Information on EC 4.1.99.14 - spore photoproduct lyase

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The expected taxonomic range for this enzyme is: Firmicutes

EC NUMBER
COMMENTARY hide
4.1.99.14
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RECOMMENDED NAME
GeneOntology No.
spore photoproduct lyase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double-helical DNA) = thymidylyl-(3'->5')-thymidylate (in double-helical DNA)
show the reaction diagram
SYSTEMATIC NAME
IUBMB Comments
spore photoproduct pyrimidine-lyase
This enzyme is a member of the 'AdoMet radical' (radical SAM) family. The enzyme binds a [4Fe-4S] cluster. The cluster is coordinated by 3 cysteines and an exchangeable SAM molecule [3]. The 5'-deoxy-adenosine radical formed after electron transfer from the [4Fe-4S] cluster to the S-adenosyl-L-methionine, initiates the repair by abstracting the C-6 hydrogen of the spore photoproduct lesion. During the second part of the repair process the SAM molecule is regenerated [3].
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
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
(5''R)-alpha-5''(6''H)-bithymine + S-adenosyl-L-methionine
thymidylyl-(3'-5')-thymidylate + 5'-deoxyadenosine + L-methionine
show the reaction diagram
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'-5')-thymidylate
show the reaction diagram
-
the enzyme establishes a complex radical transfer cascade and creates a cysteine and a tyrosyl radical dyade to establish repair. This allows the enzyme to solve topological and energetic problems associated with the radical based repair reaction
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-
?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
show the reaction diagram
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double helical DNA) + S-adenosyl-L-methionine + 2 H+
thymidylyl-(3'-5')-thymidylate (in DNA) + 5'-deoxyadenosine + L-methionine
show the reaction diagram
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine + S-adenosyl-L-methionine
thymidylyl-(3'->5')-thymidylate + 5'-deoxyadenosine + L-methionine
show the reaction diagram
(5R)-CH2-spore photoproduct + S-adenosyl-L-methionine
?
show the reaction diagram
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a dinucleotide spore photodroduct isostere (5R)-CH2SP is prepared, which contains a neutral CH2 moiety between the two thymine residues instead of a phosphate. ROESY spectroscopic, DFT computational, and enzymatic studies of this (5R)-CH2SP compound prove that it possesses similar properties with the (5R) spore photoproduct species
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-
?
(5S)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double helical DNA)
thymidylyl-(3'-5')-thymidylate (in DNA)
show the reaction diagram
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assay with a synthetic dinucleotide SP lesion substrate and with smallDNAsingle strands, which contain one SP lesion at a defined site
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-
?
5,6-dihydro-5-(thymidin-7-yl)thymidine (in double helical DNA)
thymidylyl-(3'-5')-thymidylate (in DNA)
show the reaction diagram
-
-
-
-
?
5-(alpha-thyminyl)-5,6-dihydrothymidine
thymidylyl-(3'-5')-thymidylate
show the reaction diagram
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the enzyme repairs 5-(alpha-thyminyl)-5,6-dihydrothymidine in DNA
-
-
?
5-thyminyl-5,6 dihydrothymine + S-adenosyl-L-methionine
thymidylyl-(3'-5')-thymidylate + 5'-deoxyadenosine + L-methionine
show the reaction diagram
additional information
?
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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
(5''R)-alpha-5''(6''H)-bithymine + S-adenosyl-L-methionine
thymidylyl-(3'-5')-thymidylate + 5'-deoxyadenosine + L-methionine
show the reaction diagram
-
SPL repairs specifically the 5R isomer. (5''R)-alpha-5''(6''H)-bithymine is the diastereomer produced upon UV irradiation of a TpT dinucleotide
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-
?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'-5')-thymidylate
show the reaction diagram
-
the enzyme establishes a complex radical transfer cascade and creates a cysteine and a tyrosyl radical dyade to establish repair. This allows the enzyme to solve topological and energetic problems associated with the radical based repair reaction
-
-
?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
show the reaction diagram
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double helical DNA) + S-adenosyl-L-methionine + 2 H+
thymidylyl-(3'-5')-thymidylate (in DNA) + 5'-deoxyadenosine + L-methionine
show the reaction diagram
5,6-dihydro-5-(thymidin-7-yl)thymidine (in double helical DNA)
thymidylyl-(3'-5')-thymidylate (in DNA)
show the reaction diagram
-
-
-
-
?
additional information
?
-
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the (5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine lesion does not absolutely need to be contained within a single or double-stranded DNA for recognition and repaired by the enzyme
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
S-adenosyl-L-methionine
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Fe
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iron-sulfur enzyme, 2.9 Fe per enzyme
iron-sulfur centre
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the UV-vis spectrum of the purified enzyme is characteristic of the presence of an iron-sulfur cluster. Purified enzyme contains between 2.3 and 3.1 iron atoms per protein. Electron paramagnetic resonance (EPR) spectroscopy reveals a [3Fe-4S]+ cluster accounting for 3-4% of the iron in the sample. Upon reduction, a signal characteristic of a [4Fe-4S]+ cluster is observed that accounts for 34-45% of total iron in the sample
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
aza-S-adenosyl-L-methionine
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complete inhibition
Dithionite
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0009 - 0.0113
5-(alpha-thyminyl)-5,6-dihydrothymidine
additional information
additional information
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0071
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pH 7.5, 30°C; reduced purified recombinant enzyme, pH 7.5, temperature not specified in the publication
2.6
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purified recombinant enzyme, pH and temperature not specified in the publication
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8.5
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recombinant His6-tagged mutant C141A, isoelectric focussing
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Geobacillus thermodenitrificans (strain NG80-2)
Geobacillus thermodenitrificans (strain NG80-2)
Geobacillus thermodenitrificans (strain NG80-2)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40000
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SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
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the aerobically purified recombinant apo-SplG forms a homodimer, which contains one [4Fe-4S] cluster permonomerunit after reconstitution to the holoform, secondary SplG structure, overview
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant His6-tagged mutant enzyme Y98F, with reconstituted iron-sulfur cluster, hanging drop vapor diffusion method, using 70 mM octanoyl-N-hydroxyethylglucamide, 200 mM lithium sulfate, 100 mM Tris-HCl, pH 9.0 and 19-27% w/v PEG 8000, X-ray diffraction structure determination and analysis
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purified wild-type and mutant enzymes in complex with the S-adenosyl-L-methionine cofactor and the [4Fe-4S] cluster, hanging drop vapor diffusion method, using 70 mM octanoyl-N-hydroxyethylglucamide and a reservoir solution containing 200 mM lithium sulfate, 100 mM Tris-HCl, pH 9.0, and 19-27% w/v PEG 8000., 20°C, cryoprotection of crystals by 3 mM dithiothreithol, 500 mM NaCl, and 15% v/v ethylene glycol in mother liquor, X-ray diffraction structure determination and analysis at 2.0-2.1 A resolution
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant His-tagged enzyme from Escherichia coli strain Tuner (DE3) by nickel affinity chromatography
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recombinant His-tagged enzyme from Escherichia coli Tuner(DE3)pLysS by nickel affinity chromatography to homogeneity; using a HisTrap HP 5-mL column
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recombinant His6-tagged enzyme from Escherichia coli strain pLysS (DE3) by nickel affinity chromatography and gel filtration
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recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity and cation exchange chromatography
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recombinant N- or C-terminally His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
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recombinant wild-type, selenomethionine-labeled, and mutant His6-tagged enzymes from Escherichia coli strain BL21(DE3) by nickel and heparin affinity chromatography
using Ni-NTA chromatography
E8SZR7
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli as a His-tagged fusion protein
E8SZR7
functional expression of N-terminally His6-tagged enzyme in Escherichia coli strain pLysS (DE3)
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gene GTNG_2348, expression of His6-tagged native and selenomethionine-labeled wild-type enzyme and His6-tagged mutant enzymes in Escherichia coli strain BL21(DE3)
gene splB, functional expression of His6-tagged enzyme in Escherichia coli Tuner(DE3)pLysS; heterologously expressed in Escherichia coli as a His-tagged fusion protein
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gene splB, overexpression of His6-tagged wild-type and mutant enzymes and co-expression with plasmid pDB1282, in Escherichia coli strain BL21(DE3)
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gene splB, overexpression of N- or C-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3)
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gene splB, overexpression of N-terminally His-tagged enzyme in Escherichia coli strain Tuner (DE3)
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overexpression in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Y97A
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site-directed mutagenesis, the mutation disrupts the interaction between the phenol ring of the Y97 and the adenine ring of S-adenosyl-L-methionine, subsequently affecting S-adenosyl-L-methionine binding and/or reductive cleavage, the mutant shows altered competitive kinetic isotope effects compared to the wild-type enzyme
Y97A/Y99A
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site-directed mutagenesis, inactive mutant, the Y97 mutation disrupts the interaction between the phenol ring of the Y97 and the adenine ring of S-adenosyl-L-methionine, subsequently affecting S-adenosyl-L-methionine binding and/or reductive cleavage
Y97F
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site-directed mutagenesis, the mutation disrupts the interaction between the phenol ring of the Y97 and the adenine ring of S-adenosyl-L-methionine, subsequently affecting S-adenosyl-L-methionine binding and/or reductive cleavage, the mutant shows altered competitive kinetic isotope effects compared to the wild-type enzyme
Y98A
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site-directed mutagenesis, the mutant's active site architecture, activity, and kinetics are similar to the wild-type enzyme
Y98F
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site-directed mutagenesis, the mutant's active site architecture, activity, and kinetics are similar to the wild-type enzyme
Y99A
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site-directed mutagenesis, the mutant shows altered competitive kinetic isotope effects compared to the wild-type enzyme
Y99F
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site-directed mutagenesis, the mutant shows altered competitive kinetic isotope effects compared to the wild-type enzyme
C141A
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site-directed mutagenesis, the mutant repairs spore photoproduct at a rate which is about 3fold slower than the wild-type enzyme, the mutant shows altered competitive kinetic isotope effects compared to the wild-type enzyme. S-adenosyl-L-methionine plays a non-catalytical role in the mutant in contrast to the wild-type enzyme; site-directed mutagenesis, the mutant shows altered competitive kinetic isotope effects compared to the wild-type enzyme
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Y97F
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site-directed mutagenesis, the mutation disrupts the interaction between the phenol ring of the Y97 and the adenine ring of S-adenosyl-L-methionine, subsequently affecting S-adenosyl-L-methionine binding and/or reductive cleavage, the mutant shows altered competitive kinetic isotope effects compared to the wild-type enzyme
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Y98A
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site-directed mutagenesis, the mutant's active site architecture, activity, and kinetics are similar to the wild-type enzyme
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Y99F
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site-directed mutagenesis, the mutant shows altered competitive kinetic isotope effects compared to the wild-type enzyme
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C140G
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site-directed mutagenesis, the mutant still shows catalytic turnover, but reduced activity, due to a replacement of the thiyl radical by a thermodynamically comparable glycyl radical16 in the catalytic cycle
C140S
site-directed mutagenesis, the mutant shows a similar protein and substrate binding structure compared to the wild-type enzyme, but reduced repair activity
Y98F
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the mutant shows in addition a reduced substrate binding affinity, which indicates that the phenolic hydroxyl group is important to organize the substrate in the active site
C140A
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site-directed mutagenesis, the mutant shows a similar protein and substrate binding structure compared to the wild-type enzyme, but 2.5fold reduced repair activity
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C140S
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site-directed mutagenesis, the mutant shows a similar protein and substrate binding structure compared to the wild-type enzyme, but reduced repair activity
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additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
for reconstitution of the holoenzyme with [4Fe-4S]-cluster, the recombinant apo-SplG is dissolved in 0.8 ml of reconstitution buffer containing 50 mM Tris-HCl, pH 8.0, and 5mM DTT, for 0.5 h, followed by the addition of FeCl2 and Na2S to a final concentration of 100M. The SplG is then incubated anaerobically for 16 h at 4 °C
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
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a rapid separation technique for detecting and quantitating SP by chromatography : tritiated thymine-containing photoproducts from trifluoroacetic acid-hydrolyzed DNA purified from UV-irradiated cells or spores of Bacillus subtilis are identified and isolated from paper chromatograms, subjected to HPLC on a Microsorb phenyl 5-micrometer column using 100% water as the mobile phase, and detected by scintillation counting of collected fractions
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