Federica Frascogna

• Photosynthesis stands as one of the most pivotal biological processes on our planet. To efficiently harvest light, photosynthetic organisms utilize chlorophyll antennae and carotenoids within their photosystems. However, the efficacy of light harvesting by chlorophyll and carotenoids is negligible in the green region of the light spectrum. To fully exploit light energy, several photosynthetic organisms employ specialized proteins named phycobiliproteins, which are able to harvest light in the green gap. Furthermore, most of these organisms are endowed with photoreceptors, enabling them to sense the quality and intensity of the harvested light and to modulate biological activities based on this information. Both light harvesting and light sensing functions are mediated by linear tetrapyrrole chromophores named bilins. The biosynthesis of bilins is catalyzed by enzymes known as ferredoxin-dependent bilin reductases (FDBRs). The origin of both biliproteins and this class of enzymes remain elusive. This work aimed to delineate the evolution of phycocyanobilin (PCB) biosynthesis, from emergence to primary endosymbiosis. The recent identification of FDBR-related sequences in heterotrophic, non-photosynthetic bacteria (pre-FDBRs) provided a foundation for investigating the emergence and original bioactivity of this enzyme class. Biochemical characterization of a representative from each pre-FDBR clade revealed FDBRs initially emerged as 2e- reducing enzymes yielding phytochromobilin (PΦB) production, to evolve a 4e- reducing capability in ultimately yielding phycoerythrobilin (PEB). The latter reaction represents the first described instance of PEB production occurring via PΦB as the intermediate. PCB biosynthesis catalyzed by phycocyanobilin-ferredoxin oxidoreductase (PcyA) finally evolved in α-proteobacteria and was ultimately acquired by cyanobacteria, potentially via horizontal gene transfer. The primary endosymbiosis event giving birth to the Archaeplastida supergroup resulted in a significant divergence of FDBRs, particularly related to PCB biosynthesis. While Glaucophytes retained typical cyanobacterial PCB biosynthesis catalyzed by a PCYA homolog, in Rhodophytes and Viridiplantae PCB biosynthesis underwent substantial divergence. In Rhodophytes, PCB biosynthesis is believed to rely on a chromophore isomerase rather than on specific PCB-producing FDBRs. Indeed, more than three decades ago, the biosynthesis of PCB in Galdieria sulphuraria was shown to occur first via PEB production by FDBRs and subsequent PEB to PCB isomerization. As technical problems hindered further confirmation of this scenario, the focus of this part of the project shifted to the full characterization of the activity of G. sulphuraria FDBRs to ultimately confirm only PEB production is catalyzed by these enzymes. The Viridiplantae lineage further diverged into Streptophytes and Chlorophytes. While Chlorophytes retained PCYA-mediated PCB biosynthesis, Streptophytes present an intricate situation. The Streptophyta lineage encompasses streptophyte algae and, their descendent, land plants. As land plants do not possess PCB and use PΦB-phytochromes, streptophyte algae were instead indicated to use PCB-phytochromes. However, this clade is endowed with a plant type HY2-homolog, theoretically responsible for PΦB production. The biochemical investigation of several members of the streptophyta HY2 lineage revealed that HY2 was originally a 4e- reducing FDBR, catalyzing PCB production for phytochrome incorporation in streptophyte algae, and evolved in Bryophytes to lose the second 2e- reducing step and only yield PΦB. The characterization of this FDBR lineage further proved that the activity of FDBRs cannot be solely predicted from amino acid sequence and structure model, as conserved residues and a similar overall fold can still lead to different activities. Overall, this study provides an outline of PCB biosynthesis evolution, from its emergence to primary endosymbiosis-triggered diversification. A final brief section of this work provides insights into the production of phycourobilin (PUB) in Streptophytes. The production of PUB, resulting from PEB isomerization in cyanobacteria and red algae, was solely thought to be required for light-harvesting purposes, as PUB is usually attached to PE in these organisms. However, about a decade ago, a new FDBR responsible for PUB production was discovered in Physcomitrium patens. This enzyme, named phycourobilin synthase (PUBS), catalyzes the 4e- reduction of biliverdin to PUB via 15,16-dihydrobiliverdin as the intermediate. With the constant emergence of new sequenced genomes, PUBS homologs were also identified in several streptophyte algae. The characterization of the PUBS from Klebsormidium nitens revealed a similar catalytic activity to the one of P. patens. Hitherto, PUBS remains the only FDBR whose activity and structure have not been extensively characterized. Moreover, the significance of PUB in organisms which do not perform phycobiliprotein-mediated light harvesting is still an open question.