GUN4 appeared early in cyanobacterial evolution

Photosynthesis relies on chlorophylls, which are synthesized via a common tetrapyrrole trunk pathway also leading to heme, vitamin B12, and other pigmented cofactors. The first committed step for chlorophyll biosynthesis is insertion of magnesium into protoporphyrin IX by magnesium chelatase. Magnesium chelatase is composed of H-, I-, and D-subunits, with the tetrapyrrole substrate binding to the H-subunit. This subunit is rapidly inactivated in the presence of substrate, light, and oxygen, so oxygenic photosynthetic organisms require mechanisms to protect magnesium chelatase from similar loss of function. An additional protein, GUN4, binds to the H-subunit and to tetrapyrroles. GUN4 has been proposed to serve this protective role via its ability to bind linear tetrapyrroles (bilins). In the current work, we probe the origins of bilin binding by GUN4 via comparative phylogenetic analysis and biochemical validation of a conserved bilin-binding motif. Based on our results, we propose that..., Phylogenetic data include the three files used in the workflow for teaching of the maximum likelihood phylogenies. For each analysis, a multiple sequence alignment was constructed using MAFFT v7.450 (command-line settings --genafpair --maxiterate 16 --clustalout –reorder). This file is deposited here in CLUSTAL format (.aln extension). The file was then converted to PHYLIP format with removal of gap-enriched columns (≥5%) using an in-house script. The PHYLIP format file was then used to infer a maximum-likelihood phylogeny in PhyML-3.1 with 100 bootstraps. Command-line settings for nucleic acid alignments were -m GTR -s SPR -a e -c 4 -v e -o tlr -b 100, and command-line settings for protein alignments were -m WAG -d aa -s SPR -a e -c 4 -v e -o tlr -b 100. Statistical robustness was assessed using the transfer bootstrap expectation (TBE) as calculated in booster (available at https://github.com/evolbioinfo/booster). For figure preparation, the Newick file output by Booster was processed ..., All files are reported as flat text files with unix newlines. A README.txt is associated with this submission.