Photolyases are ubiquitously occurring flavoproteins for catalyzing photo repair of UV-induced DNA damages. All photolyases described so far have a bilobal architecture with a C-terminal domain comprising flavin adenine dinucleotide (FAD) as catalytic cofactor and an N-terminal domain capable of harboring an additional antenna chromophore. Using sequence-similarity network analysis we discovered a novel subgroup of the photolyase/cryptochrome superfamily (PCSf), the NewPHLs. NewPHL occur in bacteria and have an inverted topology with an N-terminal catalytic domain and a C-terminal domain for sealing the FAD binding site from solvent access. By characterizing two NewPHL we show a photochemistry characteristic of other PCSf members as well as light-dependent repair of CPD lesions. Given their common specificity towards single-stranded DNA many bacterial species use NewPHL as a substitute for DASH-type photolyases. Given their simplified architecture and function we suggest that NewPHL are close to the evolutionary origin of the PCSf.

Photolyases are flavoproteins, which are able to repair UV‐induced DNA lesions in a light‐dependent manner. According to their substrate, they can be distinguished as CPD‐ and (6‐4) photolyases. While CPD‐photolyases repair the predominantly occurring cyclobutane pyrimidine dimer lesion, (6‐4) photolyases catalyze the repair of the less prominent (6‐4) photoproduct. The subgroup of prokaryotic (6‐4) photolyases/FeS‐BCP is one of the most ancient types of flavoproteins in the ubiquitously occurring photolyase & cryptochrome superfamily (PCSf). In contrast to canonical photolyases, prokaryotic (6‐4) photolyases possess a few particular characteristics, including a lumazine derivative as antenna chromophore besides the catalytically essential flavin adenine dinucleotide as well as an elongated linker region between the N‐terminal α/β‐domain and the C‐terminal all‐α‐helical domain. Furthermore, they can harbor an additional short subdomain, located at the C‐terminus, with a binding site for a [4Fe‐4S] cluster. So far, two crystal structures of prokaryotic (6‐4) photolyases have been reported. Within this study, we present the high‐resolution structure of the prokaryotic (6‐4) photolyase from Vibrio cholerae and its spectroscopic characterization in terms of in vitro photoreduction and DNA‐repair activity.

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