Information on EC 4.1.99.13 - (6-4)DNA photolyase

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
4.1.99.13
-
RECOMMENDED NAME
GeneOntology No.
(6-4)DNA photolyase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(6-4) photoproduct (in DNA) = 2 pyrimidine residues (in DNA)
show the reaction diagram
SYSTEMATIC NAME
IUBMB Comments
(6-4) photoproduct pyrimidine-lyase
A flavoprotein (FAD). The overall repair reaction consists of two distinct steps, one of which is light-independent and the other one light-dependent. In the initial light-independent step, a 6-iminium ion is thought to be generated via proton transfer induced by two histidines highly conserved among the (6-4) photolyases. This intermediate spontaneously rearranges to form an oxetane intermediate by intramolecular nucleophilic attack. In the subsequent light-driven reaction, one electron is believed to be transferred from the fully reduced FAD cofactor (FADH-) to the oxetane intermediate thus forming a neutral FADH radical and an anionic oxetane radical, which spontaneously fractures. The excess electron is then back-transferred to the flavin radical restoring the fully reduced flavin cofactor and a pair of pyrimidine bases [2].
CAS REGISTRY NUMBER
COMMENTARY hide
37290-70-3
not distinguished from DNA photolyase, spore photoproduct lyase
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
-
in contrast to transgenic mice expressing Potorous tridactylus CPD-photolyase transgenic mice expressing Arabidopsis thaliana (6-4) photolyase do not show any altered circadian behaviour
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(6-4) photoproduct (in DNA)
2 pyrimidine residues (in DNA)
show the reaction diagram
(6-4) photoproduct (in DNA)
pyrimidine residues (in DNA)
show the reaction diagram
-
-
-
-
?
C(6-4)C photoproduct (in DNA)
2 cytosine residues (in DNA)
show the reaction diagram
-
-
-
-
?
cyclobutadipyrimidine in DNA
2 pyrimidine residues in DNA
show the reaction diagram
-
-
?
cyclobutadipyrimidine in herring sperm DNA
2 pyrimidine residues in herring sperm DNA
show the reaction diagram
-
-
?
deoxyoligonucleotide containing (6-4) photoproduct + H2O
deoxyoligonucleotide containing 2 pyrimidine residues
show the reaction diagram
-
-
-
?
Dewar photoproduct
?
show the reaction diagram
-
although the affinity of the enzyme for the Dewar photoproduct-containing duplex is similar to that for the (6-4) photoproduct containing substrate a repair rate could not be shown. These results indicate that the (6-4) photolyase binds the DNA containing the Dewar photoproduct and induces a structural change in DNA to some extent, suggesting a difference in the binding mode compared to the (6-4) photoproduct
-
-
?
T(6-4)C photoproduct (in DNA)
2 thymidine + cytosine (in DNA)
show the reaction diagram
-
-
-
-
?
T(6-4)C photoproduct (in DNA)
T-C (in DNA)
show the reaction diagram
-
A T(6-4)C photoproduct is synthesized. Differences from T(6-4)T is formation of cytosine hydrates by UV irradiation, and acylation of the amino function with the capping reagent. The capping step is omitted to improve the yield of the desired oligonucleotides. (6-4) photolyase restores the pyrimidines in T(6-4)C to their original structures
-
-
?
T(6-4)T photoproduct (in DNA)
2 thymidine resdiues (in DNA)
show the reaction diagram
-
-
-
-
?
T(6-4)T photoproduct (in DNA)
2 thymidine residues (in DNA)
show the reaction diagram
T(6-4)T photoproduct (in DNA)
2 thymine residues (in DNA)
show the reaction diagram
-
-
-
-
?
T(6-4)T photoproduct (in DNA)
thymidine residues (in DNA)
show the reaction diagram
-
-
-
-
?
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
(6-4) photoproduct (in DNA)
2 pyrimidine residues (in DNA)
show the reaction diagram
cyclobutadipyrimidine in DNA
2 pyrimidine residues in DNA
show the reaction diagram
Q86RA1
-
-
?
T(6-4)T photoproduct (in DNA)
2 thymine residues (in DNA)
show the reaction diagram
-
-
-
-
?
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5,10-methenyltetrahydrofolate
required
6,7-dimethyl-8-ribityllumazine
the PhrB structure contains 6,7-dimethyl-8-ribityllumazine as an antenna chromophore, binding site-structure, overview
8-hydroxy-5-deazariboflavin
-
-
additional information
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Fe2+
the prokaryotic enzyme contains a a [4Fe-4S] cluster bound to the catalytic domain
additional information
-
enzyme is also active under high salinity; NH4Cl or NaH2PO4 increase activity of the enzyme. CH3COONa or Na2CO3 strongly decrease the enzymes activity. Maximum activity occurs in the presence of NaH2PO4, which is increased eight times than that in the presence of Na2CO3. When the ions that possess stronger ability to donate a proton are added to enzyme reaction buffer, the rate of photoreactivation increases
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Dithionite
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reduction of the FAD cofactor with dithionite increases the quantum yield of repair
additional information
-
for activation of Xenopus (6-4) PHR, FADox is first converted to a neutral radical form by light-induced one-electron and one-proton transfers and then into a fully reduced form by light-induced one electron transfer, four different redox states for the FAD chromophore of PHR, mechanism, overview
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8
-
assay at
10
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(6-4) photolyase in the alkaline treated algae cells exhibit much more efficient repair than that in the normally cultured algae cells
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 9
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wild-type, K281G mutant and K281R mutant show no differences in this pH range. Highest activity is reached at pH 9.0
6 - 9.5
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8.5 - 9
9 - 11
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in this pH range wild-type, K281G mutant and K281R mutant show different activities: The activity of K281G mutant declines sharply and less than 30% of the activity is retained at pH 11.0, whereas K281R mutant and the wild-type shows more tolerance to the high pH (9.0-11.0), and at pH 11.0, 78.5% and 62.3% of the activity are retained, respectively
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22
assay at room temperature
additional information
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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by Northern blot analysis it is demonstrated that the transcript is expressed at highest level only in adult ovary
Manually annotated by BRENDA team
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exposure of the cells to blue light, but not red light, for 12 h results in more than 20fold increase of the (6-4) photolyase mRNA
Manually annotated by BRENDA team
additional information
PDB
SCOP
CATH
ORGANISM
UNIPROT
Agrobacterium fabrum (strain C58 / ATCC 33970)
Agrobacterium fabrum (strain C58 / ATCC 33970)
Agrobacterium fabrum (strain C58 / ATCC 33970)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40000
-
SDS-PAGE
63000
-
SDS-PAGE
63920
calculated from amino acid sequence
67000
deduced from cDNA
110000
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molecular weight of fusion protein, determined by SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant His-tagged enzyme, hanging drop vapor diffusion method, mixing of 0.005 ml of protein in 12.5 mM Tris (hydroxymethyl) amino methane, 1.25 mM EDTA, 2.5% v/v glycerol, 75 mM sodium chloride, pH 7.8, with 0.005 ml of reservoir solution containing 2-6% w/v PEG400, 100 mM 2-(N-morpholino) ethanesulfonic acid buffer, pH 6.0, 25C, 3-7 days, X-ray diffraction structure determination and analysis at 1.45-1.95 A resolution
hanging drop vapor diffusion at 4C
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T(6-4)C lesion containing DNA duplex in complex with the (6-4) photolyase, by the hanging-drop vapour diffusion method, at 18C, to 2.95 A resolution. Lesion is flipped out of the opened DNA duplex into the active site of the enzyme
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to study how the enzyme recognizes the T(6-4)C and T(Dew)C lesion analogues in the active site, DNA duplexes are crystallized together with the (6-4) photolyase
two crystal structures of the (6-4) photolyase bound to lesion containing DNA before and after repair, repair does not involve oxetane formation before light-induced electron transfer. The histidine 369, supposed to activate the acylimine, is in a position that does not allow efficient proton donation and hence activation of this substructure
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
activity is unstable and it is lost upon storage at -20 or -70C for 2-3 months
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glycerol to a final concentration of 50% (v/v) is added to the photolyase, storage at -70C
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protein in aliquots is kept at -20C until further use or stored at -80C. The protein sample remained active after 6 months storage
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protein is stored in aliquots at -80 C after addition of glycerol to 20%
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
(6-4) photoproduct DNA photolyase activity is detected in Crotalus atrox fibroblast. Activity is considerably enhanced when a UV-damaged DNA affinity column is used for purification. However, the activity is unstable and it is lost during purification or upon storage at -20 or -70C for 2-3 months
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by fractionation of crude cell extracts with Heparin agarose and UV DNA affinity column chromatography
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by using a glutathione sepharose column and a Hi Trap Q column. Concentrated fusion protein is cleaved with thrombin from bovine plasma by incubation overnight at 4C. For chromophore determination, the eluate from the glutathione sepharose column is purified through a Q sepharose column, omitting gel filtration procedure, and concentrated by ultrafiltration
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by using a glutathione-sepharose column and UV-irradiated DNA affinity column, fusion protein is cleaved with thrombin
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by using affinity chromatography and UV-irradiated DNA attached beads
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by using glutathione-sepharose columns
fusion protein is applied to amylose column. At this point the protein is above 90% pure. Further purification can be obtained by applying the eluted material to a 10 ml heparin-agarose column. Maltose-binding protein is removed by treatment with factor Xa protease
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fusion protein is purified through a 20-ml amylose column and through heparin-agarose column
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NF-1 is isolated from nuclear extracts prepared from HeLa cell culture using a heparin column, an anion exchange Fractogel EMD TMAE-650 (S) column and a FPLC MonoS HR5/5 column
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protein is isolated from whole cell extracts from Xenopus laevis, purified by using a sepharose column and a UV-damaged DNA affinity column
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purified to near homogeneity from Drosophila embryonic cells by using NH4SO4 precipitation, SP sepharose column, UV DNA affinity beads, heparin sepharose column and Mono S column
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recombinant His-tagged enzyme from Escherichia coli by nickel affinity chromatography and gel filtration under dim light conditions or in darkness
recombinant N-terminally His-tagged enzyme from Escherichia coli strain BL21(DE3) by metal affinity and heparin affinity chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
a cDNA of (6-4)photolyase from Dunaliella salina is cloned, sequenced and its amino acid sequence is deduced
cloned and overexpressed in Escherichia coli
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due to insolubility problems (6-4) photolyases is overexpressed as a fusion protein in Escherichia coli. Plasmid pXZ1997, a derivative of pMal-c2 containing the Drosophila melanogaster phr(6-4) cDNA fused in frame to the malE gene encoding maltose-binding protein (MBP), is propagated in Escherichia coli strain UNC523 (phr::kan uvrA::Tn10) selecting for ampicillin resistance. Cells are cultured in 2 liter of LB to A600: 0.60.8. IPTG is added to 0.3 mM, and incubation continued for 6 h prior to harvesting the cells by centrifugation.
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expressed as a GST-fusion protein in Escherichia coli
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expressed as a GST-tagged fusion protein in Escherichia coli
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expressed as a GSTtagged fusion protein in Escherichia coli
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expressed as a His-tagged fusion protein
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expressed in Escherichia coli
expressed in Escherichia coli as a GST-fusion protein
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expressed in Escherichia coli as a MBP-PL-(6-4) fusion protein
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expressed in Escherichia coli, fusion protein with glutathione S-transferase
expression in Escherichia coli
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expression of N-terminally His-tagged enzyme in Escherichia coli strain BL21(DE3)
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overexpressed in Escherichia coli
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phylogenetic analysis, DNA and amino acid sequence determination and analysis, expression of isozymes in Escherichia coli strain UNC 523 from pMal-c2x vector
recombinant expression of His-tagged enzyme in Escherichia coli
recombinant protein expressed in Escherichia coli
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
mRNA level of Ds64PHR in 24 h dark-grown algae cells increases in response to the exposure of UV-C. The mRNA levels of Ds64PHR increases after alkaline treatment
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
H364A
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compared to wild-type mutant similar electron transfer dynamics in the range of 70-260 ps but decay to zero without any long plateaus, H364 is irreplaceable: Steady-state quantum yield measurements reveal a total lack of repair with the mutant
H364D
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compared to wild-type mutant similar electron transfer dynamics in the range of 70-260 ps but decay to zero without any long plateaus, H364 is irreplaceable: Steady-state quantum yield measurements reveal a total lack of repair with the mutant
H364K
-
compared to wild-type mutant similar electron transfer dynamics in the range of 70-260 ps but decay to zero without any long plateaus, H364 is irreplaceable: Steady-state quantum yield measurements reveal a total lack of repair with the mutant
H364M
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compared to wild-type mutant similar electron transfer dynamics in the range of 70-260 ps but decay to zero without any long plateaus, H364 is irreplaceable: Steady-state quantum yield measurements reveal a total lack of repair with the mutant
H364N
-
compared to wild-type mutant similar electron transfer dynamics in the range of 70-260 ps but decay to zero without any long plateaus, H364 is irreplaceable: Steady-state quantum yield measurements reveal a total lack of repair with the mutant
H364Y
-
compared to wild-type mutant similar electron transfer dynamics in the range of 70-260 ps but decay to zero without any long plateaus, H364 is irreplaceable: Steady-state quantum yield measurements reveal a total lack of repair with the mutant
H365N
mutant shows no repair activity
K281G
-
a single T(6-4)T photoproduct in a 10-mer oligonucleotide is photoreactivated by this mutant. Mutant shows similar capacity of photoreactivation compared to wild-type. Over the pH range 6-9 no difference to wild-type. Over the pH range 9-11 the activity of K281G mutant declines sharply and less than 30% of the activity is retained at pH 11.0
K281R
-
a single T(6-4)T photoproduct in a 10-mer oligonucleotide is photoreactivated by this mutant. Mutant shows similar capacity of photoreactivation compared to wild-type. Over the pH range 6-9 no difference to wild-type. Over the pH range 9-11 the K281R mutant and the wild-type show more tolerance to the high pH (9.0-11.0), and at pH 11.0, 78.5% and 62.3% of the activity are retained, respectively
L355A
-
large decrease in the affinity to the (6-4) photoproduct substrate, suggesting a hydrophobic interaction with the (6-4)photoproduct
Q288A
-
repair activity is not reduced
W291A
-
some enzymatic activity is retained
W398A
-
some enzymatic activity is retained
additional information