Literature summary for 4.1.99.13 extracted from

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 (2017), Photochem. Photobiol., 93, 304-314 .

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Crystallization (Commentary)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

Protein Variants	Comment	Organism
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
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

Metals/Ions	Comment	Organism	Structure
Fe2+	in a cubane-type Fe-S cluster	Agrobacterium fabrum

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
(6-4) photoproduct (in DNA)	Agrobacterium fabrum	-	2 pyrimidine residues (in DNA)	-	?
(6-4) photoproduct (in DNA)	Agrobacterium fabrum C58 / ATCC 33970	-	2 pyrimidine residues (in DNA)	-	?

Organism

Organism	UniProt	Comment	Textmining
Agrobacterium fabrum	A9CH39	i.e. Agrobacterium tumefaciens strain C58	-
Agrobacterium fabrum C58 / ATCC 33970	A9CH39	i.e. Agrobacterium tumefaciens strain C58	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
(6-4) photoproduct (in DNA)	-	Agrobacterium fabrum	2 pyrimidine residues (in DNA)	-	?
(6-4) photoproduct (in DNA)	-	Agrobacterium fabrum C58 / ATCC 33970	2 pyrimidine residues (in DNA)	-	?

Synonyms

Synonyms	Comment	Organism
bacterial (6-4) photolyase	-	Agrobacterium fabrum
PhrB	-	Agrobacterium fabrum

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at	Agrobacterium fabrum

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7	-	assay at	Agrobacterium fabrum

Cofactor

Cofactor	Comment	Organism
6,7-dimethyl-8-ribityllumazine	DMRL, serves as an antenna chromophore for photoreduction and DNA repair in the wild-type enzyme	Agrobacterium fabrum
FAD	-	Agrobacterium fabrum
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
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

General Information	Comment	Organism
evolution	the energy transfer from cofactor 6,7-dimethyl-8-ribityllumazine (DMRL) to FAD might represent a phylogenetically ancient process	Agrobacterium fabrum
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
physiological function	PhrB from Agrobacterium fabrum is a prokaryotic photolyase which repairs (6-4) UV DNA photoproducts	Agrobacterium fabrum