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Literature summary for 4.1.99.14 extracted from
- Insights into the activity change of spore photoproduct lyase induced by mutations at a peripheral glycine residue (2017), Front. Chem., 5, 14 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| gene spl-1 or splB, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Bacillus subtilis |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| G168A | site-directed mutagenesis, the mutant exhibits 3-4fold reduced enzyme activity compared to the wild-type enzyme, the mutant exhibits a smaller apparent kinetic isotope effect (KIE) but a bigger competitive KIE than the wild-type SPL | Bacillus subtilis |
| G168R | site-directed mutagenesis, the mutant exhibits 80fold reduced enzyme activity compared to the wild-type enzyme, the mutant exhibits a smaller apparent kinetic isotope effect (KIE) but a bigger competitive KIE than the wild-type SPL. G168R mutant also produces a large portion of the abortive repair product TpT-SO2-, the formation of which indicates that cysteine 141 is no longer well positioned as the H-donor to the thymine allylic radical intermediate | Bacillus subtilis |
| additional information | wild-type SPL, and mutant SPLs G168A and G168R exhibit indistinguishable circular dichroism spectra indicating that the mutation of glycine to alanine and arginine at the position 168 does not markedly alter the protein secondary structures | Bacillus subtilis |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | deuterium kinetic isotope effects (KIEs) of recombinant wild-type and mutant enzymes, competitive measurement, overview | Bacillus subtilis |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| (5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine | Bacillus subtilis | in double-helical DNA | thymidylyl-(3'->5')-thymidylate | - |
? |  |
| (5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine | Bacillus subtilis 168 | in double-helical DNA | thymidylyl-(3'->5')-thymidylate | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Bacillus subtilis | P37956 | - |
- |
| Bacillus subtilis 168 | P37956 | - |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and dialysis to over 95% purity | Bacillus subtilis |
Reaction
| 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) | thymine dimers can be repaired via direct reversal mechanism | Bacillus subtilis |
Source Tissue
| Source Tissue | Comment | Organism | Textmining |
|---|---|---|---|
| endospore | - |
Bacillus subtilis | - |
Specific Activity [micromol/min/mg]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| additional information | - |
SP TpT DNA is repaired to TpT DNA with 0.006/s by the wild-type and with 0.0015/s by mutant G168A or 0.00007/s by mutant G168R | Bacillus subtilis |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| (5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine | in double-helical DNA | Bacillus subtilis | thymidylyl-(3'->5')-thymidylate | - |
? | |
| (5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine | in double-helical DNA | Bacillus subtilis 168 | thymidylyl-(3'->5')-thymidylate | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| ? | x * 40000, recombinant His-tagged enzyme, SDS-PAGE | Bacillus subtilis |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| SPL | - |
Bacillus subtilis |
| spl-1 | - |
Bacillus subtilis |
| SplB | - |
Bacillus subtilis |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 22 | - |
assay at room temperature | Bacillus subtilis |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 7 | - |
assay at | Bacillus subtilis |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| S-adenosyl-L-methionine | SPL is a radical SAM enzyme | Bacillus subtilis | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| malfunction | mutation at the remote glycine 168 residue alters the enzyme 3D structure, subsequently reducing the SPL activity by changing the positions of the essential amino acids involved in the radical transfer process | Bacillus subtilis |
| additional information | a cysteine and two tyrosine residues are located in proximity and able to participate in the radical transfer process during the enzyme catalysis | Bacillus subtilis |
| physiological function | UV radiation triggers the formation of 5-thyminyl-5,6-dihydrothymine, i.e., the spore photoproduct (SP), in the genomic DNA of bacterial endospores. These SPs, if not repaired in time, may lead to genome instability and cell death. SP is mainly repaired by spore photoproduct lyase (SPL) during spore outgrowth via an unprecedented protein-harbored radical transfer pathway that is composed of at least a cysteine and two tyrosine residues. 5-thyminyl-5,6-dihydrothymine, i.e. the spore photoproduct (SP), is the dominant DNA photolesion found in bacterial endospores | Bacillus subtilis |
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