Cloned (Comment) | Organism |
---|---|
N-terminally His6-tagged fusion protein in Escherichia coli | Methanosarcina mazei |
phylogenetic analysis | Methanosarcina mazei |
Crystallization (Comment) | Organism |
---|---|
hanging drop vapour diffusion method, crystal structure of the enzyme alone and in complex with cyclobutane pyrimidine dimer lesion-containing duplex DNA | Methanosarcina mazei |
purified enzyme with or without bound cyclobutadipyrimidine-DNA, hanging drop vapour diffusion method, 7.7 mg/ml protein with 0.5 M lithium sulfate and 7.5% w/v PEG 8000, 3-5 days, 4°C, X-ray diffraction structure determination and analysis at 1.5-2.2 A resolution | Methanosarcina mazei |
Protein Variants | Comment | Organism |
---|---|---|
N403A | replacement of asparagine N403 for either a non-polar alanine or a hydrophobic leucine causes complete loss of the catalytic FAD during purification by size exclusion chromatography | Methanosarcina mazei |
N403L | replacement of asparagine N403 for either a non-polar alanine or a hydrophobic leucine causes complete loss of the catalytic FAD during purification by size exclusion chromatography | Methanosarcina mazei |
W360F | mutation of the medial tryptophan, W360, gives a 22fold decrease of photoreduction activity relative to the wild type enzyme, no major build-up of the semiquinoid FADH radical species can be observed for W360F | Methanosarcina mazei |
W381F | the mutation causes complete loss of photoreduction activity and a loss of 70% of incorporation of the catalytic FAD compared to the wild-type enzyme | Methanosarcina mazei |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
cyclobutadipyrimidine (in DNA) | Methanosarcina mazei | the enzyme catalyses light-driven DNA repair and photoreduction, but in contrast to class I enzymes lacks a high degree of binding discrimination between UV-damaged and intact duplex DNA | 2 pyrimidine residues (in DNA) | - |
? | |
cyclobutadipyrimidine (in DNA) | Methanosarcina mazei DSM 3647 | the enzyme catalyses light-driven DNA repair and photoreduction, but in contrast to class I enzymes lacks a high degree of binding discrimination between UV-damaged and intact duplex DNA | 2 pyrimidine residues (in DNA) | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Methanosarcina mazei | - |
- |
- |
Methanosarcina mazei | Q8PYK9 | - |
- |
Methanosarcina mazei DSM 3647 | Q8PYK9 | - |
- |
Methanosarcina mazei Mm0852 | - |
- |
- |
Purification (Comment) | Organism |
---|---|
- |
Methanosarcina mazei |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
cyclobutadipyrimidine (in DNA) = 2 pyrimidine residues (in DNA) | reaction mechanism, detailed overview | Methanosarcina mazei |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
cyclobutadipyrimidine (in DNA) | - |
Methanosarcina mazei | 2 pyrimidine residues (in DNA) | - |
? | |
cyclobutadipyrimidine (in DNA) | the enzyme catalyses light-driven DNA repair and photoreduction, but in contrast to class I enzymes lacks a high degree of binding discrimination between UV-damaged and intact duplex DNA | Methanosarcina mazei | 2 pyrimidine residues (in DNA) | - |
? | |
cyclobutadipyrimidine (in DNA) | - |
Methanosarcina mazei DSM 3647 | 2 pyrimidine residues (in DNA) | - |
? | |
cyclobutadipyrimidine (in DNA) | the enzyme catalyses light-driven DNA repair and photoreduction, but in contrast to class I enzymes lacks a high degree of binding discrimination between UV-damaged and intact duplex DNA | Methanosarcina mazei DSM 3647 | 2 pyrimidine residues (in DNA) | - |
? | |
additional information | the class II enzyme lacks a high degree of binding discrimination between UV-damaged and intact duplex DNA, in contrast to class I enzymes. The lesion-binding mode differs from other photolyases by a larger DNA binding site, and an unrepaired CPD lesion is found flipped into the active site and recognized by a cluster of five water molecules next to the bound 3'-thymine base. Different from other members of the photolyase-cryptochrome family, class II photolyases appear to utilize an unusual, conserved tryptophan dyad as electron transfer pathway to the catalytic FAD cofactor | Methanosarcina mazei | ? | - |
? | |
additional information | the class II enzyme lacks a high degree of binding discrimination between UV-damaged and intact duplex DNA, in contrast to class I enzymes. The lesion-binding mode differs from other photolyases by a larger DNA binding site, and an unrepaired CPD lesion is found flipped into the active site and recognized by a cluster of five water molecules next to the bound 3'-thymine base. Different from other members of the photolyase-cryptochrome family, class II photolyases appear to utilize an unusual, conserved tryptophan dyad as electron transfer pathway to the catalytic FAD cofactor | Methanosarcina mazei Mm0852 | ? | - |
? | |
thymine dimers in AnCPDI and Atcry3 complexes | the conserved MmCPDII tryptophans W305 and W421 form the L-shaped walling of the active site that clamps the CPD lesion together with the side chain of the conserved M379. Upon repair the 5'-thymine base is expected to remain in place upon breakage of the C5-C5 and C6-C6 bonds by maintaining the p-stacking interactions with the indole moiety of W305, whereas the 3'-thymine dissociates by ca. 1 A away towards the thioether group of M379 | Methanosarcina mazei | ? | - |
? | |
thymine dimers in AnCPDI and Atcry3 complexes | the conserved MmCPDII tryptophans W305 and W421 form the L-shaped walling of the active site that clamps the CPD lesion together with the side chain of the conserved M379. Upon repair the 5'-thymine base is expected to remain in place upon breakage of the C5-C5 and C6-C6 bonds by maintaining the p-stacking interactions with the indole moiety of W305, whereas the 3'-thymine dissociates by ca. 1 A away towards the thioether group of M379 | Methanosarcina mazei Mm0852 | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
More | the structure of MmCPDII is organized in an N-terminal alpha/beta subdomain and a C-terminal all-helical subdomain, the C-terminal FAD-binding subdomain contains the catalytic cofactor FAD in the U-shaped conformation | Methanosarcina mazei |
Synonyms | Comment | Organism |
---|---|---|
class II DNA photolyase | - |
Methanosarcina mazei |
class II photolyase | - |
Methanosarcina mazei |
MmCPDII | - |
Methanosarcina mazei |
MM_0852 | locus name | Methanosarcina mazei |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | the enzyme appears to utilize an unusual, conserved tryptophan dyad as electron transfer pathway to the catalytic FAD cofactor | Methanosarcina mazei | |
FAD | the enzyme is capable to photoreduce its catalytic FAD to the active FADH- form. The C-terminal FAD-binding subdomain contains the catalytic cofactor FAD in the U-shaped conformation. FAD-binding site and electron transfer pathway in class II photolyases, overview | Methanosarcina mazei |
General Information | Comment | Organism |
---|---|---|
evolution | phylogenetic analysis, comparison of substrate binding and substrate specificity of class I and class II enzymes, overview. The enzyme shows the overall fold of the photolyase cryptochrome family, surface features of the photolyase-cryptochrome family bound to DNA lesions, overview | Methanosarcina mazei |
additional information | illumination leads to the neutral semiquinoid state of the photolyase with maxima at 590 nm and 632 nm, respectively. Stabilizing role of asparagine N403 in class II photolyases. The innermost tryptophan W381 is crucial for catalytic activity, electron transfer pathway along the tryptophan catalytic triad W388-W360-W381 to FAD | Methanosarcina mazei |
physiological function | the enzyme catalyses light-driven DNA repair and photoreduction, but in contrast to class I enzymes lacks a high degree of binding discrimination between UV-damaged and intact duplex DNA | Methanosarcina mazei |