DOI: 10.5061/dryad.hhmgqnksx
title: Underlying data for characterization of phycocyanobilin (PCB) biosynthesis in Galdieria sulphuraria

description: This dataset contains data for studies of phycocyanobilin (PCB) synthesis
 in the red alga Galdieria sulphuraria. Red algae such as G.
 sulphuraria utilize phycobilisomes for light harvesting. The
 phycobilisomes of early-branching organisms such as G.
 sulphuraria or Cyanidioschyzon merolae contain PCB chromophores
 but not phycoerythrobilin (PEB), in contrast to the
 phycobilisomes of other red algae. The studies reported in this
 dataset examine biosynthesis of PCB in G. sulphuraria and C.
 merolae, starting from biliverdin IX-alpha (BV), the last known
 common precursor for PCB and PEB. In cyanobacteria, phages,
 green algae, and land plants, conversion of BV into PCB or PEB is
 carried out by a family of enzymes called ferredoxin-dependent
 bilin reductases (FDBRs). The current studies demonstrate that G.
 sulphuraria, but not C. merolae, require the action of an
 additional isomerase to synthesize PCB.Deposited data include phylogenetic
 analyses, data for in vitro characterization of recombinantly expressed
 FDBRs from G. sulphuraria and C. merolae, and data for fractionation of G.
 sulphuraria extracts and isomerase assays on the resulting enriched
 fraction. The phylogenetic analyses demonstrates that G. sulphuraria has
 two FDBRs, GsPEBA and GsPEBB, whereas C. merolae has a single FDBR,
 CmPCYA. CmPCYA would be expected to convert BV into PCB, whereas GsPEBA
 would be expected to convert BV into 15,16-dihydrobiliverdin (15,16-DHBV)
 and GsPEBB would then convert 15,16-DHBV into PEB rather than PCB. In
 vitro characterization demonstrates that all three FDBRs carry out the
 expected reactions, meaning that the G. sulphuraria enzymes produce PEB
 but the phycobilisomes contain PCB. This conundrum is resolved by
 demonstrating the existence of an isomerase in G. sulphuraria extracts
 that can convert PEB into PCB.
description: # Title: Underlying data for characterization of phycocyanobilin (PCB)
 biosynthesis in Galdieria sulphuraria ## Description of the Data and file
 structure This dataset contains data for studies of phycocyanobilin (PCB)
 synthesis in the red alga Galdieria sulphuraria. Red algae such as G.
 sulphuraria utilize phycobilisomes for light harvesting. The
 phycobilisomes of early-branching organisms such as G. sulphuraria or
 Cyanidioschyzon merolae contain PCB chromophores but not phycoerythrobilin
 (PEB), in contrast to the phycobilisomes of other red algae. The studies
 reported in this dataset examine biosynthesis of PCB in G. sulphuraria and
 C. merolae, starting from biliverdin IX-alpha (BV), the last known common
 precursor for PCB and PEB. In cyanobacteria, phages, green algae, and land
 plants, conversion of BV into PCB or PEB is carried out by a family of
 enzymes called ferredoxin-dependent bilin reductases (FDBRs). The current
 studies demonstrate that G. sulphuraria, but not C. merolae, require the
 action of an additional isomerase to synthesize PCB. Deposited data are in
 a single gzipped tarball (Galdieria_PCB_biosynthesis_version3.tar.gz) and
 include phylogenetic analyses, data for in vitro characterization of
 recombinantly expressed FDBRs from G. sulphuraria and C. merolae, and data
 for fractionation of G. sulphuraria extracts and isomerase assays on the
 resulting enriched fraction. The phylogenetic analyses demonstrate that G.
 sulphuraria has two FDBRs, GsPEBA and GsPEBB, whereas C. merolae has a
 single FDBR, CmPCYA. CmPCYA would be expected to convert BV into PCB,
 whereas GsPEBA would be expected to convert BV into
 15,16-dihydrobiliverdin (15,16-DHBV) and GsPEBB would then convert
 15,16-DHBV into PEB rather than PCB. In vitro characterization
 demonstrates that all three FDBRs carry out the expected reactions,
 meaning that the G. sulphuraria enzymes produce PEB but the phycobilisomes
 contain PCB. This conundrum is resolved by demonstrating the existence of
 an isomerase in G. sulphuraria extracts that can convert PEB into PCB.
 Data are deposited as a gzipped tarball containing a series of
 tab-delimited text files with unix newlines. After extraction, deposited
 data are in four folders: I. FDBR_phylogeny II. CmPCYA_in_vitro III.
 GsPEBA_GsPEBB_in_vitro IV. Galdieria_PEB_PCB_isomerase I. FDBR_phylogeny:
 Two maximum-likelihood phylogenies were inferred from the same alignment
 for this study. One used PhyML version 3.3; the other used IQ-TREE version
 3.0.1. Five files are deposited: the original multiple sequence alignment
 in CLUSTAL format, the input file for both phylogenies in PHYLIP format
 after removal of gap-enriched columns, and output files from the two
 software packages. The files are: NTE_118.aln (multiple sequence alingment
 in CLUSTAL format) NTE_118.05.phy (input file in PHYLIP format)
 PML_NTE_118.05.phy_phyml_tree.txt (PhyML output file)
 IQ_NTE_118.05.phy.treefile (IQ-TREE output file) IQ_NTE_118.05.phy.log
 (log file from IQ-Tree, including model search results) NTE_118.aln
 (multiple sequence alingment in CLUSTAL format) This is the original
 alignment, generated in MAFFT (v7.450) using the E-INS-i algorithm. After
 a header indicating the MAFFT version, aligned sequences are presented as
 interleaved blocks. Each block has a segment of each sequence after the
 name of the sequence, with '-' serving as the gap character. As
 an example, here are three arbitrary sequences: Protein_1
 THIS-SEQ-IS-IMAGINARY Protein_2 THAT-SEQ-VT-LMVGVNARF Protein_3
 SQATGTENGASPVMAGINARF NTE_118.05.phy (input file in PHYLIP format) For
 phylogenetic inference in PHYML, the CLUSTAL-format alignment was
 converted into the PHYLIP format with removal of gap-enriched positions.
 The resulting file is now sequential (with one protein sequence followed
 by the next). The header line is also different, indicating the number of
 sequences (3, in the above example) and positions or characters (18 above,
 after removal of gap-enriched columns). For the above example, the
 converted case looks like this: 3 18 Protein_1 THISSEQISIMAGINARY
 Protein_2 THATSEQVTLMVGVNARF Protein_3 SQATTENASVMAGINARF (noting that
 this example is arbitrary and is unrelated to the actual sequences in this
 study) PML_NTE_118.05.phy_phyml_tree.txt (PhyML output file in Newick
 format) The PhyML phylogeny is presented in the Newick format, suitable
 for use with a broad range of tree viewers but not well suited for
 analysis in a text editor or word processor. One support value is
 presented (TBE, or transfer bootstrap expectation). TBE was calculated
 from 100 bootstraps within PhyML. IQ_NTE_118.05.phy.treefile (IQ-Tree
 output file in Newick format). The IQ-Tree output file is again presented
 in Newick format. Two supports are presented: the SH-aLRT approximate
 likelihood test, followed by the ultrafast bootstrap approximation
 (UFBoot). IQ_NTE_118.05.phy.log (IQ_Tree log file as flat text file).
 IQ-Tree was run using ModelFinder to automatically choose the best
 substitution model for the alignment. The IQ_Tree log file is included to
 show the results of that step. II. CmPCYA_in_vitro: This folder contains
 data from in vitro characterization of CmPCYA protein purified after
 recombinant expression in E. coli. Two assays were used, and data from
 each data are deposited for a total of three files.
 CmPCYA_Spectroscopic_timecourse.txt This file contains a series of
 absorption spectra measured at different times after initiation of a
 CmPCYA reaction. Each spectrum (or timepoint) is one column in the file,
 with the first column providing wavelength information in nm.
 rezeroed.CmPCYA_spectra.txt This file contains the processed spectral data
 for the above file after re-zeroing and Savitsky-Golay smoothing.
 CmPCYA_HPLC_assay.txt This file contains data from an endpoint assay to
 identify reaction products via reverse-phase HPLC. After workup of the
 assay, reaction products were run on a C18 column along with controls. BV
 and PCB were detected using a diode arrray detector set for 650 nm. The
 file contains paired time/absorption values for three HPLC runs: one with
 CmPCYA reaction products, one with cyanobacterial PcyA (SynPcyA from
 Synechocystis sp. PCC6803) as a positive control for PCB formation, and
 one with BV substrate alone as a negative control). III.
 GsPEBA_GsPEBB_in_vitro: This folder contains data from in vitro
 characterization of purified GsPEBA and GsPEBB. Each enzyme was
 characterized by itself using the spectroscopic and HPLC assays also used
 for CmPCYA. The action of the two enzymes together was also studied using
 the same assays. The deposited files are:
 GsPEBA_Spectroscopic_timecourse.txt This file contains a series of
 absorption spectra measured at different times after initiation of a
 GsPEBA reaction. Each spectrum (or timepoint) is one column in the file,
 with the first column providing wavelength information in nm. BV was
 substrate. rezeroed.GsPEBA_Spectra.txt This file contains the processed
 spectral data for the above file after re-zeroing and Savitsky-Golay
 smoothing. GsPEBB_Spectroscopic_timecourse.txt This file contains a series
 of absorption spectra measured at different times after initiation of a
 GsPEBB reaction. Each spectrum (or timepoint) is one column in the file,
 with the first column providing wavelength information in nm. 15,16-DHBV
 was substrate. rezeroed.GsPEBB_1516_spectra.txt This file contains the
 processed spectral data for the above file after re-zeroing and
 Savitsky-Golay smoothing. rezeroed.GsPEBB_BV_spectra.txt This file
 contains the processed spectral data for a similar reaction with GsPEBB
 using BV as substrate rather than 15,15-DHBV.
 GsPEBA_GsPEBB_individual_HPLC.txt This file contains data from endpoint
 assays using reverse-phase HPLC. After workup, reaction products were run
 on a C18 column along with controls. Bilin chromophores were detected
 using a diode arrray detector. The file contains paired time/absorption
 values for four HPLC runs: one with GsPEBA reaction products, one with
 GsPEBB reaction products, and one each with 15,16-DHBV and PEB reference
 standards. combined_GsPEBAB_spectroscopic_timecourse.txt This file
 contains a series of absorption spectra measured at different times after
 initiation of a reaction using GsPEBA and GsPEBB in combination, with BV
 as substrate. Each spectrum (or timepoint) is one column in the file, with
 the first column providing wavelength information in nm.
 rezeroed.combined_GsPEBAB_spectra.txt This file contains the processed
 spectral data for the above file after re-zeroing and Savitsky-Golay
 smoothing. combined_GsPEBAB_HPLC.txt This file contains data from endpoint
 assays using GsPEBB paired with either GsPEBA or cyanobacterial PebA from
 Synechococcus sp. WH8020. Data are deposited as paired time/absorption
 values for both HPLC runs. IV. Galdieria_PEB_PCB_isomerase: This folder
 contains data for fractionation of G. sulphuraria extracts and for
 assaying isomerization of PEB to PCB using the resulting fraction,
 demonstrating the existence of an isomerase activity in G. sulphuraria.
 The deposited files are: Affinity_SEC_chromatography.txt
 Spectroscopic_Isomerase_Assay.txt HPLC_Isomerase_Assay.txt
 Spectroscopic_Assay_HeatTreated.txt HPLC_Assay_HeatTreated.txt
 Spectroscopic_Assay_NoProtein.txt HPLC_Assay_NoProtein.txt
 Affinity_SEC_chromatography.txt This file contains paired time/absorption
 values at 280 nm (protein absorbance) for two fractionation steps with a
 soluble G. sulphuraria extract: affinity chromatography on Blue Sepharose
 and size exclusion chromatography (SEC) on Superdex 75. Data for bovine
 serum albumin (BSA) on SEC is included as a size control. After SEC, the
 resulting fraction was assayed using spectroscopic and HPLC assays, along
 with controls (no protein and heat-treated protein).
 Spectroscopic_Isomerase_Assay.txt This file contains a series of
 absorption spectra measured at different times after initiation of the
 isomerase reaction. Each spectrum (or timepoint) is one column in the
 file, with the first column providing wavelength information in nm. PEB
 was substrate. HPLC_Isomerase_Assay.txt This file contains data from the
 isomerase assay using reverse-phase HPLC. After workup, reaction products
 were run on a C18 column and monitored using a diode arrray detector set
 to track 560 nm (reporting the starting PEB substrate) and 650 nm
 (reporting the PCB product). The file contains time/A560/A650 values and
 can be compared to standards and controls in other folders.
 Spectroscopic_Assay_HeatTreated.txt This file contains a series of
 absorption spectra measured at different times after initiation of the
 isomerase reaction. The protein fraction was heat-treated in this
 experiment. HPLC_Assay_HeatTreated.txt This file contains data from the
 isomerase assay using reverse-phase HPLC. The protein fraction was
 heat-treated in this experiment. Spectroscopic_Assay_NoProtein.txt This
 file contains a series of absorption spectra measured at different times
 after initiation of the isomerase reaction. The protein fraction was
 replaced with a buffer control in this experiment.
 HPLC_Assay_NoProtein.txt This file contains data from the isomerase assay
 using reverse-phase HPLC. The protein fraction was replaced with a buffer
 control in this experiment. ### Change Log: **Version 1** is the initial
 submission approved 18 April 2025. **Version 2** replaces the earlier
 phylogenetic analysis with two analyses of a more extensive sequence
 alignment including additional sequences that have been recently
 identified. **Version 3** adds re-processed spectroscopic files to remove
 an artifact in the original Savitsky-Golay smoothing procedure and adds
 one additional spectroscopic asset as a control to show that GsPEBB does
 not use biliverdin IX-alpha as a substrate. ## Sharing/access Information
 Links to other publicly accessible locations of the data: none Was data
 derived from another source? no If yes, list source(s): n/a