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Information on EC 4.2.3.20 - (R)-limonene synthase
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4.2.3.20 (R)-limonene synthaseIUBMB Comments
Forms the first step of carvone biosynthesis in caraway. The enzyme from Carum carvi (caraway) seeds requires a divalent metal ion (preferably Mn2+) for catalysis. This enzyme occurs in Citrus, Carum (caraway) and Anethum (dill); (-)-limonene, however, is made in the fir, Abies, and mint, Mentha, by EC 4.2.3.16, (4S)-limonene synthase.
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Word Map
- 4.2.3.20
-
monoterpene
-
terpene
- citrus
-
volatile
- orange
- peel
-
+-limonene
- linalool
- tenuifolia
-
schizonepeta
- digitatum
- gpp
-
mentha
-
repelling
- spicata
- labiatae
-
lavandula
- isoprene
- beta-pinene
- gamma-terpinene
- angustifolia
-
frugivores
- lavender
- drug development
The enzyme appears in viruses and cellular organisms
Synonyms
d-limonene synthase, (+)-limonene synthase, citmtse1, cl(+)lims2, lalims, (+)-ls, cl(+)lims1, 
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
(+)-limonene synthase
(+)-LS
CitMTSE1
Cl(+)LIMS1
d-limonene synthase
limonene synthase
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
geranyl diphosphate = (R)-limonene + diphosphate
requires a divalent metal ion (preferably Mn2+) for catalysis
geranyl diphosphate = (R)-limonene + diphosphate
forms the first step of carvone biosynthesis in caraway
-
geranyl diphosphate = (R)-limonene + diphosphate
-
-
-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
cyclization
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PATHWAY SOURCE
PATHWAYS
MetaCyc
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SYSTEMATIC NAME
IUBMB Comments
geranyl-diphosphate diphosphate-lyase [cyclizing, (+)-(4R)-limonene-forming]
Forms the first step of carvone biosynthesis in caraway. The enzyme from Carum carvi (caraway) seeds requires a divalent metal ion (preferably Mn2+) for catalysis. This enzyme occurs in Citrus, Carum (caraway) and Anethum (dill); (-)-limonene, however, is made in the fir, Abies, and mint, Mentha, by EC 4.2.3.16, (4S)-limonene synthase.
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CAS REGISTRY NUMBER
COMMENTARY
155807-65-1
-
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
geranyl diphosphate
(1R,5R)-(+)-alpha-pinene + diphosphate
-
-
?
geranyl diphosphate
(R)-limonene
-
products are 39% (R)-limonene, 22% terpinolene, 16% (1R,5S)-camphene, 14% (1R,5R)-(+)-alpha-pinene, 8% betamyrcene and traces of alpha-phellandrene. Product pattern changes markedly to 46% limonene, 9% terpinolene, 23% alpha-pinene, 5% beta-myrcene and 4% alpha-phellandrene, when Mn2+ is supplied instead of Mg2+
?
geranyl diphosphate
D-limonene + diphosphate
single step in plants
D-limonene shows cancer preventative and cancer therapeutic activities in vitro and in vivo, its modulates apoptosis-related genes and proteins in cultured cells
?
geranyl diphosphate
(+)-(R)-limonene + diphosphate
-
-
high stereoselectivity, 98.4% of (+)-product
?
geranyl diphosphate
(+)-(R)-limonene + diphosphate
-
product is exclusively R-(+)-limonene
?
additional information
?
-
no substrates: farnesyl diphosphate, geranylgeranyl diphosphate
-
?
additional information
?
-
-
no substrates: farnesyl diphosphate, geranylgeranyl diphosphate
-
?
additional information
?
-
a concomitant increase in the level of apoptosis-related protein B-cell lymphoma protein 2 and decreases in the levels of Bcl-2 antagonist of cell death and phosphorylated c-Jun N-terminal kinase are observed in limonene-producing cells
-
?
additional information
?
-
limonene can be synthesized in animal cells using their own mevalonate pathway and limonene-producing cells may provide a new system for the elucidation of limonene function
-
?
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
geranyl diphosphate
D-limonene + diphosphate
single step in plants
D-limonene shows cancer preventative and cancer therapeutic activities in vitro and in vivo, its modulates apoptosis-related genes and proteins in cultured cells
-
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Mg2+
Mn2+
additional information
Mg2+
may replace Mn2+ but with much lower efficiency (maximum activity at 20 mM)
Mn2+
the proportions of the products change significantly when Mn2+ is supplied instead of Mg2+
Mn2+
or Mg2+, required. Optimum concentration 0.2 mM, 68% of the efficieny with Mg2+. Product pattern changes markedly to 46% limonene, 9% terpinolene, 23% alpha-pinene, 5% beta-myrcene and 4% alpha-phellandrene, when Mn2+ is supplied instead of Mg2+
additional information
DDxxD motif, binding site for substrate-divalent metal cation complex
additional information
-
DDxxD motif, binding site for substrate-divalent metal cation complex
additional information
DDxxD motif, binding site for substrate-divalent metal cation complex
additional information
-
DDxxD motif, binding site for substrate-divalent metal cation complex
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
geranyl diphosphate
substrate inhibition above 0.01 mM; substrate inhibition above 0.01 mM
K+
maximum inhibition above 100 mM; maximum inhibition above 100 mM
Mn2+
above 0.6 mM, activation below; above 0.6 mM, activation below
Mn2+
the enzyme is strongly inhibited at concentrations of more than 1 mM
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0131
geranyl diphosphate
at pH 8.0, temperature not specified in the publication
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.00054
2-Fluorogeranyl diphosphate
-
pH and temperature not specified in the publication
0.00024
2-fluoroneryl diphosphate
-
pH and temperature not specified in the publication
0.186
geranyl diphosphate
at pH 8.0, temperature not specified in the publication
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0024
2-Fluorogeranyl diphosphate
-
pH and temperature not specified in the publication
0.0395
2-fluoroneryl diphosphate
-
pH and temperature not specified in the publication
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.5
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
30
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
production of the monoterpene limonene and modulation of apoptosis-related proteins in embryonic-mouse NIH 3T3 fibroblast cells by introduction of the limonene synthase gene isolated from japanese catnip
SwissProt
brenda
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
malfunction
malfunction
-
Drastic down-regulation of D-limonene synthase gene expression in the peel of transgenic oranges leads to fruit resistance against Penicillium digitatum, Xanthomonas citri subsp. citri and other specialized pathogens
malfunction
-
enzyme down-regulation in orange fruit induces resistance against the fungus Phyllosticta citricarpa through enhanced accumulation of monoterpene alcohols and activation of defence
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UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). RLC1_CITLI
606
0
70348
Swiss-Prot
Chloroplast (Reliability: 5)
RLC1_CITSI
607
0
70354
Swiss-Prot
Chloroplast (Reliability: 2)
LALIM_LAVAN
602
0
70345
Swiss-Prot
Chloroplast (Reliability: 3)
B9S898_RICCO
192
0
22128
TrEMBL
other Location (Reliability: 4)
B9RH09_RICCO
599
0
69129
TrEMBL
Chloroplast (Reliability: 4)
B9SXQ2_RICCO
351
0
40294
TrEMBL
other Location (Reliability: 3)
B9S899_RICCO
226
0
26444
TrEMBL
other Location (Reliability: 3)
B9RXW6_RICCO
180
0
20877
TrEMBL
Mitochondrion (Reliability: 4)
B9T537_RICCO
257
0
29569
TrEMBL
other Location (Reliability: 1)
B9S897_RICCO
123
0
14157
TrEMBL
other Location (Reliability: 3)
A0A2G9GEG6_9LAMI
606
0
70951
TrEMBL
other Location (Reliability: 4)
B9TL72_RICCO
143
0
17319
TrEMBL
other Location (Reliability: 2)
A0A2G9HPL8_9LAMI
607
0
70197
TrEMBL
Chloroplast (Reliability: 2)
B9S8A3_RICCO
222
0
26283
TrEMBL
other Location (Reliability: 4)
B9S893_RICCO
250
0
28930
TrEMBL
other Location (Reliability: 4)
B9RPN5_RICCO
288
0
32558
TrEMBL
Secretory Pathway (Reliability: 5)
A0A2G9GQ38_9LAMI
606
0
70759
TrEMBL
Mitochondrion (Reliability: 4)
B9RPM0_RICCO
596
0
68363
TrEMBL
Chloroplast (Reliability: 2)
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PDB
SCOP
CATH
UNIPROT
ORGANISM
Citrus aurantium var. sinensis
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
62000
x * 62000, SDS-PAGE, x * 64000, deduced from gene sequence
64000
x * 62000, SDS-PAGE, x * 64000, deduced from gene sequence
70000
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
monomer
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
apoenzyme, sitting drop vapor diffusion method, using 12-16% (w/v) PEG 8000 as precipitant
in complex with 2-fluorogeranyl diphosphate or 2-fluoroneryl diphosphate, vapor diffusion method
-
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21-CodonPlus(DE3)-RIL cells
expressed in Saccharomyces cerevisiae strain AE9 K197G and Escherichia coli BL21 cells
into the pGEM-T vector for sequencing and into the pGEX-T-1 vector for expression in Escherichia coli cells
introduction of limonene synthase cDNA with three different sorting signals (to localize either in cytosol or the endoplasmic reticulum) in Nicotiana tabacum. Full-length and modified enzyme are subcloned in a binary vector under the El2 promoter, which is a strong constitutive promoter, to yield pBin-FullLC1, pBin-DEltaLC1 and pBin.ERLC1 to plastidial, cytosolic and endoplasmic localization, respectively. These plasmids are introduced via Agrobacterium tumefaciens into tobacco. More than 10 transgenic tobacco plants for each construct, i.e. 15 plastid localization, 17 cytosol localization, 11 endoplasmic reticulum localization are established
-
limonene cyclase enzyme Cl(+)LIMS2 produced exclusively (+)-limonene as the major product of the enzymatic catalysis. In a chimeric enzyme, substitution of ClgammaTS domain III and domain IV by their counterparts from Cl(+)LIMS2 changed product specificity to that of Cl(+)LIMS2, (+)-limonene being the major product formed
-
The cDNA of D-limonene synthase obtained from Japanese catnip, modified by deletion of the transit peptide sequence and addition of a new ATG initiation codon, is cloned. Establishment of transgenic mouse embryonic NIH 3T3 fibroblast cells that produce limonene by introducing the D-limonene synthase gene. Apoptosis is not observed in the limonene-producing cells.
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
drug development
D-limonene is effective in cancer prevention in animal cancer models, some clinical applications have been performed
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Bouwmeester, H.J.; Gershenzon, J.; Konings, M.C.J.M.; Croteau, R.
Biosynthesis of the monoterpenes limonene and carvone in the fruit of caraway. I. Demonstration of enzyme activities and their changes with development
Plant Physiol.
117
901-912
1998
Carum carvi
brenda
Lucker, J.; El Tamer, M.K.; Schwab, W.; Verstappen, F.W.; van der Plas, L.H.; Bouwmeester, H.J.; Verhoeven, H.A.
Monoterpene biosynthesis in lemon (Citrus limon). cDNA isolation and functional analysis of four monoterpene synthases
Eur. J. Biochem.
269
3160-3171
2000
Citrus limon (Q8L5K1), Citrus limon (Q8L5K3)
brenda
Maruyama, T.; Ito, M.; Kiuchi, F.; Honda, G.
Molecular cloning, functional expression and characterization of D-limonene synthase from Schizonepeta tenuifolia
Biol. Pharm. Bull.
24
373-377
2001
Schizonepeta tenuifolia (Q9FUW5), Schizonepeta tenuifolia
brenda
Maruyama, T.; Saeki, D.; Ito, M.; Honda, G.
Molecular cloning, functional expression and characterization of D-limonene synthase from Agastache rugosa
Biol. Pharm. Bull.
25
661-665
2002
Agastache rugosa (Q940E7), Agastache rugosa
brenda
El Tamer, M.K.; Lucker, J.; Bosch, D.; Verhoeven, H.A.; Verstappen, F.W.A.; Schwab, W.; van Tunen, A.J.; Voragen, A.G.J.; de Maagd, R.A.; Bouwmeester, H.J.
Domain swapping of Citrus limon monoterpene synthases: impact on enzymatic activity and product specificity
Arch. Biochem. Biophys.
411
196-203
2003
Citrus limon
brenda
Ohara, K.; Ujihara, T.; Endo, T.; Sato, F.; Yazaki, K.
Limonene production in tobacco with Perilla limonene synthase cDNA
J. Exp. Bot.
54
2635-2642
2003
Perilla frutescens
brenda
Shimada, T.; Endo, T.; Fujii, H.; Hara, M.; Ueda, T.; Kita, M.; Omura, M.
Molecular cloning and functional characterization of four monoterpene synthase genes from Citrus unshiu Marc
Plant Sci.
166
49-58
2004
Citrus unshiu, Citrus unshiu Marc.
brenda
Landmann, C.; Fink, B.; Festner, M.; Dregus, M.; Engel, K.H.; Schwab, W.
Cloning and functional characterization of three terpene synthases from lavender (Lavandula angustifolia)
Arch. Biochem. Biophys.
465
417-429
2007
Lavandula angustifolia (Q2XSC6), Lavandula angustifolia
brenda
Satomi, Y.; Ohara, K.; Yazaki, K.; Ito, M.; Honda, G.; Nishino, H.
Production of the monoterpene limonene and modulation of apoptosis-related proteins in embryonic-mouse NIH 3T3 fibroblast cells by introduction of the limonene synthase gene isolated from Japanese catnip (Schizonepeta tenuifolia)
Biotechnol. Appl. Biochem.
52
185-190
2009
Schizonepeta tenuifolia (Q9FUW5)
brenda
Nishikawa, F.; Endo, T.; Shimada, T.; Fujii, H.; Shimizu, T.; Omura, M.
Characterization of the 5 flanking region of the citrus D-limonene synthase gene, which shows a quantitatively preferential expression in peel
J. Jpn. Soc. Hort. Sci.
78
84-89
2009
Citrus unshiu (Q6F5H2)
-
brenda
Morehouse, B.R.; Kumar, R.P.; Matos, J.O.; Olsen, S.N.; Entova, S.; Oprian, D.D.
Functional and structural characterization of a (+)-limonene synthase from Citrus sinensis
Biochemistry
56
1706-1715
2017
Citrus sinensis (A0A1C9J6A7), Citrus sinensis
brenda
Kumar, R.P.; Morehouse, B.R.; Matos, J.O.; Malik, K.; Lin, H.; Krauss, I.J.; Oprian, D.D.
Structural characterization of early Michaelis complexes in the reaction catalyzed by (+)-limonene synthase from Citrus sinensis using fluorinated substrate analogues
Biochemistry
56
1716-1725
2017
Citrus sinensis
brenda
Rodriguez, A.; Peris, J.E.; Redondo, A.; Shimada, T.; Pena, L.
Principal component analysis (PCA) of volatile terpene compounds dataset emitted by genetically modified sweet orange fruits and juices in which a D-limonene synthase was either up- or down-regulated vs. empty vector controls
Data Brief
9
355-361
2016
Citrus sinensis
brenda
Rodriguez, A.; Peris, J.E.; Redondo, A.; Shimada, T.; Costell, E.; Carbonell, I.; Rojas, C.; Pena, L.
Impact of D-limonene synthase up- or down-regulation on sweet orange fruit and juice odor perception
Food Chem.
217
139-150
2017
Citrus sinensis
brenda
Turner, G.; Lange, B.
Ultrastructure of grapefruit secretory cavities and immunocytochemical localization of (+)-limonene synthase
Int. J. Plant Sci.
176
643-661
2015
Citrus x paradisi
-
brenda
Rodriguez, A.; Kava, V.; Latorre-Garcia, L.; da Silva, G.J.; Pereira, R.G.; Glienke, C.; Ferreira-Maba, L.S.; Vicent, A.; Shimada, T.; Pena, L.
Engineering D-limonene synthase down-regulation in orange fruit induces resistance against the fungus Phyllosticta citricarpa through enhanced accumulation of monoterpene alcohols and activation of defence
Mol. Plant Pathol.
19
2077-2093
2018
Citrus sinensis
brenda
Rodriguez, A.; Shimada, T.; Cervera, M.; Redondo, A.; Alquezar, B.; Rodrigo, M.J.; Zacarias, L.; Palou, L.; Lopez, M.M.; Pena, L.
Resistance to pathogens in terpene down-regulated orange fruits inversely correlates with the accumulation of D-limonene in peel oil glands
Plant Signal. Behav.
10
e1028704
2015
Citrus sinensis
brenda
Jongedijk, E.; Cankar, K.; Ranzijn, J.; van der Krol, S.; Bouwmeester, H.; Beekwilder, J.
Capturing of the monoterpene olefin limonene produced in Saccharomyces cerevisiae
Yeast
32
159-171
2015
Citrus limon (Q8L5K3), Citrus limon
brenda
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