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Information on EC 4.1.99.13 - (6-4)DNA photolyase and Organism(s) Agrobacterium fabrum and UniProt Accession A9CH39
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4.1.99.13 (6-4)DNA photolyaseIUBMB Comments
A flavoprotein (FAD). The overall repair reaction consists of two distinct steps, one of which is light-independent and the other one light-dependent. In the initial light-independent step, a 6-iminium ion is thought to be generated via proton transfer induced by two histidines highly conserved among the (6-4) photolyases. This intermediate spontaneously rearranges to form an oxetane intermediate by intramolecular nucleophilic attack. In the subsequent light-driven reaction, one electron is believed to be transferred from the fully reduced FAD cofactor (FADH-) to the oxetane intermediate thus forming a neutral FADH radical and an anionic oxetane radical, which spontaneously fractures. The excess electron is then back-transferred to the flavin radical restoring the fully reduced flavin cofactor and a pair of pyrimidine bases .
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Word Map
- 4.1.99.13
- photoproducts
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cyclobutane
-
uv-induced
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cryptochromes
-
photoreactivation
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light-dependent
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pyrimidone
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blue-light
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photorepair
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oxetane
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pyrimidine-pyrimidone
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photoreduction
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photolesion
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dash
- ostreococcus
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four-membered
- fadox
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photoreception
- cry-dashs
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dewar
- cryptochrome-dash
The taxonomic range for the selected organisms is: Agrobacterium fabrum
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Synonyms
(6-4) photolyase, (6-4) phr, (6-4)photolyase, 6-4 photolyase, nf-10, otcpf1, 6-4ciphr, prokaryotic (6-4) photolyase, 6-4pp-photolyase, h64prh, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
bacterial (6-4) photolyase
PhrB
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
(6-4) photoproduct (in DNA) = 2 pyrimidine residues (in DNA)
photoreduction of PhrB differs from the typical pattern because the amino acid of the electron cascade next to FAD is a tyrosine (Tyr391), whereas photolyases and cryptochromes of other groups have a tryptophan as direct electron donor of FAD. Residues Trp342 and Trp390 are essential for charge transfer, Trp342 is located at the periphery of PhrB, while Trp390 connects Trp342 and Tyr391. Charge transfer occurs via the triad 391-390-342. Charge transfer simulations reveal an unusual stabilization of the positive charge on site 391 compared to other photolyases or cryptochromes. Water molecules near Tyr391 offer a polar environment which stabilizes the positive charge on this site, thereby lowering the energetic barrier intrinsic to tyrosine. This opens a second charge transfer channel in addition to tunnelling through the tyrosine barrier, based on hopping and therefore transient oxidation of Tyr391, which enables a fast charge transfer similar to proteins utilizing a tryptophan-triad
SYSTEMATIC NAME
IUBMB Comments
(6-4) photoproduct pyrimidine-lyase
A flavoprotein (FAD). The overall repair reaction consists of two distinct steps, one of which is light-independent and the other one light-dependent. In the initial light-independent step, a 6-iminium ion is thought to be generated via proton transfer induced by two histidines highly conserved among the (6-4) photolyases. This intermediate spontaneously rearranges to form an oxetane intermediate by intramolecular nucleophilic attack. In the subsequent light-driven reaction, one electron is believed to be transferred from the fully reduced FAD cofactor (FADH-) to the oxetane intermediate thus forming a neutral FADH radical and an anionic oxetane radical, which spontaneously fractures. The excess electron is then back-transferred to the flavin radical restoring the fully reduced flavin cofactor and a pair of pyrimidine bases [2].
CAS REGISTRY NUMBER
COMMENTARY
37290-70-3
not distinguished from DNA photolyase, spore photoproduct lyase
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(6-4) photoproduct (in DNA)
2 pyrimidine residues (in DNA)
-
-
-
?
(6-4) photoproduct (in DNA)
2 pyrimidine residues (in DNA)
-
(6-4)TT
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(6-4) photoproduct (in DNA)
2 pyrimidine residues (in DNA)
-
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
6,7-dimethyl-8-ribityllumazine
DMRL, serves as an antenna chromophore for photoreduction and DNA repair in the wild-type enzyme
FAD
-
transition from the oxidized to the semi-reduced form of FAD is characterized by absorbance decreases at around 450 nm and 370 nm, and an increase at 580 nm
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
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
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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
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Fe2+
Fe2+
-
Fe-S cluster, the Fe-S cluster is required for structural stability, integrity and correct folding of PhrB
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
25
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
malfunction
physiological function
additional information
evolution
-
PhrB from Agrobacterium fabrum represents a distinct group of prokaryotic (6-4) photolyases which contain an iron-sulfur cluster and a DMRL chromophore. The family of photolyases and cryptochromes may be divided into seven major phylogenetic groups: CPD photolyases class I, II and III, Cry-DASH proteins, eukaryotic (6-4) photolyases and animal cryptochromes, plant cryptochromes and prokaryotic FeS-BCP (Fe-S bacterial cryptochromes and photolyases) proteins. The terms CPD- and (6-4) photolyases refer to the kind of lesions that are repaired by these proteins, which are cyclopyrimidine dimers and (6-4) photoproducts, respectively. Both kinds of repair are triggered by a rapid electron transfer from the excited flavin adenine dinucleotide (FAD) chromophore to the DNA lesion. A second light reaction, termed photoreduction, results in the transition of oxidized or semi-reduced FAD to fully reduced FAD in photolyases or from oxidized to semi reduced FAD in plant cryptochromes. During photoreduction, electrons are transmitted from the surface via Trp or Tyr residues of the protein to the FAD chromophore. The classical photoreduction pathways in which electrons travel via three conserved Trp residues is realized in most photolyases and in cryptochromes. The group of FeS-BCP proteins is most distantly related to the other members of the cryptochrome/photolyase family. Two members of this group are CryB from Rhodobacter sphaeroides and PhrB from Agrobacterium fabrum. Among FeS-BCP members, amino acid residues in the active center are highly conserved. Loss of the cluster during the early evolution of the other photolyases
evolution
the bacterial (6-4) photolyase PhrB belongs to a phylogenetically ancient group. Photoreduction of PhrB differs from the typical pattern because the amino acid of the electron cascade next to FAD is a tyrosine (Tyr391), whereas photolyases and cryptochromes of other groups have a tryptophan as direct electron donor of FAD. Evolution of the first site of the redox chain has just been possible by tuning the protein structure and environment to manage a downhill hole transfer process from FAD to solvent
evolution
the energy transfer from cofactor 6,7-dimethyl-8-ribityllumazine (DMRL) to FAD might represent a phylogenetically ancient process
malfunction
-
mutants on cysteines that coordinate the Fe-S cluster of PhrB are either insoluble or not expressed. The same result is found for proteins with a truncated C-terminus, in which one of the Fe-S binding cysteines is mutated and for expression in minimal medium with limited Fe concentrations. The replacement of the highly conserved His366 results in loss of DNA repair activity. Leu370, which is also highly conserved, is not essential for repair, the L370M mutant has a lower repair activity. Mutants in which Tyr430 is replaced are characterized by a lower DNA repair activity. Tyr424 mutants are inactive
malfunction
replacement of Tyr391 by phenylalanine does not block photoreduction, while replacement by alanine blocks photoreduction, replacement of Tyr391 by Trp results in loss of FAD and DMRL chromophores
physiological function
-
photolyases are flavoproteins that repair UV-damaged DNA in a light-dependent fashion, photolyases can repair pyrimidine dimers on the DNA that are formed during UV irradiation
physiological function
PhrB from Agrobacterium fabrum is a prokaryotic photolyase which repairs (6-4) UV DNA photoproducts
additional information
-
role of particular amino acid residues in photorepair and photoreduction, during which the FAD chromophore converts from the oxidized to the enzymatically active, reduced form. Important function of highly conserved tyrosines in prokaryotic (6-4) photolyases, Tyr424 is essential for lesion binding and repair, and Tyr430 is required for efficient repair. Residues Trp342 and Trp390 as electron transmitters. Significant role of His366 in the protonation of the lesion during DNA repair
additional information
tunnelling matrix calculations show that tyrosine or phenylalanine can be involved in a productive bridged electron transfer between FAD and Trp390, in line with experimental findings, structure modeling of wild-type and mutant enzymes. Unusual stabilization of the positive charge on site 391 compared to other photolyases or cryptochromes. Mutational analyses of oligonucleotide sequences for DNA repair studies. Charge migration pathway from the protein surface Trp342 to FAD via Trp390 and Tyr391, light induced consecutive electron transfers, structures. Model structures and molecular dynamics simulations, overview
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
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
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
C350S
-
site-directed mutagenesis, mutation of a Cys residue of the Fe-S cluster, the mutant protein is not expressed in Escherichia coli under conditions where the wild-type protein is expressed as soluble protein
C438S
-
site-directed mutagenesis, mutation of a Cys residue of the Fe-S cluster, the mutant protein is not expressed in Escherichia coli under conditions where the wild-type protein is expressed as soluble protein
C441S
-
site-directed mutagenesis, mutation of a Cys residue of the Fe-S cluster, the mutant protein is not expressed in Escherichia coli under conditions where the wild-type protein is expressed as soluble protein
H366A
-
site-directed mutagenesis, in the mutant repair activity is lost
H366N/L370M
-
site-directed mutagenesis, in the mutant repair activity is lost
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
W342F
-
site-directed mutagenesis, the mutant shows highly reduced light-induced spectral changes compared to wild-type
W390F
-
site-directed mutagenesis, the mutant shows highly reduced light-induced spectral changes compared to wild-type
W390F/Y391F
-
site-directed mutagenesis, the mutant shows highly reduced light-induced spectral changes compared to wild-type
Y391A
site-directed mutagenesis, Tyr391 replacement by alanine blocks photoreduction
Y391F
Y391W
site-directed mutagenesis, replacement of Tyr391 by Trp results in loss of FAD and DMRL chromophores, Trp might participate in the electron transfer cascade
Y399F
Y424F
Y430F
-
site-directed mutagenesis, photoreduction of the mutant is indistinguishable from the wild-type, DNA binding assays are performed with single-stranded oligonucleotides with or without (-4)TT lesion, the mutant repair activity is 70% reduced compared to wild-type
Y460F
-
site-directed mutagenesis, photoreduction of the mutant is indistinguishable from the wild-type, DNA binding assays are performed with single-stranded oligonucleotides with or without (6-4)TT lesion, the mutant repair activity is unaltered compared to wild-type
additional information
-
mutants on cysteines that coordinate the Fe-S cluster of PhrB are either insoluble or not expressed. The same result is found for proteins with a truncated C-terminus, in which one of the Fe-S binding cysteines is mutated and for expression in minimal medium with limited Fe concentrations. Construction of two truncated versions of PhrB, designated PhrB-C and PhrB-D, which consist of amino acids 1-432 and 1-476, respectively. In PhrB-C, three of the Fe-S coordinating Cys residues aremissing, in PhrB-D, all Cys residues are present but the two C-terminal helices are missing. PhrB-C is insoluble under all tested conditions, whereas PhrB-D is partially soluble. With expression of PhrB by Escherichia coli cells growing in minimal medium without iron, PhrB is also insoluble. Thus, all conditions that might lead to a protein without Fe-S cluster result in very poor protein expression or insoluble protein
Y391F
-
site-directed mutagenesis, the mutant Y391F shows wild-type-like light-induced spectral changes
Y391F
site-directed mutagenesis, Tyr391 replacement by phenylalanine does not block photoreduction
Y399F
-
site-directed mutagenesis, the mutant shows highly reduced light-induced spectral changes compared to wild-type
Y399F
-
site-directed mutagenesis, the mutant Y391F shows slightly reduced light-induced spectral changes compared to wild-type
Y424F
-
site-directed mutagenesis, photoreduction of the mutant is indistinguishable from the wild-type, DNA binding assays are performed with single-stranded oligonucleotides with or without (6-4)TT lesion, the mutant repair activity is lost
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
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain ER2566 by nickel affinity chromatography and gel filtration
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene phrB, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain ER2566
recombinant expression of wild-type and mutant enzymes in Escherichia coli
-
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Holub, D.; Ma, H.; Krauss, N.; Lamparter, T.; Elstner, M.; Gillet, N.
Functional role of an unusual tyrosine residue in the electron transfer chain of a prokaryotic (6-4) photolyase
Chem. Sci.
9
1259-1272
2018
Agrobacterium fabrum (A9CH39), Agrobacterium fabrum C58 / ATCC 33970 (A9CH39)
Zhang, F.; Ma, H.; Bowatte, K.; Kwiatkowski, D.; Mittmann, E.; Qasem, H.; Krauss, N.; Zeng, X.; Ren, Z.; Scheerer, P.; Yang, X.; Lamparter, T.
Crystal structures of bacterial (6-4) photolyase mutants with impaired DNA repair activity
Photochem. Photobiol.
93
304-314
2017
Agrobacterium fabrum (A9CH39), Agrobacterium fabrum, Agrobacterium fabrum C58 / ATCC 33970 (A9CH39)
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