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Literature summary for 4.1.99.3 extracted from

  • Kiontke, S.; Geisselbrecht, Y.; Pokorny, R.; Carell, T.; Batschauer, A.; Essen, L.O.
    Crystal structures of an archaeal class II DNA photolyase and its complex with UV-damaged duplex DNA (2011), EMBO J., 30, 4437-4449.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
N-terminally His6-tagged fusion protein in Escherichia coli Methanosarcina mazei
phylogenetic analysis Methanosarcina mazei

Crystallization (Commentary)

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

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/ Products (Substrates)

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)
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?
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)
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?

Organism

Organism UniProt Comment Textmining
Methanosarcina mazei
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-
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Methanosarcina mazei Q8PYK9
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Methanosarcina mazei DSM 3647 Q8PYK9
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Methanosarcina mazei Mm0852
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-

Purification (Commentary)

Purification (Comment) Organism
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Methanosarcina mazei

Reaction

Reaction Comment Organism Reaction ID
cyclobutadipyrimidine (in DNA) = 2 pyrimidine residues (in DNA) reaction mechanism, detailed overview Methanosarcina mazei

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
cyclobutadipyrimidine (in DNA)
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Methanosarcina mazei 2 pyrimidine residues (in DNA)
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?
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)
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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)
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?
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 ?
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?
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

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

Synonyms Comment Organism
class II DNA photolyase
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Methanosarcina mazei
class II photolyase
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Methanosarcina mazei
MmCPDII
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Methanosarcina mazei
MM_0852 locus name Methanosarcina mazei

Cofactor

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

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