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Literature summary extracted from
- Crystal structures of bacterial (6-4) photolyase mutants with impaired DNA repair activity (2017), Photochem. Photobiol., 93, 304-314 .
Crystallization (Commentary)
| EC Number | Crystallization (Comment) | Organism |
|---|---|---|
| 4.1.99.13 | purified recombinant PhrBI51W mutant, is crystallized by hanging drop vapor diffusion method, mixing of 5 mg/ml protein in 12.5 mM Tris, 1.25 mM EDTA, 2.5% w/v glycerol, and 75 mM sodium chloride, pH 7.8, with reservoir solution containing 5% PEG 400 and 100 mM 2-(N-morpholino) ethanesulfonic acid, pH 6.5, in a 1:1 ratio, X-ray diffraction structure determination and analysis at 2.15 A resolution, molecular replacement using the wild-type PhrBWT structure, PDB ID 4DJA, as the initial search model. Purified recombinant PhrBY424F mutant is crystallized by sitting drop vapor diffusion method, mixing 0.004 ml of 4-6 mg/ml protein in 12.5 mM Tris, 1.25 mM EDTA, 2.5% w/v glycerol, and 75 mM sodium chloride, pH 7.8, with an equal volume of reservoir solution containing 5% w/v PEG 400, 100 mM 2-(N-morpholino) ethanesulfonic acid, pH 6.0, and equilibration against 1 mLl of reservoir solution at 16°C, in darkness, 3-7 days, X-ray diffraction structure determination and analysis at 2.50 A resolution | Agrobacterium fabrum |
Protein Variants
| EC Number | Protein Variants | Comment | Organism |
|---|---|---|---|
| 4.1.99.13 | I51W | site-directed mutagenesis, the mutant PhrBI51W shows loss of the DMRL chromophore (due to structural rearrangements of the residues in the DMRL binding pocket), reduced photoreduction, and reduced DNA repair capacity compared to wild-type. The mutation only affects local protein environments, whereas the overall fold remains unchanged. The crystal structure of PhrBI51W shows how the bulky Trp leads to structural rearrangements in the DMRL chromophore pocket. Structure analysis of mutant PhrBI51W | Agrobacterium fabrum |
| 4.1.99.13 | Y424F | site-directed mutagenesis, the PhrBY424F mutant shows reduced binding of lesion DNA and loss of DNA repair compared to wild-type. The mutation only affects local protein environments, whereas the overall fold remains unchanged. The crystal structure of PhrBY424F reveals a water network extending to His366, which are part of the lesion-binding site. Structure analysis of mutant PhrBY424F | Agrobacterium fabrum |
Metals/Ions
| EC Number | Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|---|
| 4.1.99.13 | Fe2+ | in a cubane-type Fe-S cluster | Agrobacterium fabrum |  |
Natural Substrates/ Products (Substrates)
| EC Number | Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 4.1.99.13 | (6-4) photoproduct (in DNA) | Agrobacterium fabrum | - |
2 pyrimidine residues (in DNA) | - |
? | |
| 4.1.99.13 | (6-4) photoproduct (in DNA) | Agrobacterium fabrum C58 / ATCC 33970 | - |
2 pyrimidine residues (in DNA) | - |
? | |
Organism
| EC Number | Organism | UniProt | Comment | Textmining |
|---|---|---|---|---|
| 4.1.99.13 | Agrobacterium fabrum | A9CH39 | i.e. Agrobacterium tumefaciens strain C58 | - |
| 4.1.99.13 | Agrobacterium fabrum C58 / ATCC 33970 | A9CH39 | i.e. Agrobacterium tumefaciens strain C58 | - |
Substrates and Products (Substrate)
| EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 4.1.99.13 | (6-4) photoproduct (in DNA) | - |
Agrobacterium fabrum | 2 pyrimidine residues (in DNA) | - |
? | |
| 4.1.99.13 | (6-4) photoproduct (in DNA) | - |
Agrobacterium fabrum C58 / ATCC 33970 | 2 pyrimidine residues (in DNA) | - |
? | |
Synonyms
| EC Number | Synonyms | Comment | Organism |
|---|---|---|---|
| 4.1.99.13 | bacterial (6-4) photolyase | - |
Agrobacterium fabrum |
| 4.1.99.13 | PhrB | - |
Agrobacterium fabrum |
Temperature Optimum [°C]
| EC Number | Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|---|
| 4.1.99.13 | 25 | - |
assay at | Agrobacterium fabrum |
pH Optimum
| EC Number | pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|---|
| 4.1.99.13 | 7 | - |
assay at | Agrobacterium fabrum |
Cofactor
| EC Number | Cofactor | Comment | Organism | Structure |
|---|---|---|---|---|
| 4.1.99.13 | 6,7-dimethyl-8-ribityllumazine | DMRL, serves as an antenna chromophore for photoreduction and DNA repair in the wild-type enzyme | Agrobacterium fabrum | |
| 4.1.99.13 | FAD | - |
Agrobacterium fabrum | |
| 4.1.99.13 | Fe-S cluster | a cubane-type Fe-S cluster, residue Tyr424 of PhrB is part of the DNA-binding site and provides an electron link to the Fe-S cluster | Agrobacterium fabrum | |
| 4.1.99.13 | additional information | the protein harbors three cofactors: the enzymatically active FAD chromophore, a second chromophore, 6,7-dimethyl-8-ribityllumazine (DMRL) and a cubane-type Fe-S cluster | Agrobacterium fabrum |
General Information
| EC Number | General Information | Comment | Organism |
|---|---|---|---|
| 4.1.99.13 | evolution | the energy transfer from cofactor 6,7-dimethyl-8-ribityllumazine (DMRL) to FAD might represent a phylogenetically ancient process | Agrobacterium fabrum |
| 4.1.99.13 | additional information | two highly conserved Tyr residues at positions 424 and 430 form an electron bridge between the DNA lesion and the Fe-S cluster, Tyr424 of PhrB is part of the DNA-binding site | Agrobacterium fabrum |
| 4.1.99.13 | physiological function | PhrB from Agrobacterium fabrum is a prokaryotic photolyase which repairs (6-4) UV DNA photoproducts | Agrobacterium fabrum |
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