Crystal Structures of Bacterial (6‐4) Photolyase Mutants with Impaired DNA Repair Activity

Abstract

PhrB from Agrobacterium fabrum is the first prokaryotic photolyase which repairs (6-4) UV DNA photoproducts. 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. Tyr424 of PhrB is part of the DNA-binding site and could provide an electron link to the Fe-S cluster. The PhrB_Y_424F mutant showed reduced binding of lesion DNA and loss of DNA repair. The mutant PhrB_I_51W is characterized by the loss of the DMRL chromophore, reduced photoreduction and reduced DNA repair capacity. We have determined the crystal structures of both mutants and found that both mutations only affect local protein environments, whereas the overall fold remained unchanged. The crystal structure of PhrB_Y_424F revealed a water network extending to His366, which are part of the lesion-binding site. The crystal structure of PhrB_I_51W shows how the bulky Trp leads to structural rearrangements in the DMRL chromophore pocket. Spectral characterizations of PhrB_I_51W suggest that DMRL serves as an antenna chromophore for photoreduction and DNA repair in the wild type. The energy transfer from DMRL to FAD could represent a phylogenetically ancient process.