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
-
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
-
-
-
-
(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
reaction mechanism, overview
-
(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
the enzyme is a radical S-adenosyl-L-methionine enzyme, which uses a [4Fe-4S]1+ cluster to reduce the S-adenosyl-L-methionine, generating a catalytic 5'-deoxyadenosyl radical. This in turn abstracts an H atom from spore product, generating an spore product radical that undergoes beta scission to form a repaired 5'-thymine and a 3'-thymine allylic radical. A conserved cysteine donates an H atom to the thymine radical, resulting in a putative thiyl radical. Two conserved tyrosines are also critical in enzyme catalysis. One, Y99, is downstream of the cysteine, suggesting that the enzyme uses a hydrogen atom transfer pathway with a pair of cysteine-tyrosine residues to regenerate S-adenosyl-L-methionine. The other tyrosine, Y97, has a structural role to facilitate S-adenosyl-L-methionine binding. It may also contribute to the S-adenosyl-L-methionine regeneration process by interacting with the putative Y99 radical and/or 5-dA radical intermediates to lower the energy barrier for the second H-abstraction step. Irreversible first step and tightly coupled radical relay mechanism
-
(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
the enzyme catalyzes the spore photoproduct repair reaction via a radical mediated direct reverse mechanism. At the 1+ oxidation state, the [4Fe-4S] cluster provides an electron to the S-adenosyl-L-methionine, which binds to the cluster in a bidentate manner as the fourth and fifth ligands, to reductively cleave the C-S bond associated with the sulfonium ion in S-adenosyl-L-methionine, generating a reactive 5'-deoxyadenosyl radical. This 5'-dA radical abstracts the proR hydrogen atom from the C6 carbon of spore photoproduct to initiate the repair process. The resulting spore photoproduct radical subsequently fragments to generate a putative thymine methyl radical, which accepts a back-donated H atom to yield the repaired thymidylyl-(3'->5')-thymidylate. Cys141 is involved in the catalytic mechanism as the potential H atom donor to the thymine methyl radical, reaction mechanism, detailed overview. A a thiyl radical is subsequently generated on Cys141
-
(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
reaction mechanism, two different routes, overview
-
(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
the enzyme catalyzes the spore photoproduct repair reaction and combines specific features of radical S-adenosyl-L-methionine and DNA repair enzymes to enable a complex radical-based repair reaction to take place
A4IQU1
(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
reaction mechanism, overview; the enzyme catalyzes the spore photoproduct repair reaction via a radical mediated direct reverse mechanism. At the 1+ oxidation state, the [4Fe-4S] cluster provides an electron to the S-adenosyl-L-methionine, which binds to the cluster in a bidentate manner as the fourth and fifth ligands, to reductively cleave the C-S bond associated with the sulfonium ion in S-adenosyl-L-methionine, generating a reactive 5'-deoxyadenosyl radical. This 5'-dA radical abstracts the proR hydrogen atom from the C6 carbon of spore photoproduct to initiate the repair process. The resulting spore photoproduct radical subsequently fragments to generate a putative thymine methyl radical, which accepts a back-donated H atom to yield the repaired thymidylyl-(3'->5')-thymidylate. Cys141 is involved in the catalytic mechanism as the potential H atom donor to the thymine methyl radical, reaction mechanism, detailed overview. A a thiyl radical is subsequently generated on Cys141; the enzyme is a radical S-adenosyl-L-methionine enzyme, which uses a [4Fe-4S]1+ cluster to reduce the S-adenosyl-L-methionine, generating a catalytic 5'-deoxyadenosyl radical. This in turn abstracts an H atom from spore product, generating an spore product radical that undergoes beta scission to form a repaired 5'-thymine and a 3'-thymine allylic radical. A conserved cysteine donates an H atom to the thymine radical, resulting in a putative thiyl radical. Two conserved tyrosines are also critical in enzyme catalysis. One, Y99, is downstream of the cysteine, suggesting that the enzyme uses a hydrogen atom transfer pathway with a pair of cysteine-tyrosine residues to regenerate S-adenosyl-L-methionine. The other tyrosine, Y97, has a structural role to facilitate S-adenosyl-L-methionine binding. It may also contribute to the S-adenosyl-L-methionine regeneration process by interacting with the putative Y99 radical and/or 5-dA radical intermediates to lower the energy barrier for the second H-abstraction step. Irreversible first step and tightly coupled radical relay mechanism
Bacillus subtilis 168
-
-
(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
the enzyme catalyzes the spore photoproduct repair reaction and combines specific features of radical S-adenosyl-L-methionine and DNA repair enzymes to enable a complex radical-based repair reaction to take place
-
-
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
-
-
-
SPL
Bacillus subtilis 168
-
-
-
spore photoproduct lyase
-
-
spore photoproduct lyase
-
-
spore photoproduct lyase
Clostridium acetobutylicum ATCC824D
-
-
-
spore photoproduct lyase
-
-
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
Bacillus subtilis 168
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
evolution
-
the enzyme is a member of the radical SAM superfamily
evolution
-
the enzyme is the first member of the radical SAM superfamily (comprising more than 44000 members) to bear a catalytically operating hydrogen atom transfer chain that is essential for S-adenosyl-L-methionine regeneration after the catalytic cycle
evolution
-
the enzyme is a member of the so-called radical SAM superfamily, which is defined by the characteristic CXXXCXXC motif. The three cysteine residues serve as ligands respectively for three irons in the [4Fe-4S] cluster, with the fourth iron being coordinated by the S-adenosylmethionine in a bi-dentate manner, with its amino and carboxylate moieties serving as the fourth and fifth ligands to the cluster. SPL and the DNA photolyase, EC 4.1.99.3, show amino acid sequence homolgy and might have descended from a common ancestral protein
evolution
A4IQU1
unlike DNA photolyases, EC 4.1.99.3, SP lyase belongs to the emerging superfamily of radical S-adenosyl-L-methionine (SAM) enzymes
evolution
Bacillus subtilis 168
-
the enzyme is a member of the radical SAM superfamily; the enzyme is a member of the so-called radical SAM superfamily, which is defined by the characteristic CXXXCXXC motif. The three cysteine residues serve as ligands respectively for three irons in the [4Fe-4S] cluster, with the fourth iron being coordinated by the S-adenosylmethionine in a bi-dentate manner, with its amino and carboxylate moieties serving as the fourth and fifth ligands to the cluster. SPL and the DNA photolyase, EC 4.1.99.3, show amino acid sequence homolgy and might have descended from a common ancestral protein; the enzyme is the first member of the radical SAM superfamily (comprising more than 44000 members) to bear a catalytically operating hydrogen atom transfer chain that is essential for S-adenosyl-L-methionine regeneration after the catalytic cycle
-
evolution
-
unlike DNA photolyases, EC 4.1.99.3, SP lyase belongs to the emerging superfamily of radical S-adenosyl-L-methionine (SAM) enzymes
-
malfunction
-
the enzyme C141A mutant produces thymidylyl-(3'->5')-thymidylate likely via an unprecedented thymine radical cation reduction (proton coupled electron transfer) mechanism
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
physiological function
-
spore photoproduct lyase repairs a covalent UV-induced thymine dimer, spore photoproduct, in germinating endospores and is responsible for endospores' strong UV resistance. The spore photoproduct is rapidly repaired by the metalloenzyme spore photoproduct lyase when spores start germinating
physiological function
-
spore photoproduct lyase repairs a covalent UV-induced thymine dimer, spore photoproduct, in germinating endospores and is responsible for strong UV resistance of endospores
physiological function
-
spore photoproduct lyase repairs a special thymine dimer 5-thyminyl-5,6-dihydrothymine, which is commonly called spore photoproduct at the bacterial early germination phase. Spore photoproduct is the exclusive DNA photo-damage product in bacterial endospores. Its generation and swift repair by the enzyme are responsible for the spores' extremely high UV resistance
physiological function
Bacillus subtilis 168
-
spore photoproduct lyase repairs a covalent UV-induced thymine dimer, spore photoproduct, in germinating endospores and is responsible for endospores' strong UV resistance. The spore photoproduct is rapidly repaired by the metalloenzyme spore photoproduct lyase when spores start germinating; spore photoproduct lyase repairs a covalent UV-induced thymine dimer, spore photoproduct, in germinating endospores and is responsible for strong UV resistance of endospores; spore photoproduct lyase repairs a special thymine dimer 5-thyminyl-5,6-dihydrothymine, which is commonly called spore photoproduct at the bacterial early germination phase. Spore photoproduct is the exclusive DNA photo-damage product in bacterial endospores. Its generation and swift repair by the enzyme are responsible for the spores' extremely high UV resistance
-
malfunction
Bacillus subtilis 168
-
the enzyme C141A mutant produces thymidylyl-(3'->5')-thymidylate likely via an unprecedented thymine radical cation reduction (proton coupled electron transfer) mechanism
-
additional information
-
spectral analysis of the purified recombinant enzyme, overview
additional information
-
residue C141 is solvent exposable and no other protein residue locates between the thymidylyl-(3'->5')-thymidylate radical and the C141 in the wild--type enzyme reaction pathway
additional information
-
the enzyme protects at least 9 nucleotides in the spore photoproduct containing DNA strand with 5 nucleotides 3' to and 2 nucleotides 5' to the spore photoproduct damage, suggesting that the phosphates included in this region may be involved in the binding interaction with the enzyme
additional information
-
residues Cys140 and Tyr98 are important for establishing catalytic turnover. While the allyl radical is situated and reduced at the 3'-side, transfer of the radical center back to the 5'-dAdoH requires moving the radical back to the 50-part in the active side over a distance of roughly 10 A. This creates next to a topological problem also an energetic obstacle, because regeneration of the adenosyl radical by the thiyl radical would be endothermic. The enzyme uses a further tyrosyl radical intermediate to solve the energetic and topological problem
additional information
A4IQU1
active site structure, DNA lesion recognition, and substrate binding which involve a beta-hairpin structure, overview. S-adenosyl-L-methionine and a conserved cysteine residue are perfectly positioned in the active site for hydrogen atom abstraction from the dihydrothymine residue of the lesion and donation to the alpha-thyminyl radical moiety, respectively. Structure comparison of wild-type and C140 mutant enzymes, overview
additional information
Bacillus subtilis 168
-
residue C141 is solvent exposable and no other protein residue locates between the thymidylyl-(3'->5')-thymidylate radical and the C141 in the wild--type enzyme reaction pathway; the enzyme protects at least 9 nucleotides in the spore photoproduct containing DNA strand with 5 nucleotides 3' to and 2 nucleotides 5' to the spore photoproduct damage, suggesting that the phosphates included in this region may be involved in the binding interaction with the enzyme
-
additional information
Clostridium acetobutylicum ATCC824D
-
spectral analysis of the purified recombinant enzyme, overview
-
additional information
-
active site structure, DNA lesion recognition, and substrate binding which involve a beta-hairpin structure, overview. S-adenosyl-L-methionine and a conserved cysteine residue are perfectly positioned in the active site for hydrogen atom abstraction from the dihydrothymine residue of the lesion and donation to the alpha-thyminyl radical moiety, respectively. Structure comparison of wild-type and C140 mutant enzymes, overview
-
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
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
thymidylyl-(3'->5')-thymidylate
show the reaction diagram
-
i.e. spore photoproduct, an in situ monomerization
-
-
?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
show the reaction diagram
-
the reaction is highly stereo-selective
-
-
?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
show the reaction diagram
Bacillus subtilis 168
-
-, the reaction is highly stereo-selective
-
-
?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
show the reaction diagram
Bacillus subtilis 168
-
-
-
-
?
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine
thymidylyl-(3'->5')-thymidylate
show the reaction diagram
Bacillus subtilis 168
-
i.e. spore photoproduct, an in situ monomerization
-
-
?
(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-(alpha-thyminyl)-5,6-dihydrothymidine
thymidylyl-(3'-5')-thymidylate
show the reaction diagram
-
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
-
-
-
-
?
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
-
-
?
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
-
-
-
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
-
-
-
additional information
?
-
Bacillus subtilis, Bacillus subtilis 168
-
the enzyme recognizes and repairs a range of spore photoproduct containing DNA substrates, ranging from dinucleotide and dinucleoside spore photoproduct to spore photoproduct containing single- and double-stranded DNA. The fastest reaction rate employs a single-stranded spore photoproduct containing GGSPGG 6-mer as the substrate, while the double-stranded spore photoproduct-containing plasmid DNA also supports a fast repair reaction
-
-
-
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
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
thymidylyl-(3'->5')-thymidylate
show the reaction diagram
Bacillus subtilis 168
-
-
-
-
?
(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
-
-
S-adenosyl-L-methionine
-
a radical S-adenosyl-L-methionine enzyme that uses a [4Fe-4S]1+ cluster to reduce the S-adenosyl-L-methionine generating a catalytic 5'-deoxyadenosyl radical
S-adenosyl-L-methionine
-
a radical S-adenosyl-L-methionine enzyme, S-adenosyl-L-methionine is suggested to be regenerated at the end of each catalytic cycle, and only a catalytic amount of S-adenosyl-L-methionine is needed in the enzyme reaction. The H atom source for the back donation step is suggested to be a cysteine residue 141, and the H-atom transfer reaction leaves a thiyl radical behind on the protein. This thiyl radical thus must participate in the S-adenosyl-L-methionine regeneration process, the thiyl radical abstracts an H atom from the 5'-deoxyadenosyl radical to regenerate S-adenosyl-L-methionine
S-adenosyl-L-methionine
-
a radical SAM enzyme
S-adenosyl-L-methionine
A4IQU1
a radical S-adenosyl-L-methionine enzyme that uses a [4Fe-4S]1+ cluster and S-adenosyl-L-methionine to initiate the repair reaction
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
Fe2+
-
metalloenzyme, the enzyme uses a [4Fe-4S]1+ cluster to reduce the S-adenosyl-L-methionine generating a catalytic 5'-deoxyadenosyl radical
Fe2+
-
the enzyme uses a [4Fe-4S]1+ cluster to reduce the S-adenosyl-L-methionine generating a catalytic 5'-deoxyadenosyl radical
Fe2+
-
utilizes a tricysteine CXXXCXXC motif to harbor a [4Fe-4S] cluster
Fe2+
-
contains a [4Fe-4S] cluster
Fe2+
A4IQU1
the enzyme uses a [4Fe-4S]1+ cluster and S-adenosyl-L-methionine to initiate the repair reaction
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
0.0009
-
5-(alpha-thyminyl)-5,6-dihydrothymidine
-
wild-type enzyme, pH and temperature not specified in the publication
-
0.0011
-
5-(alpha-thyminyl)-5,6-dihydrothymidine
-
mutant C140A, pH and temperature not specified in the publication
-
0.0113
-
5-(alpha-thyminyl)-5,6-dihydrothymidine
-
mutant Y98F, pH and temperature not specified in the publication
-
additional information
-
additional information
-
kinetics
-
additional information
-
additional information
-
steady-state kinetics and competitive kinetic isotope effects of recombinant wild-type and mutant enzymes, overview
-
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
8
-
-
assay at
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
20
-
E8SZR7
assay at
22
-
-
assay at room temperature
22
-
A4IQU1
assay at room temperature
37
-
-
assay at
37
-
E8SZR7
assay at
pI VALUE
pI VALUE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
8.5
-
-
recombinant His6-tagged mutant C141A, isoelectric focussing
SOURCE TISSUE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
SOURCE
Bacillus subtilis 168
-
germinating
-
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
Geobacillus thermodenitrificans (strain NG80-2)
Geobacillus thermodenitrificans (strain NG80-2)
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
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
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
-
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
A4IQU1
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
recombinant His-tagged enzyme from Escherichia coli strain Tuner (DE3) by nickel affinity chromatography
-
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity and cation exchange chromatography
-
recombinant N- or C-terminally His6-tagged enzyme from Escherichia coli strain BL21(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
recombinant wild-type, selenomethionine-labeled, and mutant His6-tagged enzymes from Escherichia coli strain BL21(DE3) by nickel and heparin affinity chromatography
A4IQU1
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
gene splB, overexpression of His6-tagged wild-type and mutant enzymes and co-expression with plasmid pDB1282, in Escherichia coli strain BL21(DE3)
-
gene splB, overexpression of N- or C-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3)
-
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)
-
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)
A4IQU1
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
C141A
-
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
Y97A
-
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
-
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
-
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
-
site-directed mutagenesis, the mutant's active site architecture, activity, and kinetics are similar to the wild-type enzyme
Y98F
-
site-directed mutagenesis, the mutant's active site architecture, activity, and kinetics are similar to the wild-type enzyme
Y99A
-
site-directed mutagenesis, the mutant shows altered competitive kinetic isotope effects compared to the wild-type enzyme
Y99F
-
site-directed mutagenesis, the mutant shows altered competitive kinetic isotope effects compared to the wild-type enzyme
Y97F
Bacillus subtilis 168
-
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
Bacillus subtilis 168
-
site-directed mutagenesis, the mutant's active site architecture, activity, and kinetics are similar to the wild-type enzyme
-
Y99F
Bacillus subtilis 168
-
site-directed mutagenesis, the mutant shows altered competitive kinetic isotope effects compared to the wild-type enzyme
-
C140A
-
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
C140A
A4IQU1
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
C140G
-
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
A4IQU1
site-directed mutagenesis, the mutant shows a similar protein and substrate binding structure compared to the wild-type enzyme, but reduced repair activity
Y98F
-
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
-
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
-
C140S
-
site-directed mutagenesis, the mutant shows a similar protein and substrate binding structure compared to the wild-type enzyme, but reduced repair activity
-
C141A
-
site-directed mutagenesis, the mutant shows altered competitive kinetic isotope effects compared to the wild-type enzyme
additional information
-
compared to tyrosine, phenylalanine retains the aromatic ring, but does not support the radical propagation reaction due to the loss of the OH moiety. [4Fe-4S] clusters remains intact in the Y->F mutants
C141A
Bacillus subtilis 168
-
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
-
additional information
Bacillus subtilis 168
-
compared to tyrosine, phenylalanine retains the aromatic ring, but does not support the radical propagation reaction due to the loss of the OH moiety. [4Fe-4S] clusters remains intact in the Y->F mutants
-
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
-
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