SummaryThe pink open-chain tetrapyrrole pigment phycoerythrobilin (PEB) is employed by marine cyanobacteria, red algae and cryptophytes as a light-harvesting chromophore in phycobiliproteins. Genes encoding biosynthesis proteins for PEB have also been discovered in cyanophages, viruses that infect cyanobacteria, and mimic host pigment biosynthesis with the exception of PebS which combines the enzymatic activities of two host enzymes. In this study, we have identified novel members of the PEB biosynthetic enzyme families, heme oxygenases and ferredoxin-dependent bilin reductases. Encoding genes were found in metagenomic datasets and could be traced back to bacteriophage but not cyanophage origin. While the heme oxygenase exhibited standard activity, a new bilin reductase with highest homology to the teal pigment producing enzyme PcyA revealed PEB biosynthetic activity. Although PcyX possesses PebS-like activity both enzymes share only 9% sequence identity and likely catalyze the reaction via two independent mechanisms. Our data point towards the presence of phycobilin biosynthetic genes in phages that probably infect alphaproteobacteria and, therefore, further support a role of phycobilins outside oxygenic phototrophs.
Phytochromes are biliprotein photoreceptors present in plants, algae, certain bacteria and fungi. Land plant phytochromes use phytochromobilin (PΦB) as the bilin chromophore. Phytochromes of streptophyte algae, the clade from 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 IXα (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 PΦB by phytochromobilin synthase (HY2). However, phylogenetic studies proved the absence in streptophyte algae of any ortholog of PcyA and the presence of only PΦB 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. To provide the direct evidence of an intrinsic FDBR activity of this enzyme, we overexpressed and purified a His6-tagged variant of K. nitens HY2 (KflaHY2) in E. coli. In anaerobic bilin reductase activity assays, we found that KflaHY2 is a functional FDBR yielding mostly 3(Z)-PCB as the reaction product. Via coupled phytochrome assembly assays, we identified 3(Z)-phytochromobilin and 181,182-dihydrobiliverdin (181,182-DHBV) as intermediates of the reaction. With the help of site-directed mutagenesis two aspartate residues were identified to be critical for catalysis. Overall, our study gives insight into the evolution of the HY2 lineage of FDBRs.
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