Cloned (Comment) | Organism |
---|---|
gene splB, overexpression of His6-tagged wild-type and mutant enzymes and co-expression with plasmid pDB1282, in Escherichia coli strain BL21(DE3) | Bacillus subtilis |
Crystallization (Comment) | Organism |
---|---|
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 | Bacillus subtilis |
Protein Variants | Comment | Organism |
---|---|---|
C141A | site-directed mutagenesis, the mutant shows altered competitive kinetic isotope effects compared to the wild-type enzyme | Bacillus subtilis |
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 | Bacillus subtilis |
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 | Bacillus subtilis |
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 | Bacillus subtilis |
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 | Bacillus subtilis |
Y98A | site-directed mutagenesis, the mutant's active site architecture, activity, and kinetics are similar to the wild-type enzyme | Bacillus subtilis |
Y98F | site-directed mutagenesis, the mutant's active site architecture, activity, and kinetics are similar to the wild-type enzyme | Bacillus subtilis |
Y99A | site-directed mutagenesis, the mutant shows altered competitive kinetic isotope effects compared to the wild-type enzyme | Bacillus subtilis |
Y99F | site-directed mutagenesis, the mutant shows altered competitive kinetic isotope effects compared to the wild-type enzyme | Bacillus subtilis |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | steady-state kinetics and competitive kinetic isotope effects of recombinant wild-type and mutant enzymes, overview | Bacillus subtilis |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Fe2+ | the enzyme uses a [4Fe-4S]1+ cluster to reduce the S-adenosyl-L-methionine generating a catalytic 5'-deoxyadenosyl radical | Bacillus subtilis |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine | Bacillus subtilis | - |
thymidylyl-(3'->5')-thymidylate | - |
? | |
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine | Bacillus subtilis 168 | - |
thymidylyl-(3'->5')-thymidylate | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Bacillus subtilis | - |
gene splB | - |
Bacillus subtilis 168 | - |
gene splB | - |
Purification (Comment) | Organism |
---|---|
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity and cation exchange chromatography | Bacillus subtilis |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine (in double-helical DNA) = thymidylyl-(3'->5')-thymidylate (in double-helical DNA) | 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 |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine | - |
Bacillus subtilis | thymidylyl-(3'->5')-thymidylate | - |
? | |
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine | - |
Bacillus subtilis 168 | thymidylyl-(3'->5')-thymidylate | - |
? |
Synonyms | Comment | Organism |
---|---|---|
SPL | - |
Bacillus subtilis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
22 | - |
assay at room temperature | Bacillus subtilis |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7 | - |
assay at | Bacillus subtilis |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
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 | Bacillus subtilis |
General Information | Comment | Organism |
---|---|---|
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 | Bacillus subtilis |
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 | Bacillus subtilis |