Literature summary for 4.1.99.13 extracted from

Graf, D.; Wesslowski, J.; Ma, H.; Scheerer, P.; Krauss, N.; Oberpichler, I.; Zhang, F.; Lamparter, T.
Key amino acids in the bacterial (6-4) photolyase PhrB from Agrobacterium fabrum (2015), PLoS ONE, 10, e0140955 .

Search for more literature for 4.1.99.13

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of wild-type and mutant enzymes in Escherichia coli	Agrobacterium fabrum

Protein Variants

Protein Variants	Comment	Organism
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	Agrobacterium fabrum
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	Agrobacterium fabrum
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	Agrobacterium fabrum
H366A	site-directed mutagenesis, in the mutant repair activity is lost	Agrobacterium fabrum
H366N/L370M	site-directed mutagenesis, in the mutant repair activity is lost	Agrobacterium fabrum
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	Agrobacterium fabrum
W342F	site-directed mutagenesis, the mutant shows highly reduced light-induced spectral changes compared to wild-type	Agrobacterium fabrum
W390F	site-directed mutagenesis, the mutant shows highly reduced light-induced spectral changes compared to wild-type	Agrobacterium fabrum
W390F/Y391F	site-directed mutagenesis, the mutant shows highly reduced light-induced spectral changes compared to wild-type	Agrobacterium fabrum
Y391F	site-directed mutagenesis, the mutant Y391F shows wild-type-like light-induced spectral changes	Agrobacterium fabrum
Y399F	site-directed mutagenesis, the mutant shows highly reduced light-induced spectral changes compared to wild-type	Agrobacterium fabrum
Y399F	site-directed mutagenesis, the mutant Y391F shows slightly reduced light-induced spectral changes compared to wild-type	Agrobacterium fabrum
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	Agrobacterium fabrum
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	Agrobacterium fabrum
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	Agrobacterium fabrum

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Fe2+	Fe-S cluster, the Fe-S cluster is required for structural stability, integrity and correct folding of PhrB	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)	-	?

Organism

Organism	UniProt	Comment	Textmining
Agrobacterium fabrum	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant wild-type and mutant enzymes from Escherichia coli	Agrobacterium fabrum

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)	(6-4)TT	Agrobacterium fabrum	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
20	-	assay at	Agrobacterium fabrum

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7	7.5	assay at	Agrobacterium fabrum

Cofactor

Cofactor	Comment	Organism	Structure
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	Agrobacterium fabrum
Fe-S cluster	the Fe-S cluster is required for protein stability	Agrobacterium fabrum

General Information

General Information	Comment	Organism
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	Agrobacterium fabrum
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	Agrobacterium fabrum
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	Agrobacterium fabrum
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	Agrobacterium fabrum

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Release 2023.1 (January 2023)