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Information on EC 1.23.5.1 - violaxanthin de-epoxidase and Organism(s) Spinacia oleracea and UniProt Accession Q9SM43
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1.23.5.1 violaxanthin de-epoxidaseIUBMB Comments
Along with EC 1.14.15.21, zeaxanthin epoxidase, this enzyme forms part of the xanthophyll (or violaxanthin) cycle for controlling the concentration of zeaxanthin in chloroplasts. It is activated by a low pH of the thylakoid lumen (produced by high light intensity). Zeaxanthin induces the dissipation of excitation energy in the chlorophyll of the light-harvesting protein complex of photosystem II. In higher plants the enzyme reacts with all-trans-diepoxides, such as violaxanthin, and all-trans-monoepoxides, but in the alga Mantoniella squamata, only the diepoxides are good substrates.
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Word Map
- 1.23.5.1
-
vertical
-
xanthophyll
- zeaxanthin
- thylakoids
-
photoelectron
-
de-epoxidation
-
photosystem
-
photoprotection
-
non-photochemical
-
adiabatic
- antheraxanthin
-
photoinhibition
-
ccsdt
-
coupled-cluster
- monogalactosyldiacylglycerol
-
barrier-free
-
photodetachment
-
lowest-energy
- diadinoxanthin
-
pi-scei
-
transthylakoid
-
deepoxidase
The taxonomic range for the selected organisms is: Spinacia oleracea
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Reaction Schemes
+
2
=
+
2
+
2
Synonyms
violaxanthin de-epoxidase, csvde, vx de-epoxidase, zmvde1, ahvde, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
PATHWAY SOURCE
PATHWAYS
BRENDA
SYSTEMATIC NAME
IUBMB Comments
violaxanthin:ascorbate oxidoreductase
Along with EC 1.14.15.21, zeaxanthin epoxidase, this enzyme forms part of the xanthophyll (or violaxanthin) cycle for controlling the concentration of zeaxanthin in chloroplasts. It is activated by a low pH of the thylakoid lumen (produced by high light intensity). Zeaxanthin induces the dissipation of excitation energy in the chlorophyll of the light-harvesting protein complex of photosystem II. In higher plants the enzyme reacts with all-trans-diepoxides, such as violaxanthin, and all-trans-monoepoxides, but in the alga Mantoniella squamata, only the diepoxides are good substrates.
CAS REGISTRY NUMBER
COMMENTARY
57534-73-3
-
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
antheraxanthin + L-ascorbate
zeaxanthin + L-dehydroascorbate + H2O
-
-
-
?
violaxanthin + 2 L-ascorbate
zeaxanthin + 2 L-dehydroascorbate + 2 H2O
overall reaction
-
-
?
violaxanthin + L-ascorbate
antheraxanthin + L-dehydroascorbate + H2O
-
-
-
?
all-trans-neoxanthin + ascorbate
? + dehydroascorbate + H2O
-
2.5% of the activity with violaxanthin
-
-
?
cryptoxanthin-5,6,5',6'-di-epoxide + ascorbate
? + dehydroascorbate + H2O
-
54% of the activity with violaxanthin
-
-
?
cryptoxanthin-5,6-epoxide + ascorbate
? + dehydroascorbate + H2O
-
12.5% of the activity with violaxanthin
-
-
?
diadinoxanthin + ascorbate
? + dehydroascorbate + H2O
-
69% of the activity with violaxanthin
-
-
?
lutein-5,6-epoxide + ascorbate
? + dehydroascorbate + H2O
-
110% of the activity with violaxanthin
-
-
?
violaxanthin + ascorbate
antheraxanthin + dehydroascorbate + H2O
-
-
-
-
?
antheraxanthin + ascorbate
zeaxanthin + dehydroascorbate + H2O
-
-
-
-
?
antheraxanthin + ascorbate
zeaxanthin + dehydroascorbate + H2O
-
146% of the activity with violaxanthin
-
-
?
additional information
?
-
spinach VDE is able to de-epoxidize violaxanthin bound to spinach or Mantoniella squamata light harvesting complexes in a comparable manner, rate constants for first and second reaction step, overview
-
-
?
additional information
?
-
by measuring the initial formation of the product, enzyme VDE is found to convert a large number of violaxanthin molecules to antheraxanthin before producing any zeaxanthin, favoring a model where violaxanthin is bound non-symmetrically in VDE, overview
-
-
?
additional information
?
-
-
no activity with 9-cis neoxanthin and 9-cis violaxanthin
-
-
?
additional information
?
-
-
only the epoxy-oxygen at the 5,6(5',6') position of xanthophylls are cleaved by the VDE, whereas ring-spanning epoxides at position 3,6(3',6') are not accessible to the enzyme. The structure and chemical ligands of the second jonon ring are insignificant for the de-epoxidation of the 5,6-epoxy groups of the first ring. The epoxy-free second jonon ring is not involved in the binding of the xanthophyll to the catalytic center and does not affect the enzyme reaction. Due to steric hindrance, any tested cis-configuration in the polyene chain of the xanthophylls, as well as the 8-oxy group, in fucoxanthin, prevents the de-epoxidation
-
-
?
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
antheraxanthin + L-ascorbate
zeaxanthin + L-dehydroascorbate + H2O
-
-
-
?
violaxanthin + 2 L-ascorbate
zeaxanthin + 2 L-dehydroascorbate + 2 H2O
overall reaction
-
-
?
violaxanthin + L-ascorbate
antheraxanthin + L-dehydroascorbate + H2O
-
-
-
?
antheraxanthin + ascorbate
zeaxanthin + dehydroascorbate + H2O
-
-
-
-
?
violaxanthin + ascorbate
antheraxanthin + dehydroascorbate + H2O
-
-
-
-
?
additional information
?
-
spinach VDE is able to de-epoxidize violaxanthin bound to spinach or Mantoniella squamata light harvesting complexes in a comparable manner, rate constants for first and second reaction step, overview
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
o-phenanthroline
-
0.025 mM, 50% inhibition. The rate of conversion of violaxanthin to antheraxanthin is relatively unchanged whereas the conversion of antheraxanthin to zeaxanthin is 50-80% inhibited
pepstatin
-
50% inhibition at 0.12 mM, reversible, protonation-induced structural change of the enzyme
additional information
-
replacement of monogalactosyldiacylglycerol with digalactosyldiacylglycerol or phosphatidylcholine (PC) in the assay medium results in a strong inhibition of xanthophyll cycle pigments, violaxanthin and diadinoxanthin de-epoxidation. VDE and DDE activity depends not on the chemical character of lipids but on the kind of structure they form in water environment
-
dithiothreitol
-
0.055 mM, 50% inhibition. The rate of conversion of violaxanthin to antheraxanthin is relatively unchanged whereas the conversion of antheraxanthin to zeaxanthin is 50-80% inhibited
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
-
the gel to liquid-crystalline phase transition in the single lipid component systems strongly enhances both the solubilization of the xanthophyll cycle pigment violaxanthin in the membrane and the activity of the violaxanthin de-epoxidase. This phase transition has a significantly stronger impact on violaxanthin de-epoxidase activity than the transition from the liquid-crystalline phase to the inverted hexagonal phase. Especially at higher temperatures increased violaxanthin de-epoxidase reaction rates are detected in the presence of the liquid-crystalline phase compared to those in the presence of HII phase forming lipids. The HII phase is better suited to maintain high violaxanthin de-epoxidase activities at lower temperatures
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
2.3
ascorbate
-
at pH 5.0, with 0.0004 mM violaxanthin and 0.0116 mM monogalactosyldiacylglycerol in reaction medium containing 10 mM KCl, 5 mM MgCl2 and 40 mM morpholinoethanesulfonic acid
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
5 - 5.2
-
irrespective of the presence of high or low ascorbate concentrations
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
an increase of the hydrodynamic radius of wild-type VDE is observed when pH is lowered toward the pH required for activity, consistent with a pH-dependent oligomerization
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
4.79
recombinant N-terminally truncated enzyme mutant, isoelectric focusing
5.24
recombinant mix of N-terminal and C-terminal domain, isoelectric focusing
5.36
recombinant C-terminally truncated enzyme mutant, isoelectric focusing
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
-
regulation of the violaxanthin de-epoxidase involves a conformational change at low lumenal pH, followed by binding of the enzyme to the thylakoid membrane. Protonation of His at low pH induces the conformational change of the enzyme, and hence indirectly regulates binding of the enzyme to the thylakoid membrane
-
the enzyme is mobile within the thylakoid lumen at neutral pH values which occur under in-vivo conditions in the dark. However, upon strong illumination, when the lumen pH drops below pH 6.5 due to formation of a proton gradient, the properties of the de-epoxidase are altered and the enzyme becomes tightly bound to the membrane thus gaining access to its substrate
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
malfunction
mutational reduction of the disulfides in the enzyme results in loss of a rigid structure and a decrease in thermal stability of 15°C
physiological function
additional information
VDE enzyme activity is possible without the C-terminal domain but not without the N-terminal domain. The N-terminal domain shows no VDE activity by itself, but when separately expressed domains are mixed, VDE activity is possible
physiological function
photosynthetic organisms need protection against excessive light. By using non-photochemical quenching, where the excess light is converted into heat, the organism can survive at higher light intensities. This process is partly initiated by the formation of zeaxanthin, which is achieved by the de-epoxidation of violaxanthin and antheraxanthin to zeaxanthin. This reaction is catalyzed by violaxanthin de-epoxidase (VDE)
physiological function
violaxanthin de-epoxidase (VDE) catalyses the conversion of violaxanthin to zeaxanthin at the lumen side of the thylakoids during exposure to intense light
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). VDE_SPIOL
472
0
53667
Swiss-Prot
other Location (Reliability: 3)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
enzyme VDE consists of a cysteine-rich N-terminal domain, a lipocalin-like domain and a negatively charged C-terminal domain. A disulphide pattern in VDE of C9-C27, C14-C21, C33-C50, C37-C46, C65-C72 and C118-C284 is obtained after digestion of VDE with thermolysin followed by mass spectroscopy analysis of reduced versus non-reduced samples. Reduction of the disulfides results in loss of a rigid structure and a decrease in thermal stability of 15°C. Peptide mapping, mass spectroscopy, overview
additional information
-
enzyme VDE consists of a cysteine-rich N-terminal domain, a lipocalin-like domain and a negatively charged C-terminal domain. A disulphide pattern in VDE of C9-C27, C14-C21, C33-C50, C37-C46, C65-C72 and C118-C284 is obtained after digestion of VDE with thermolysin followed by mass spectroscopy analysis of reduced versus non-reduced samples. Reduction of the disulfides results in loss of a rigid structure and a decrease in thermal stability of 15°C. Peptide mapping, mass spectroscopy, overview
additional information
enzyme VDE consists of three domains with the central lipocalin-like domain. VDE enzyme activity is possible without the C-terminal domain but not without the N-terminal domain. The N-terminal domain shows no VDE activity by itself, but when separately expressed domains are mixed, VDE activity is possible. Presence of alpha-helical structure in both the N- and C-terminal domains
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
C09S
site-directed mutagenesisthe mutant shows slightly decreased activity compared to the wild-type enzyme
C248S
site-directed mutagenesis, the mutant shows over 95% reduced activity compared to the wild-type enzyme
C27S
site-directed mutagenesis, the mutant shows about 70% reduced activity compared to the wild-type enzyme
C33S
site-directed mutagenesis, the mutant shows about 90% reduced activity compared to the wild-type enzyme
C37S
site-directed mutagenesis, the mutant shows about 50% reduced activity compared to the wild-type enzyme
C46S
site-directed mutagenesis, the mutant shows about 45% reduced activity compared to the wild-type enzyme
C50S
site-directed mutagenesis, the mutant shows 90% reduced activity compared to the wild-type enzyme
C65S
site-directed mutagenesis, the mutant shows 85% reduced activity compared to the wild-type enzyme
C72S
site-directed mutagenesis, the mutant shows over 95% reduced activity compared to the wild-type enzyme
C7S
site-directed mutagenesis, the mutant shows 2fold increased activity compared to the wild-type enzyme
H121A/H124A
-
considerably lower pH dependence for binding than wild-type, cooperativity value around 2 compared to wild-type value of 3.7. Km-value for ascorbate is 3.2 mM compared to 1.9 mM for wild-type enzyme
H124R
H134A
-
considerably lower pH dependence for binding than wild-type, cooperativity value around 2 compared to wild-type value of 3.7
H167A/H173A
-
considerably lower pH dependence for binding than wild-type, cooperativity value of 1.6 compared to wild-type value of 3.7. Km-value for ascorbate is 8.3 mM compared to 1.9 mM for wild-type enzyme
H167R/H173R
-
considerably lower pH dependence for binding than wild-type, cooperativity value around 2 compared to wild-type value of 3.7. Km-value for ascorbate is 6.3 mM compared to 1.9 mM for wild-type enzyme
additional information
the C-terminally truncated VDE does not show such an oligomerization, is relatively more active at higher pH, but does not alter the KM for ascorbate
H124R
-
considerably lower pH dependence for binding than wild-type, cooperativity value around 2 compared to wild-type value of 3.7. Km-value for ascorbate is 2.1 mM compared to 1.9 mM for wild-type enzyme
H124R
-
considerably lower pH dependence for binding than wild-type, cooperativity value around 2 compared to wild-type value of 3.7.Km-value for ascorbate is 1.5 mM compared to 1.9 mM for wild-type enzyme
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
an increase of the hydrodynamic radius of wild-type VDE is observed when pH is lowered toward the pH required for activity, consistent with a pH-dependent oligomerization
745996
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme from Escherichia coli strain BL21(DE3) solubilized from inclusion bodies
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) solubilized from inclusion bodies
in presence of Tween 20. The enzyme has more than one disulfide bond and takes multiple forms depending on the extent of the reduction
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene VDE1, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) in inclusion bodies
gene VDE1, recombinant expression of wild-type and truncated mutants in Escherichia coli strain BL21(DE3) in inclusion bodies
RENATURED/Commentary
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme from Escherichia coli strain BL21(DE3) inclusion bodies by solibilization with 8 M urea, 60 mM Tris-HCl, pH 8.0, 60 mM NaCl, and 0.6 mM EDTA, followed by dialysis and ultracentrifugation
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) inclusion bodies by solibilization with 8 M urea, 60 mM Tris-HCl, pH 8.0, 60 mM NaCl, and 0.6 mM EDTA, followed by dialysis and ultracentrifugation
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Kawano, M.; Kuwabara, T.
pH-dependent reversible inhibition of violaxanthin de-epoxidase by pepstatin related to protonation-induced structural change of the enzyme
FEBS Lett.
481
101-104
2000
Spinacia oleracea
Grotz, B.; Molnar, P.; Stransky, H.; Hager, A.
Substrate specificity and functional aspects of violaxanthin-de-epoxidase, an enzyme of the xanthophyll cycle
J. Plant Physiol.
154
437-446
1999
Spinacia oleracea
-
Arvidsson, P.O.; Eva Bratt, C.; Carlsson, M.; Aakerlund, H.E.
Purification and identification of the violaxanthin deepoxidase as a 43 kDa protein
Photosynth. Res.
49
119-129
1996
Spinacia oleracea
Emanuelsson, A.; Eskling, M.; Akerlund, H.E.
Chemical and mutational modification of histidines in violaxanthin de-epoxidase from Spinacia oleracea
Physiol. Plant.
119
97-104
2003
Spinacia oleracea (Q9SM43)
-
Gisselsson, A.; Szilagyi, A.; Akerlund, H.E.
Role of histidines in the binding of violaxanthin de-epoxidase to the thylakoid membrane as studied by site-directed mutagenesis
Physiol. Plant.
122
337-343
2004
Spinacia oleracea, Triticum aestivum
-
Kuwabara, T.; Hasegawa, M.; Kawano, M.; Takaichi, S.
Characterization of violaxanthin de-epoxidase purified in the presence of Tween 20: effects of dithiothreitol and pepstatin A
Plant Cell Physiol.
40
1119-1126
1999
Spinacia oleracea
Havir, E.A.; Tausta, S.L.; Peterson, R.B.
Purification and properties of violaxanthin de-epoxidase from spinach
Plant Sci.
123
57-66
1997
Spinacia oleracea
-
Hager, A.; Holocher, K.
Localization of the xanthophyll-cycle enzyme violaxanthin de-epoxidase within the thylakoid lumen and abolition of its mobility by a (light-dependent) pH decrease
Planta
192
581-589
1994
Spinacia oleracea
-
Frommolt, R.; Goss, R.; Wilhelm, C.
The de-epoxidase and epoxidase reactions of Mantoniella squamata (Prasinophyceae) exhibit different substrate-specific reaction kinetics compared to spinach
Planta
213
446-456
2001
Mantoniella squamata, Spinacia oleracea
Goss, R.
Substrate specificity of the violaxanthin de-epoxidase of the primitive green alga Mantoniella squamata (Prasinophyceae)
Planta
217
801-812
2003
Spinacia oleracea, Mantoniella squamata
Grouneva, I.; Jakob, T.; Wilhelm, C.; Goss, R.
Influence of ascorbate and pH on the activity of the diatom xanthophyll cycle-enzyme diadinoxanthin de-epoxidase
Physiol. Plant.
126
205-211
2006
Spinacia oleracea
Vieler, A.; Scheidt, H.A.; Schmidt, P.; Montag, C.; Nowoisky, J.F.; Lohr, M.; Wilhelm, C.; Huster, D.; Goss, R.
The influence of phase transitions in phosphatidylethanolamine models on the activity of violaxanthin de-epoxidase
Biochim. Biophys. Acta
1778
1027-1034
2008
Spinacia oleracea
Latwoski, D.; Goss, R.; Grzyb, J.; Akerlund, H.; Burda, K.; Kruk, J.; Strzalka, K.
De-epoxidases of xanthophyll cycles require non-bilayer lipids for their activity
Biologija (Vilnius)
53
16-20
2007
Spinacia oleracea
-
Goss, R.; Opitz, C.; Lepetit, B.; Wilhelm, C.
The synthesis of NPQ-effective zeaxanthin depends on the presence of a transmembrane proton gradient and a slightly basic stromal side of the thylakoid membrane
Planta
228
999-1009
2008
Spinacia oleracea
Schaller, S.; Latowski, D.; Jemiola-Rzeminska, M.; Quaas, T.; Wilhelm, C.; Strzalka, K.; Goss, R.
The investigation of violaxanthin de-epoxidation in the primitive green alga Mantoniella squamata (Prasinophyceae) indicates mechanistic differences in xanthophyll conversion to higher plants
Phycologia
51
359-370
2012
Mantoniella squamata, Spinacia oleracea (Q9SM43), Mantoniella squamata CCAP 1965/1
-
Hallin, E.I.; Guo, K.; Akerlund, H.E.
Violaxanthin de-epoxidase disulphides and their role in activity and thermal stability
Photosynth. Res.
124
191-198
2015
Spinacia oleracea (Q9SM43), Spinacia oleracea
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