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Key Amino Acids in the Bacterial (6-4) Photolyase PhrB from Agrobacterium fabrum

Graf, D.; Wesslowski, J.; Ma, H.; Scheerer, P.; Krauß, Norbert; Oberpichler, I.; Zhang, F.; Lamparter, Tilman

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. ... mehr

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Volltext §
DOI: 10.5445/IR/1000050816
DOI: 10.1371/journal.pone.0140955
Zitationen: 13
Web of Science
Zitationen: 13
Zugehörige Institution(en) am KIT Botanisches Institut und Botanischer Garten (BOTANIK)
Publikationstyp Zeitschriftenaufsatz
Jahr 2015
Sprache Englisch
Identifikator ISSN: 1932-6203
KITopen-ID: 1000050816
Erschienen in PLoS one
Band 10
Heft 10
Seiten e0140955
Bemerkung zur Veröffentlichung Gefördert durch den KIT-Publikationsfonds
Nachgewiesen in Scopus
Web of Science
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