## 3 Results and Discussion  
### Identification of a Z-ISO Homology in Synechocystis  
To our knowledge, at the time of writing, the experimental verification of a cyanobacterial Z-ISO has only been reported for the enzyme from the filamentous, non-N\({}_{2}\)-fixing cyanobacterium _Arthrospira platensis_ NIES-39, where activity was demonstrated by heterologous production in an _E. coli_ strain that accumulated 9,15,9'-tri-_cis-_c-carotene in darkness [11]. The same authors also identified Z-ISO homologs in other _Arthrospira_ species, the model cyanobacteria _Synechocystis_ and _Synechococcus_ sp. PCC 7002 (hereafter _Synechococcus_ 7002), and _Prochlorococcus marinus_ str. SS355 [11]. [PERSON] et al [41] had earlier identified the same 238 amino acid protein (SYNPCC7002_A1197) as the likely \(\zeta\)-carotene isomerase in _Synechococcus_ 7002, and the locus predicted to encode Z-ISO in the original genome-sequenced _Synechocystis_ Kazusa substrain [42] is slr1599 [43].  
In the UniProt ([[https://www.uniprot.org/uniprot/P72984](https://www.uniprot.org/uniprot/P72984)]([https://www.uniprot.org/uniprot/P72984](https://www.uniprot.org/uniprot/P72984))), KEGG ([[https://www.genome.jp/dbget-bin/www_bget?syn:slr1599](https://www.genome.jp/dbget-bin/www_bget?syn:slr1599)]([https://www.genome.jp/dbget-bin/www_bget?syn:slr1599](https://www.genome.jp/dbget-bin/www_bget?syn:slr1599))), and CYORF ([[http://cyano.genome.jp/cgi-bin/cyorf_view.cgi?ORG=syn&ACCESSION=slr1599](http://cyano.genome.jp/cgi-bin/cyorf_view.cgi?ORG=syn&ACCESSION=slr1599)]([http://cyano.genome.jp/cgi-bin/cyorf_view.cgi?ORG=syn&ACCESSION=slr1599](http://cyano.genome.jp/cgi-bin/cyorf_view.cgi?ORG=syn&ACCESSION=slr1599))) databases (accessed on 1 June 2022), slr1599 is predicted to encode a 188 amino acid protein, \(\sim\)50 residues smaller than other cyanobacterial Z-ISO homologs [11; 41]. This is due to an apparent misidentification of the start codon; the translation of the DNA immediately upstream of slr1599 in combination with analysis of other _Synechocystis_ substrains (where the protein is predicted to range from 229-247 amino acids) and an alignment with known Z-ISO enzymes from other organisms indicates that the gene likely starts 162 bp upstream of the annotated GTG start codon, encoding a 242 amino acid protein (Figure 2A). Like other Z-ISO enzymes, Slr1599 is annotated as an NnrU domain-containing protein [8], and similar to other cyanobacterial Z-ISO homologs it is predicted to contain six TMHs (Figure 2B), compared to seven for the plant enzymes [5; 12].  
Slr1599 that is'missing' in some databases is indicated in red font. Note that the eukaryotic enzymes (Eg, At and Zm) are N-terminally truncated and, where appropriate, the sequence corresponding to their chloroplast transit peptides is shown in blue. The position of predicted TMHs in Slr1599 (six; note that helices 4 and 5 are consecutive) and the _Z. mays_ (seven) enzyme are highlighted in green and yellow, respectively. Residue numbers are shown on the left and the degree of conservation is shown below the sequence alignment, where \"* indicates the residue is identical, '.' = a conservative substitution, and '.' = a semi-conservative substitution. (**B**) Schematic showing the predicted number of TMHs for Slr1599 in comparison to the _Z. mays_ Z-ISO. One fewer TMH in the _Synechocystis_ enzyme means that the N-terminus is predicted to be localized to the thylakoid lumen, in contrast to the plant Z-ISO, where the extra TMH leads to a predicted location for the N-terminus on the stromal side of the membrane. Four residues previously implicated as having potential structural/functional roles in the maize Z-ISO enzyme [5; 6], and investigated here in Slr1599 (Section 3.4), are indicated on the schematics.