the Related Elements, 194:7, 698-700, DOI:
10.1080/10426507.2019.1603704
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PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
2019, VOL. 194, NO. 7, 698–700
https://doi.org/10.1080/10426507.2019.1603704
Discovery of overlooked enzyme in onion and its application
Takahiro Kamoi
Research & Development Headquarters, House Foods Group Inc, Yotsukaido, Chiba, Japan
ABSTRACT
ARTICLE HISTORY
Received 28 March 2019
Accepted 1 April 2019
When onions (Allium cepa) are chopped, cells are broken, propanthial S-oxide (lachrymatory factor;
LF) is released and it makes our eyes water. LF had long been believed to be formed non-enzy-
matically from 1-propenesulfenic acid (PSA), a putative reaction product of alliinase acting on
trans-(þ)-S-(1-propenyl)-L-cysteine sulfoxide (1-PRENCSO). During the course of our study for the
discoloration of Allium plants, however, we got a clue that some unknown enzyme responsible for
the LF formation should be present in the crude onion alliinase preparation. In this study, we
report the discovery of this new enzyme called lachrymatory factor synthase (LFS), and its applica-
tion such as non-lachrymatory onions.
KEYWORDS
Onion; lachrymatory factor
synthase; alliinase
GRAPHICAL ABSTRACT
Introduction
During the course of our study for the discoloration, we
got a clue that some unknown factors responsible for the
lachrymatory factor (LF) formation should be present in the
crude onion alliinase. Brodnitz identified the LF as propane-
thial S-oxide more than 45 years ago.[3] Like other sulfur
compounds, such as thiosulfinate, the LF had long been
believed to form spontaneously from 1-propenelsulfenic
acid, a putative reaction product derived from trans-(þ)-S-
(1-propenyl)-L-cysteine sulfoxide (PRENCSO) by alliinase.[4]
In this study, we report the discovery, isolation and cDNA
cloning of a novel enzyme, which we named Lachrymatory
The discoloration of garlic and onion is a well-known phe-
nomenon, and a three-step reaction scheme was proposed
for “pinking” of onion.[1,2] Although chemical constituents
proposed by earlier studies for the discoloration reactions
were generally in agreement, the detailed reaction mecha-
nisms and the chemical structures of the pigments or those
of the intermediates had not been fully elucidated. We,
therefore, attempted to resolve those remaining issues by
establishing a model reaction system that comprised only
well defined, highly purified constituents.
CONTACT Takahiro Kamoi
t-kamoi@housefoods.co.jp
Research & Development Headquarters, House Foods Group Inc, Yotsukaido, Chiba, 284-0033, Japan.
ß 2019 Taylor & Francis Group, LLC

PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
699
O-
S+
NH2
O-
S+
NH2
H2C
COOH
COOH
H3C
alliin
PRENCSO
alliinase
OH
OH
S
S
H2C
H3C
O-
S+
O-
LFS
S+
O-
S+
S
S
CH3
H3C
CH2
CH3
CH2
LF
allicin
H3C
CH3
other
beneficial
compounds
N
NH2
R
COOH
H3C
R
COOH
PPs
Amino acids
H3C
CH3
CH3
N+
N
R
COOH
COOH
R
PUR-1
Figure 1. Reaction mechanism of blue-green discoloration of a mixture of chopped onion and garlic.
Factor Synthase (LFS), and consequences of suppressing the Structure of pigment precursors (PPs) and a reddish-
enzyme activity in onion.
purple pigment (PUR-1)
Pigment precursors were prepared by mixing PRENCSO,
alliinase, and an amino acid. When we used valine for the
amino acid, PP-Val was formed. The PP was isolated from
reaction mixture, and the structure was determined as 2-
(3,4-dimethylpyrrolyl)-3-methylbutanoic acid.[6]
Next, we prepared pigment from a heat-treated solution
containing PP-Val and allicin. When heated, the mixture
turned reddish purple first, then to blue to dark blue, and
finally, formed green-colored precipitate. We isolated a red-
dish purple pigment (PUR-1) from the mixture and, its
structure was determined as (1E)-1-(1-((1S)-1-carboxy-2-
methylpropyl)-3,4-dimethyl-1H-pyrrol-2-yl)-prop-1-enylene-
3- (1-((1S)-1-carboxy-2-methylpropyl)-3,4-dimethyl-1H-pyr-
rol-2-ylidenium).[6] From these results, it was suggested that
PP molecules had been connected by an allyl group of alli-
cin to form conjugated pigments. Proposed reaction mech-
Results and discussion
Model reaction system of discoloration
Since the vivid-blue color was produced easily by mixing
juices from heated onion and unheated garlic, we isolated
compounds responsible for color formation, and succeeded
in inducing the pigment using a model reaction system
comprising only four compounds. The first compound was
PRENCSO, which was isolated from heat-treated onion. The
second and the third compounds were allicin and alliinase
isolated from raw garlic. The fourth compound was an
amino acid, which was present in both onion and garlic.[5]
Alliin was used instead of allicin because allicin is formed
from alliin by the action of alliinase. Using this model reac-
tion system, we determined the structures of a pigment pre- anism leading to the pigment formation in the model
cursor, PP-Val, and a pigment, PUR-1. reaction system is shown in Figure 1.

700
T. KAMOI
Effect of crude alliinase source on pigment and
LF formation
in onions. LFS enzyme activity as well as endogenous lfs
transcripts levels was reduced significantly. In consequence,
LF was decreased and thiosulfinates levels were increased
dramatically and simultaneously.[8] Moreover, we deter-
mined the structure of one of the thiosulfinates, increased in
the non-lachrymatory onion, as S-3,4-dimethyl-5-hydroxy-
thiolane-2-yl 1-propenethiosulfinate, and showed its inhibi-
tory activity against cyclooxygenase-1 in vitro.[9]
Mutagenesis was also used to silence the endogenous
gene. By irradiating heavy-ion beams, we succeeded in gen-
erating non-lachrymatory onions in which alliinase enzyme
activity as well as endogenous alliinase transcripts levels was
decreased significantly.[10]
By using our model reaction system comprising PRENCSO,
alliin, glycine, and alliinase, we found that crude onion allii-
nase was less efficient in blue color formation than crude
garlic enzyme showing equal alliinase activity. Moreover, the
amount of pigment formation did not increase if the activity
of crude onion alliinase added to the model reaction system
was increased by about 2-fold. According to the previous
studies of Allium chemistry, treating PRENCSO with allii-
nase should give rise to both the LF and di-1-propenyl thio-
sulfinate. Since the LF was thought not to be involved in the
color forming reactions, we confirmed whether the amount
of LF would differ by the source of the crude alliinase.
Although the crude onion alliinase yielded LF, the LF was
not formed at all when PRENCSO was mixed with crude
garlic alliinase. From these results, we speculated that some
unknown factors responsible for the LF formation should be
present in the crude onion alliinase.
Conclusions
These non-lachrymatory onions would be a useful resource
for understanding not only organosulfur chemistry of the
genus Allium but also the role of specific sulfur secondary
metabolites in plant biology and in human health.
Isolation of LF forming factor and identification of
lfs cDNA
References
[1] Shannon, S.; Yamaguchi, M.; Howard, F. D. Reactions Involved
in Formation of a Pink Pigment in Onion Purees. J. Agric. Food
Chem. 1967, 15, 417–422. DOI: 10.1021/jf60151a026.
[2] Shannon, S.; Yamaguchi, M.; Howard, F. D. Precursors
Involved in the Formation of Pink Pigments in Onion Purees.
J. Agric. Food Chem. 1967, 15, 423–426. DOI: 10.1021/
jf60151a027.
We used a reaction system consisting of PRENCSO, purified
garlic alliinase, and a fractionated crude onion alliinase to
evaluate the LF forming activity. If the unknown factors were
present in the fractionated sample, LF would be detected. We
succeeded in isolating the active protein which was named LFS.
We used the RACE technique with degenerate primers
deduced from the N-terminal sequence of LFS to obtain a
complete cDNA sequence (GenBank accession no.
AB089203). The full-length cDNA consisted of 737 base
pairs, with a predicted gene product of 169 amino acids.
[3] Brodnitz, M. H.; Pascale, J. V. Thiopropanal S-Oxide:
A
Lachrymatory Factor in Onions. J. Agric. Food Chem. 1971, 19,
269–272. DOI: 10.1021/jf60174a009.
[4] Block, E.; Penn, R. E.; Revelle, L. K. Structure and Origin of the
Onion Lachrymatory Factor. A Microwave Study. J. Am. Chem.
Soc. 1979, 101, 2200–2201. DOI: 10.1021/ja00502a044.
[5] Imai, S.; Akita, K.; Tomotake, M.; Sawada, H. Model Studies on
Precursor System Generating Blue Pigment in Onion and
Garlic. J. Agric. Food Chem. 2006, 54, 848–852. DOI: 10.1021/
jf051980f.
[6] Imai, S.; Akita, K.; Tomotake, M.; Sawada, H. Identification of
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Involved in the Blue-Green Discoloration of Onion and Garlic.
J. Agric. Food Chem. 2006, 54, 843–847. DOI: 10.1021/
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[7] Imai, S.; Tsuge, N.; Tomotake, M.; Nagatome, Y.; Sawada, H.;
Nagata, T.; Kumagai, H. An Onion Enzyme That Makes the
Eyes Water. Nature 2002, 419, 685. DOI: 10.1038/419685a.
[8] Eady, C. C.; Kamoi, T.; Kato, M.; Porter, N. G.; Davis, S.; Shaw,
M.; Kamoi, A.; Imai, S. Silencing Onion Lachrymatory Factor
Synthase Causes a Significant Change in the Sulfur Secondary
Metabolite Profile. Plant Physiol. 2008, 147, 2096–2106. DOI:
10.1104/pp.108.123273.
[9] Aoyagi, M.; Kamoi, T.; Kato, M.; Sasako, H.; Tsuge, N.; Imai, S.
Structure and Bioactivity of Thiosulfinates Resulting from
Suppression of Lachrymatory Factor Synthase in Onion. J.
Agric. Food Chem. 2011, 59, 10893–10900. DOI: 10.1021/
jf202446q.
LF forming activity of LFS
When we expressed the LFS gene in Escerichia coli, the
resulting recombinant protein exhibited the expected LF
forming activity. The LF was detected only when all three
factors, namely, PRENCSO, purified alliinase, and recombin-
ant LFS were mixed in the reaction system, and the system
lacking any of these factors did not give LF at all. This result
provides a direct proof that LFS is essential in producing LF
from PRENCSO.[7]
Production of non-lachrymatory onions
The discovery of LFS led to the proposal that non-lachryma-
tory onions unable to produce LF should be possible by
silencing LFS enzyme activity. In the absence of LFS, by
stopping conversion of 1-propenesulfenic acid to the LF, the
unstable sulfenic acid would be predicted to undergo spon-
taneous self-condensation to thiosulfinates and other com-
pounds responsible for the onion’s characteristic flavor and
their bioactivity. We succeeded in suppressing the endogen-
ous lfs gene expression by using RNA interference silencing
[10] Kato, M.; Masamura, N.; Shono, J.; Okamoto, D.; Abe, T.; Imai,
S. Production and Characterization of Tearless and Non-
Pungent Onion. Sci. Rep. 2016, 6, 23779. DOI: 10.1038/
srep23779.