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Literature summary for 4.1.99.13 extracted from
- Rhodobacter sphaeroides CryB is a bacterial cryptochrome with (6-4) photolyase activity (2016), FEBS J., 283, 4291-4309 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| gene RSP_3077 or cryB, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strains BL21(DE3)pLysS or Rosetta(DE3)pRARE | Cereibacter sphaeroides |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| C434A/C437A | site-directed mutagenesis, the mutant cells show altered survival rates compaired to wild-type cells | Cereibacter sphaeroides |
| E37F | site-directed mutagenesis, the mutant cells show altered survival rates compaired to wild-type cells | Cereibacter sphaeroides |
| E37F/L366H | site-directed mutagenesis, the mutant cells show altered survival rates compaired to wild-type cells | Cereibacter sphaeroides |
| E37F/W388F | site-directed mutagenesis, the mutant cells show altered survival rates compaired to wild-type cells | Cereibacter sphaeroides |
| H362A | site-directed mutagenesis of an active site residue, the mutant cells show altered survival rates compaired to wild-type cells, in vivo and in vitro inactive mutant, no influence of an altered cofactor composition on the lack of repair. The position of H362 in the active centre next to FAD and the amino acids is involved in FAD reduction | Cereibacter sphaeroides |
| L366A | site-directed mutagenesis, the mutant cells show altered survival rates compaired to wild-type cells, the mutant shows reduced in vitro repair activity compared to wild-type, and highly reduced FAD content | Cereibacter sphaeroides |
| L366F | site-directed mutagenesis, the mutant cells show altered survival rates compaired to wild-type cells, the mutant shows reduced in vitro repair activity compared to wild-type, and highly reduced FAD content | Cereibacter sphaeroides |
| L366H | site-directed mutagenesis, the mutant cells show altered survival rates compaired to wild-type cells, the mutant shows reduced in vitro repair activity compared to wild-type, and highly reduced FAD content | Cereibacter sphaeroides |
| additional information | construction of a knockout strain of cryB, termed 2.4.1DELTAcryB, which shows no detectable photoreduction of its FAD cofactor in vitro, but is able to maintain photoreactivation of Rhodobacter sphaeroides cells, in contrast, its in vitro repair activity is lost due to the inability to accumulate the repair-competent FADH- state under the assay conditions. In vivo photoreactivation is also functional when binding of FAD or the antenna chromophore is impaired, or when only low amounts of both are present. The impairment of one of the two light absorbing cofactors at a time through mutagenesis strongly influences the kinetics of photoreactivation, and the combination of both mutation completely abolishes CryB-dependent photoreactivation. The misfolding of the protein when trying to remove the iron-sulfur cluster by amino acid exchanges demonstrates its structural relevance for the protein. Survival rates of different Rhodobacter sphaeroides mutant strains after UV light treatment, and absorbance spectra of purified His-tagged CryB variants, overview | Cereibacter sphaeroides |
| Q302A | site-directed mutagenesis of an active site residue, the mutant cells show altered survival rates compaired to wild-type cells | Cereibacter sphaeroides |
| S431V | removal of the C-terminal roof-like subdomain of CryB including the [4Fe4S] cluster, two variants: variant CryBDELTAD432-V508 and CryBS431VDELTAS431-P507, both variants show highly reduced production and activity compared to wild-type | Cereibacter sphaeroides |
| W388F | site-directed mutagenesis, the mutant cells show altered survival rates compaired to wild-type cells. The cofactor composition of CryB W338F does not differ from that of the wild-type protein. Considering the requirement of W338 for full reduction of FAD in vitro, the in vivo functionality of CryB W338F is remarkable | Cereibacter sphaeroides |
| Y387F | site-directed mutagenesis, the mutant cells show altered survival rates compaired to wild-type cells, the mutant has a slightly reduced FAD content compared to wild-type | Cereibacter sphaeroides |
| Y391F | site-directed mutagenesis, the mutant cells show altered survival rates compaired to wild-type cells, the mutant has a slightly reduced FAD content compared to wild-type | Cereibacter sphaeroides |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Fe2+ | in an [4Fe4S] cluster | Cereibacter sphaeroides |  |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| (6-4) photoproduct (in DNA) | Cereibacter sphaeroides | - |
2 pyrimidine residues (in DNA) | - |
? | |
| (6-4) photoproduct (in DNA) | Cereibacter sphaeroides ATCC 17023 / 2.4.1 / NCIB 8253 / DSM 158 | - |
2 pyrimidine residues (in DNA) | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Cereibacter sphaeroides | Q3IXP1 | - |
- |
| Cereibacter sphaeroides ATCC 17023 / 2.4.1 / NCIB 8253 / DSM 158 | Q3IXP1 | - |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant His-tagged wild-type and mutant enzymes from Escherichia coli by nickel affinity and heparin affinity chromatography, followed by gel filtration | Cereibacter sphaeroides |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| (6-4) photoproduct (in DNA) | - |
Cereibacter sphaeroides | 2 pyrimidine residues (in DNA) | - |
? | |
| (6-4) photoproduct (in DNA) | - |
Cereibacter sphaeroides ATCC 17023 / 2.4.1 / NCIB 8253 / DSM 158 | 2 pyrimidine residues (in DNA) | - |
? | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| CryB | - |
Cereibacter sphaeroides |
| deoxyribodipyrimidine photolyase-related protein | UniProt | Cereibacter sphaeroides |
| RSP_3077 | - |
Cereibacter sphaeroides |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 15 | - |
assay at | Cereibacter sphaeroides |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 8.3 | - |
assay at | Cereibacter sphaeroides |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| 6,7-dimethyl-8-ribityllumazine | a light absorbing cofactor | Cereibacter sphaeroides | |
| FAD | a light absorbing cofactor | Cereibacter sphaeroides | |
| [4Fe-4S] cluster | the iron-sulfur cluster is an indispensable cofactor for the structural integrity of the enzyme protein | Cereibacter sphaeroides | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| evolution | photolyases are efficient DNA repair enzymes that specifically repair either cyclobutane pyrimidine dimers or (6-4) photoproducts in a light-dependent cleavage reaction. The closely related classical cryptochrome blue light photoreceptors do not repair DNA lesions, instead they are involved in regulatory processes. CryB of Rhodobacter sphaeroides has been described as a cryptochrome that affects light-dependent and singlet oxygen-dependent gene expression and is unusual in terms of its cofactor composition. Evidence for a repair activity of (6-4) photoproducts by CryB is described suggesting a dual character combining the functions of cryptochromes and photolyases | Cereibacter sphaeroides |
| malfunction | impairment of one of the two light absorbing cofactors, FAD or 6,7-dimethyl-8-ribityllumazine, only marginally affect the final survival rate but strongly decelerate photoreactivation kinetics. The impairment of both of them together through mutagenesis decreases CryB-dependent photoreactivation to the level of the DELTAcryB knockout strain. Survival rates of different Rhodobacter sphaeroides mutant strains after UV light treatment, overview | Cereibacter sphaeroides |
| additional information | effects of crucial amino acids involved in cofactor or DNA lesion binding on the light-dependent recovery of cells after UV light exposure (in vivo photoreactivation), overview. The iron-sulfur cluster is essential for the structural integrity of CryB | Cereibacter sphaeroides |
| physiological function | photolyases are efficient DNA repair enzymes that specifically repair either cyclobutane pyrimidine dimers or (6-4) photoproducts in a light-dependent cleavage reaction. CryB of Rhodobacter sphaeroides has been described as a cryptochrome that affects light-dependent and singlet oxygen-dependent gene expression and is unusual in terms of its cofactor composition. Evidence for a repair activity of (6-4) photoproducts by CryB is described suggesting a dual character combining the functions of cryptochromes and photolyases. The reduction of FAD via the conserved tryptophan W338, which is crucial for in vitro reduction and consequently DNA repair, is not required for in vivo photoreactivation, suggesting that this reduction pathway to FAD is dispensable in the cellular environment | Cereibacter sphaeroides |
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