Die erste funktionelle Charakterisierung einer eukaryot kodierten Bilin Lyase

The nucleomorph genome of the cryptophyte Guillardia theta is an exemplary model for nuclear compaction and is one of the smallest eukaryotic genomes presently known. That 30 of its genes encode plastid proteins might be a reason why the nucleomorph is still maintained. Characterisation of as yet plastid proteins of unknown function is of great interest. In this thesis, the nucleomorph-encoded protein Orf222 could be identified as a functional ortholog to a specific bilin lyase from a cyanobacterium. Additionally, it could be shown with the complementation of the deleted ortholog with the gene orf222 that the one missing bilin in the insertion mutant is responsible for the complex phenotype. The protein Orf222 functions in the heterologous system of the cyanobacterium Synechocystis sp. PCC6803 as a lyase, which is responsible for the attachment of a phycocyanobilin to position cystein-155 in the phycocyanin β-subunit. Despite its divergent pigment composition, localisation and organisation, a lyase function must also be postulated in the cryptophyte. The protein Orf222 is supposed to attach a phycoerythrobilin to the homologous position cystein-158 in the phycoerythrin β-subunit. Designated as CpeT, Orf222 shows a clear regioselectivity in regard to homologous binding sites as shown by its functional activity in the heterologous system, albeit a lowered specificity in relation to the respective bilins and apoproteins. These features are defining qualities of cyanobacterial T-type lyases, of which this protein is one. A phylogenetic analysis shows a closer relationship between the protein Orf222 and its homologs from cryptophytes and rhodophytes than with homologs from cyanobacteria or plants. Homologs from cyanobacteria can be divided into four monophyletic groups which correlate to a specific biliprotein and to the genetic context of the corresponding gene. The nucleomorph-encoded protein CpeT (Orf222) from Guillardia theta is the first functionally characterised eukaryotic bilin lyase and contributes significant insight into nucleomorph-encoded proteins.