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
4.1.99.14
-
RECOMMENDED NAME
GeneOntology No.
spore photoproduct lyase
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
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
-
-
-
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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].
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
SP lyase
Clostridium acetobutylicum ATCC824D
-
-
-
spore photoproduct lyase
-
-
spore photoproduct lyase
-
-
spore photoproduct lyase
Clostridium acetobutylicum ATCC824D
-
-
-
additional information
-
the SP lyase is an S-adenosyl-L-methionine-dependent iron-sulfur protein that belongs to the radical S-adenosylmethionine superfamily
additional information
-
SP lyase is a member of the radical S-adenosyl-L-methionine superfamily of enzymes
additional information
Clostridium acetobutylicum ATCC824D
-
SP lyase is a member of the radical S-adenosyl-L-methionine superfamily of enzymes
-
additional information
-
the thermophilic spore photoproduct lyase belongs to the family of radical S-adenosylmethionine enzymes
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
gene splB
-
-
Manually annotated by BRENDA team
Clostridium acetobutylicum ATCC824D
gene splB
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
physiological function
-
the overwhelming majority of DNA photoproducts in UV-irradiated spores is a unique thymine dimer called spore photoproduct, SP, or 5-thymine-5,6-dihydrothymine. This lesion is repaired by the spore photoproduct lyase enzyme that directly reverts 5-thymine-5,6-dihydrothymine to two unmodified thymines. The SP lyase enzyme demonstrates an aspect of the diversity of DNA repair mechanisms in living organisms
additional information
-
spectral analysis of the purified recombinant enzyme, overview
additional information
Clostridium acetobutylicum ATCC824D
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spectral analysis of the purified recombinant enzyme, overview
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SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
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, SPL repairs specifically the 5R isomer
-
-
?
(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 (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 (in double helical DNA) + S-adenosyl-L-methionine + 2 H+
thymidylyl-(3'-5')-thymidylate (in DNA) + 5'-deoxyadenosine + L-methionine
show the reaction diagram
-
assay with a synthetic dinucleotide SP lesion substrate and with smallDNAsingle strands, which contain one SP lesion at a defined site
-
-
?
(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
-
the reduced enzyme rapidly and completely repairs the 5R-diastereomer of a synthetic dinucleotide SP, whereas no repair occurs with the 5S-diastereomer
-
-
?
(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
Clostridium acetobutylicum ATCC824D
-
-, the reduced enzyme rapidly and completely repairs the 5R-diastereomer of a synthetic dinucleotide SP, whereas no repair occurs with the 5S-diastereomer
-
-
?
(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
Clostridium acetobutylicum, Clostridium acetobutylicum ATCC824D
-
no repair is observed for the (5S) diasteroisomer
-
-
?
(5R)-CH2-spore photoproduct + S-adenosyl-L-methionine
?
show the reaction diagram
-
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
-
-
?
(5S)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double helical DNA)
thymidylyl-(3'-5')-thymidylate (in DNA)
show the reaction diagram
-
assay with a synthetic dinucleotide SP lesion substrate and with smallDNAsingle strands, which contain one SP lesion at a defined site
-
-
?
5,6-dihydro-5-(thymidin-7-yl)thymidine (in double helical DNA)
thymidylyl-(3'-5')-thymidylate (in DNA)
show the reaction diagram
-
-
-
-
?
5-thyminyl-5,6 dihydrothymine + S-adenosyl-L-methionine
thymidylyl-(3'-5')-thymidylate + 5'-deoxyadenosine + L-methionine
show the reaction diagram
-
-
-
-
?
5-thyminyl-5,6 dihydrothymine + S-adenosyl-L-methionine
thymidylyl-(3'-5')-thymidylate + 5'-deoxyadenosine + L-methionine
show the reaction diagram
-
via organic synthesis and DNA photochemistry, the two H-atoms at the C6 carbon (6-HproS or 6-HproR position) are selectively labeled with a deuterium in a dinucleotide spore photoproduct TpT substrate. Monitoring the deuterium migration in enzyme catalysis reveals that it is the 6-HproR atom of spore photoproduct that is abstracted by the 5'-dA radical. The abstracted deuterium is not returned to the resulting TpT after enzymatic catalysis, an H-atom from the aqueous buffer is incorporated into TpT instead
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-
?
additional information
?
-
E8SZR7
synthesis and DNA incorporation of a DNA SP lesion analogue lacking the phosphodiester backbone is reported. Repair studies show that the 5'-3' (5R) analogue, incorporated in oligonucleotides, is efficiently repaired
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-
-
additional information
?
-
-
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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-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
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
-
-
?
(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 (in double helical DNA) + S-adenosyl-L-methionine + 2 H+
thymidylyl-(3'-5')-thymidylate (in DNA) + 5'-deoxyadenosine + L-methionine
show the reaction diagram
Clostridium acetobutylicum, Clostridium acetobutylicum ATCC824D
-
-
-
-
?
5,6-dihydro-5-(thymidin-7-yl)thymidine (in double helical DNA)
thymidylyl-(3'-5')-thymidylate (in DNA)
show the reaction diagram
-
-
-
-
?
additional information
?
-
-
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
-
-
-
COFACTOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
S-adenosyl-L-methionine
-
the enzyme is an S-adenosylmethionine-dependent iron-sulfur protein that belongs to the radical S-adenosylmethionine superfamily
S-adenosyl-L-methionine
-
-
S-adenosyl-L-methionine
-
-
S-adenosyl-L-methionine
-
-
METALS and IONS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
Fe
-
iron-sulfur enzyme, 2.9 Fe per enzyme
Fe2+
-
the enzyme is an S-adenosylmethionine-dependent [4Fe-4S]2+ protein, spectral analysis, overview
Fe2+
-
iron-sulfur protein, the aerobically purified apo-SplG forms a homodimer, which contains one [4Fe-4S] cluster per monomer unit after reconstitution to the holoform
Fe2+
-
the purified enzyme contains between 2.3 and 3.1 iron atoms per protein molecule. A [3Fe-4S]+ cluster accounts for 3-4% of the iron, and a [4Fe-4S]+ cluster accounts for 34-45% of total iron. The SP lyase can be photoreduced under anaerobic conditions in the presence of DTT, tris(hydroxymethyl)aminomethane, and 5-deazariboflavin
iron-sulfur centre
-
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 ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
aza-S-adenosyl-L-methionine
-
complete inhibition
ACTIVATING COMPOUND
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
additional information
-
additional information
-
kinetics
-
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
0.0071
-
-
pH 7.5, 30°C; reduced purified recombinant enzyme, pH 7.5, temperature not specified in the publication
2.6
-
-
purified recombinant enzyme, pH and temperature not specified in the publication
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
7
-
-
assay at
8
-
-
assay at
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
20
-
E8SZR7
assay at
37
-
-
assay at
37
-
E8SZR7
assay at
PDB
SCOP
CATH
ORGANISM
Geobacillus thermodenitrificans (strain NG80-2)
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
40000
-
-
SDS-PAGE
41000
-
-
SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
?
-
x * 41000, recombinant His-tagged enzyme, SDS-PAGE
?
Clostridium acetobutylicum ATCC824D
-
x * 41000, recombinant His-tagged enzyme, SDS-PAGE
-
homodimer
-
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
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
recombinant His-tagged enzyme from Escherichia coli strain Tuner (DE3) by nickel affinity chromatography
-
recombinant His-tagged enzyme from Escherichia coli Tuner(DE3)pLysS by nickel affinity chromatography to homogeneity; using a HisTrap HP 5-mL column
-
recombinant His6-tagged enzyme from Escherichia coli strain pLysS (DE3) by nickel affinity chromatography and gel filtration
-
using Ni-NTA chromatography
E8SZR7
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
gene splB, overexpression of N-terminally His-tagged enzyme in Escherichia coli strain Tuner (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
-
overexpression in Escherichia coli
-
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)
-
Renatured/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
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 ACCESSION NO.
COMMENTARY
LITERATURE
analysis
-
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