Key Amino Acids in the Bacterial (6-4) Photolyase PhrB from Agrobacterium fabrum.
Photolyases can repair pyrimidine dimers on the DNA that are formed during UV irradiation. PhrB from Agrobacterium fabrum represents a new group of prokaryotic (6-4) photolyases which contain an iron-sulfur cluster and a DMRL chromophore. We performed site-directed mutagenesis in order to assess the 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. Our study showed that Trp342 and Trp390 serve as electron transmitters. In the H366A mutant repair activity was lost, which points to a significant role of His366 in the protonation of the lesion, as discussed for the homolog in eukaryotic (6-4) photolyases. Mutants on cysteines that coordinate the Fe-S cluster of PhrB were either insoluble or not expressed. The same result was found for proteins with a truncated C-terminus, in which one of the Fe-S binding cysteines was mutated and for expression in minimal medium with limited Fe concentrations. We therefore assume that the Fe-S cluster is required for protein stability. We further mutated conserved tyrosines that are located between the DNA lesion and the Fe-S cluster. Mutagenesis results showed that Tyr424 was essential for lesion binding and repair, and Tyr430 was required for efficient repair. The results point to an important function of highly conserved tyrosines in prokaryotic (6-4) photolyases.
Karlsruhe Institute of Technology (KIT), Botanical Institute, Kaiserstr. 2, D-76131 Karlsruhe, Germany.
Dominik
D
Graf
Karlsruhe Institute of Technology (KIT), Botanical Institute, Kaiserstr. 2, D-76131 Karlsruhe, Germany.
Janine
J
Wesslowski
Karlsruhe Institute of Technology (KIT), Botanical Institute, Kaiserstr. 2, D-76131 Karlsruhe, Germany.
Hongju
H
Ma
Charité-University Medicine Berlin, Institute of Medical Physics and Biophysics (CC2), AG Protein X-ray Crystallography and Signal Transduction, Charitéplatz 1, D-10117 Berlin, Germany.
Patrick
P
Scheerer
Queen Mary University of London, School of Biological and Chemical Sciences, London E1 4NS, United Kingdom.
Norbert
N
Krauß
Karlsruhe Institute of Technology (KIT), Botanical Institute, Kaiserstr. 2, D-76131 Karlsruhe, Germany.
Inga
I
Oberpichler
Karlsruhe Institute of Technology (KIT), Botanical Institute, Kaiserstr. 2, D-76131 Karlsruhe, Germany.
Fan
F
Zhang
Karlsruhe Institute of Technology (KIT), Botanical Institute, Kaiserstr. 2, D-76131 Karlsruhe, Germany.
Tilman
T
Lamparter
D060054:Agrobacterium / Q000201:enzymology* / Q000235:genetics*; D000595:Amino Acid Sequence / Q000235:genetics; D000596:Amino Acids / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics*; D004249:DNA Damage / Q000235:genetics; D004260:DNA Repair / Q000235:genetics; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000235:genetics*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed
D000596:Amino Acids; D001426:Bacterial Proteins; D004255:Deoxyribodipyrimidine Photo-Lyase