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Characterization of pre-PcyA ferredoxin-dependent bilin reductases in vivo and in vitro

Rockwell, Nathan1; Frankenberg-Dinkel, Nicole2; Frascogna, Federica2

Published Jun 30, 2026 on Dryad. https://doi.org/10.5061/dryad.fqz612k74

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Abstract

This dataset contains data for studies of pre-PcyA proteins from diverse bacteria. The ferredoxin-dependent bilin reductase (FDBR) PcyA is used by cyanobacteria to synthesize phycocyanobilin (PCB) from biliverdin IX-alpha (BV) in a 4-electron reduction. PCB is critical for proper function of the phycobilisome light-harvesting antenna used by most cyanobacteria, but it has been shown to be derived from pre-PcyA proteins that are not found in cyanobacteria and that do not synthesize PCB. Instead, pre-PcyA proteins studied to date instead synthesize structurally related phytochromobilin (PFB) and/or phycoerythrobilin (PEB) from BV, based on an in vivo assay that reconstitutes activity in E. coli cells with a bilin-binding acceptor protein. Pre-PcyA proteins studied to date fall into 3 lineages: pre-1, pre-2, and pre-3; previous studies demonstrated robust activity for examples of the pre-2 and pre-3 groups, but not for pre-1. The current work extends these studies by examining an alternative pre-1 protein, which is found to exhibit much higher activity, and by characterizing examples of all 3 groups in vitro using spectroscopic and HPLC assays.

Deposited data include an updated phylogenetic analysis of FDBRs, HPLC and spectroscopic data from direct in vitro FDBR assays, and spectroscopic data from in vivo and in vitro assays with bilin-binding acceptor proteins. The phylogenetic analysis confirms that the alternative pre-1 protein targeted in this study is a representative member of this lineage. The direct in vitro assays demonstrate that pre-1 and pre-2 synthesize 3E-PFB, whereas pre-3 generates a mix of 3E-PFB and 3E-PEB. These results contrast with the synthesis of the 3Z-PEB isomer by the known FDBR PebS, and data with a bilin-binding acceptor protein confirm the presence of spectroscopically distinct PEB populations after co-expression with either the pre-3 protein or PebS. The results thus demonstrate that the pre-PcyA proteins in this study generate the predicted products based on previous studies while providing a robust characterization of a pre-1 protein and identifying the isomer produced by these proteins.

However, attempts to modulate the behavior of these enzymes using site-directed mutagenesis gave rise to unpredictable results. Mutagenesis of a conserved acidic residue to its uncharged equivalent (Glu to Gln or Asp to Asn) resulted in formation of the 3Z-PFB isomer by pre-1 but had no effect on pre-2 and resulted in loss of activity in pre-3. These results are consistent with other studies demonstrating that effects of such mutations can be unpredictable in FDBRs, perhaps indicating that subtle effects on substrate positioning relative to the active site cannot be uncoupled from direct roles in catalysis.