Biology subjects

Frascogna, F.

Publications and source records attributed to Frascogna, F..

3 recordsLinked to original sources

bioRxiv2026

Evolutionary insights into bilin biosynthesis: Functional characterization of pre-PcyA enzymes

Biosynthesis of the linear tetrapyrrole phycocyanobilin (PCB) by the ferredoxin-dependent bilin reductase (FDBRs) PcyA is essential for light-harvesting and regulatory processes in diverse photosynthetic organisms, yet its evolutionary origins are not fully understood. PcyA evolved from pre-PcyA proteins found in diverse bacteria. Three lineages of pre-PcyA proteins were identified: Pre-1, Pre-2 and Pre-3. Using an in vivo co-expression assay, Pre-2 and Pre-3 proteins were shown to be active FDBRs that did not synthesize PCB, whereas Pre-1 activity was apparently low. In refining these results, we noted a discrepancy between phycoerythrobilin populations generated by Pre-3 and by the distantly related FDBR PebS. We therefore examined the properties of pre-PcyA enzymes in vitro, using an updated pre-PcyA phylogeny to select an alternative pre-1 target. Biochemical analyses revealed that Pre-1 and Pre-2 catalyze the two-electron reduction of biliverdin (BV) to 3E-phytochromobilin (3E-P[FE]B), in contrast to the known synthesis of 3Z-phytobilins by other FDBRs. Pre-3 can also carry out an additional two-electron reduction to yield 3E-phycoerythrobilin (3E-PEB), again distinct from the 3Z-PEB produced by PebS. We then used comparative sequence and structure analysis to target candidate catalytic residues for site-directed mutagenesis. Variant Pre-1 exhibited altered product stereochemistry, but no effects on Pre-2 were observed and Pre-3 variants unexpectedly gained the ability to bind cyclic tetrapyrroles. These findings underscore the plasticity and promiscuity of this enzyme family. Together, this work illustrates how the flexible catalytic potential of ancestral enzymes shaped the evolution and diversification of bilin biosynthetic pathways.

biochemistry↗

bioRxiv2025

Phycocyanobilin biosynthesis in Galdieria sulphuraria requires isomerization of phycoerythrobilin synthesized by bilin reductases

Cyanobacteria and red algae (Rhodophyta) utilize phycobilisomes as light-harvesting antennae to carry out oxygenic photosynthesis. Phycobilisomes are large protein complexes comprised of phycobiliproteins and associated linker proteins, with phycobiliproteins carrying one or more linear tetrapyrrole (bilin) chromophores. Bilin biosynthesis is typically catalyzed by ferredoxin-dependent bilin reductases (FDBRs). Cyanobacterial genomes encode one FDBR, PcyA, for biosynthesis of phycocyanobilin (PCB), which is required both for harvesting red light and for coupling phycobilisomes to photosynthetic reaction centers via energy transfer. Some cyanobacteria also contain two additional FDBRs (PebA and PebB) for synthesis of the green-absorbing phycoerythrobilin (PEB). Most rhodophytes contain only PebA and PebB homologs (PEBA and PEBB), so the biosynthesis of PCB in such organisms is not understood. This process is especially relevant in the early-branching rhodophyte Galdieria sulphuraria, which contains PCB as the main light-harvesting chromophore yet contains the PEBA and PEBB FDBRs for PEB biosynthesis. In this organism, PCB biosynthesis is thought to be dependent both on FDBRs and on a yet-to-be identified bilin isomerase. Our current work reaffirms these findings. Phylogenetic analysis clearly places G. sulphuraria FDBRs in the PEBA and PEBB clades, in contrast to the PCYA found in Cyanidioschyzon merolae, another early-branching rhodophyte. All three rhodophyte FDBRs were found to have the expected substrate preferences and enzymatic activities after recombinant expression, affinity purification, and in vitro characterization. Therefore, bilin biosynthesis in G. sulphuraria results in biosynthesis of PEB rather than PCB. However, a previous procedure allowed partial purification of a PEB:PCB isomerase, clearly demonstrating the existence of such an activity and adding to our understanding of bilin biosynthesis and algal light harvesting.

plant biology↗

bioRxiv2022

On the evolution of the plant phytochrome chromophore

Phytochromes are biliprotein photoreceptors present in plants, algae, certain bacteria and fungi. Land plant phytochromes use phytochromobilin (P{Phi}B) as the bilin chromophore. Phytochromes of streptophyte algae, the clade within which land plants evolved, employ phycocyanobilin (PCB), leading to a more blue-shifted absorption spectrum. Both chromophores are synthesized by ferredoxin-dependent bilin reductases (FDBRs) starting from biliverdin IX (BV). In cyanobacteria and chlorophyta, BV is reduced to PCB by the FDBR phycocyanobilin:ferredoxin oxidoreductase (PcyA), whereas, in land plants, BV is reduced to P[FE]B by phytochromobilin synthase (HY2). However, phylogenetic studies suggested the absence of any ortholog of PcyA in streptophyte algae and the presence of only P[FE]B biosynthesis related genes (HY2). The HY2 of the early diverging streptophyte alga Klebsormidium nitens (formerly Klebsormidium flaccidum) was already indirectly indicated to be involved in PCB biosynthesis. Here, we overexpressed and purified a His6-tagged variant of K. nitens HY2 (KflaHY2) in E. coli. Employing anaerobic bilin reductase activity assays and coupled phytochrome assembly assays, we were able to confirm the product and to identify intermediates of the reaction. Site-directed mutagenesis revealed two aspartate residues critical for catalysis. While it was not possible to convert KflaHY2 into a P{Phi}B-producing enzyme by simply exchanging the catalytic pair, the biochemical investigation of two additional members of the HY2 lineage enabled us to define two distinct clades, the PCB-HY2 and the P{Phi}B-HY2 clade. Overall, our study gives insight into the evolution of the HY2 lineage of FDBRs.

plant biology↗