Strukturelle und funktionelle Charakterisierung eines neuartigen bakteriellen Cryptochroms sowie Analysen von mikrobiellen Photolyasen

Cryptochromes and photolyases constitute a group of highly distributed, FAD-binding, bluelight dependent signaling proteins ans enzymes, which together form the photolyase/cryptochrome-superfamily (PCSf). Photolyases recognize UV-induced DNA lesions between adjacent pyrimidine bases, namely cyclobutanpyrimidindimers (CPD) and (6-4)-pyrimidine-pyrimidone-photoproducts ((6-4)), and repair them blue-light dependently, whereas cryptochromes rather show regulatory functions in vivo. The plant representatives are involved in responses to blue-light stimuli and take influence on growth, development and the circadian clock of the plant. Cryptochromes in animals participate in the circadian clock as blue-light sensors (type I), or light-independently as part of the central oscillator (type II). Cryptochromes from bacteria are still poorly understood, the best characterized representatives from Synechocystis sp. PCC6803 belongs to the CryDASH family whose exact biological function is still unknown. This work dealt with functional and the first structural characterization of a true bacterial cryptochrome,cryptochrome B from Rhodobacter sphaeroides (RsCryB). RsCryB shows no DNA repair activity and participates in the light-dependent and oxygen-dependent regulation of photosynthesis genes in R. sphaeroides. It defines a new family of proteins in the PCSf, predominantly occurring in proteobacteria, the proteobacterial cryptochromes (CryPro). Despite low sequence identity to the other representatives of the PCSf, the structure of the CryPro family is analogous to the conserved overall fold of the superfamily. Surprisingly, one [4Fe-4S] clusters was identified, which is along with the catalytic cofactor FAD the defining element of RsCryB's C-terminus. This cluster is structurally and chemically related to known clusters of eukaryotic primase subunits, as was shown by EPR experiments. Moreover 6,7- dimethyl-8-ribityl-lumazine was identified as antenna chromophore of RsCryB, yet unknown inside the PCSf. These studies are complemented by an DNA binding analysis of the class II CPD photolyase from Methanosarcina mazei (MmCPDII), the model photolyase for plant homologs, as well as an analysis of the fully reduced state of MmCPDII by ultrafast spectroscopy. In the third part of the project the native antenna chromophore of the (6-4)-photolyase from Dunaliella salina was identified as 8-hydroxy-5-deazaflavin and the in vitro repair of (6-4)-damages by the enzyme was shown.