Cloned (Comment) | Organism |
---|---|
gene GTNG_2348, sequence comparisons, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Geobacillus thermodenitrificans |
Crystallization (Comment) | Organism |
---|---|
purified recombinant reconstituted C140A/S76C mutant SP lyase, hanging drop vapor diffusion method, the enzyme is incubated with Se-SAM, crystallization in the presence of 70 mM octanoyl-N-hydroxyethylglucamide and mixing with a reservoir solution containing 200 mM lithium sulfate, 100 mM Tris-HCl, pH 9.0, and 19-27% w/v PEG 8000 under anaerobic conditions, 20°C, X-ray diffraction structure determination and analysis at 2.1-2.6 A resolution | Geobacillus thermodenitrificans |
Protein Variants | Comment | Organism |
---|---|---|
C140A | site-directed mutagenesis | Geobacillus thermodenitrificans |
C140A/S76C | site-directed mutagenesis | Geobacillus thermodenitrificans |
additional information | by rational engineering, the enzyme's HAT pathway is rewired. Possible development of improved catalysts based on the radical SAM enzyme scaffold | Geobacillus thermodenitrificans |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Fe2+ | [4Fe-4S] cluster | Geobacillus thermodenitrificans |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine | Geobacillus thermodenitrificans | in double-helical DNA | thymidylyl-(3'->5')-thymidylate | - |
? | |
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine | Geobacillus thermodenitrificans NG80-2 | in double-helical DNA | thymidylyl-(3'->5')-thymidylate | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Geobacillus thermodenitrificans | A4IQU1 | - |
- |
Geobacillus thermodenitrificans NG80-2 | A4IQU1 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity and heparin affinity chromatography. The iron-sulfur cluster of the purified proteins is then reconstituted | Geobacillus thermodenitrificans |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine | in double-helical DNA | Geobacillus thermodenitrificans | thymidylyl-(3'->5')-thymidylate | - |
? | |
(5R)-5,6-dihydro-5-(thymidin-7-yl)thymidine | in double-helical DNA | Geobacillus thermodenitrificans NG80-2 | thymidylyl-(3'->5')-thymidylate | - |
? | |
additional information | activity of the reconstituted wild-type enzyme and the enzyme mutants is measured using a 13-mer oligonucleotide containing the dinucleoside SP (5'-CAGCGGT-TGCAGG-3') as substrate, mass spectrometric poduct analysis, overview. The repair of the SP containing DNA leads to two oligonucleotides: the 7 mer (5'-CAGCGGT-3') and the 6 mer (5'-TGCAGG-3') | Geobacillus thermodenitrificans | ? | - |
? | |
additional information | activity of the reconstituted wild-type enzyme and the enzyme mutants is measured using a 13-mer oligonucleotide containing the dinucleoside SP (5'-CAGCGGT-TGCAGG-3') as substrate, mass spectrometric poduct analysis, overview. The repair of the SP containing DNA leads to two oligonucleotides: the 7 mer (5'-CAGCGGT-3') and the 6 mer (5'-TGCAGG-3') | Geobacillus thermodenitrificans NG80-2 | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
More | enzyme secondary structure comparisons using the crystal structures of the substrate-free wild-type SP lyase from Geobacillus thermodenitrificans, PDB ID 4FHC, of enzyme mutant C140A mutant (PDB code 4FHF), and the enzyme double mutant SP lyase | Geobacillus thermodenitrificans |
Synonyms | Comment | Organism |
---|---|---|
Gt SP lyase | - |
Geobacillus thermodenitrificans |
GTNG_2348 | - |
Geobacillus thermodenitrificans |
SP lyase | - |
Geobacillus thermodenitrificans |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Geobacillus thermodenitrificans |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Geobacillus thermodenitrificans |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
S-adenosyl-L-methionine | SPL is a radical SAM enzyme | Geobacillus thermodenitrificans | |
[4Fe-4S] cluster | - |
Geobacillus thermodenitrificans |
General Information | Comment | Organism |
---|---|---|
physiological function | the radical SAM enzyme, spore photoproduct lyase, requires an H-atom transfer (HAT) pathway to catalyze DNA repair | Geobacillus thermodenitrificans |