Literature summary for 1.3.7.6 extracted from

Ledermann, B.; Schwan, M.; Sommerkamp, J.A.; Hofmann, E.; Beja, O.; Frankenberg-Dinkel, N.
Evolution and molecular mechanism of four-electron reducing ferredoxin-dependent bilin reductases from oceanic phages (2018), FEBS J., 285, 339-356 .

Cloned(Commentary)

Cloned (Comment)	Organism
gene pcyX, phylogenetic analysis and tree, recombinant overexpression of synthetic construct of GST-tagged enzyme	uncultured marine phage
gene pcyX, phylogenetic analysis and tree, recombinant overexpression of synthetic constructs with the original sequences of the genes derived from metagenomic data (termed PcyX-EBQ, PcyX-ECK and PcyX-actino) in Escherichia coli. PcyX-EBQ and PcyX-ECK are functional FDBRs catalysing the reduction of BV via DHBV to PEB, recombinant expression of GST-tagged enzymes in Escherichia coli strain BL21(DE3)	uncultured actinobacterium

Cloned (Comment)

Organism

gene pcyX, phylogenetic analysis and tree, recombinant overexpression of synthetic construct of GST-tagged enzyme

uncultured marine phage

gene pcyX, phylogenetic analysis and tree, recombinant overexpression of synthetic constructs with the original sequences of the genes derived from metagenomic data (termed PcyX-EBQ, PcyX-ECK and PcyX-actino) in Escherichia coli. PcyX-EBQ and PcyX-ECK are functional FDBRs catalysing the reduction of BV via DHBV to PEB, recombinant expression of GST-tagged enzymes in Escherichia coli strain BL21(DE3)

uncultured actinobacterium

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant substrate free form of PhiPcyX, sitting drop vapour diffusion method, mixing of 100 nl of 10-16.5 mg/ml protein in 20 mM TES-KOH pH 7.5, and 20 mM KCl, with 100 nl of reservoir solution containing 0.1 M Tris-HCl, pH 8.5, 0.2 M trimethylamine N-oxide (TMAO), and 20% w/v PEG MME 2000, at 4 °C. Final crystals used for structure determination grow at 4 °C via hanging drop vapour diffusion with 0.001 ml of 10 mg/ml protein in 20 mM TES-KOH pH 7.5, and 20 mM KCl mixed with 0.001 ml of 0.1 M Tris-HCl, pH 8.5, 0.05 M, TMAO, and 15% w/v PEG MME 2000 as reservoir solution, X-ray diffraction structure determination and analysis at 2.2 A resolution	uncultured marine phage

Crystallization (Comment)

Organism

purified recombinant substrate free form of PhiPcyX, sitting drop vapour diffusion method, mixing of 100 nl of 10-16.5 mg/ml protein in 20 mM TES-KOH pH 7.5, and 20 mM KCl, with 100 nl of reservoir solution containing 0.1 M Tris-HCl, pH 8.5, 0.2 M trimethylamine N-oxide (TMAO), and 20% w/v PEG MME 2000, at 4 °C. Final crystals used for structure determination grow at 4 °C via hanging drop vapour diffusion with 0.001 ml of 10 mg/ml protein in 20 mM TES-KOH pH 7.5, and 20 mM KCl mixed with 0.001 ml of 0.1 M Tris-HCl, pH 8.5, 0.05 M, TMAO, and 15% w/v PEG MME 2000 as reservoir solution, X-ray diffraction structure determination and analysis at 2.2 A resolution

uncultured marine phage

Protein Variants

Protein Variants	Comment	Organism
C71A	site-directed mutagenesis, mutant shows reduced activity compared to wild-type	uncultured actinobacterium
D55N	site-directed mutagenesis	uncultured actinobacterium
D86N	site-directed mutagenesis, inactive mutant	uncultured actinobacterium
H200Q	site-directed mutagenesis, the PcyX mutant shows a faster turnover compared with to wild-type enzyme	uncultured actinobacterium
H69Q	site-directed mutagenesis, altered substrate biliverdin binding compared to wild-type, the mutant shows highly reduced activity compared to wild-type	uncultured actinobacterium
M67I	site-directed mutagenesis, mutant shows highly reduced activity compared to wild-type	uncultured actinobacterium
additional information	a conserved aspartate-histidine pair is critical for activity. The same residues are part of a catalytic Asp-His-Glu triad in PcyA (EC 1.3.7.2), including an additional Glu. While this Glu residue is replaced by Asp in PcyX, it is not involved in catalysis. Substitution back to a Glu fails to convert PcyX to a PcyA	uncultured actinobacterium
N198D	site-directed mutagenesis, inactive mutant	uncultured actinobacterium

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Fe2+	in cofactor ferredoxin, [2Fe-2S]-center	uncultured actinobacterium
Fe2+	in cofactor ferredoxin, [2Fe-2S]-center	uncultured marine phage

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
biliverdin IXalpha + 2 reduced ferredoxin	uncultured actinobacterium	-	(3Z)-phycoerythrobilin + 2 oxidized ferredoxin	-	?
biliverdin IXalpha + 2 reduced ferredoxin	uncultured marine phage	-	(3Z)-phycoerythrobilin + 2 oxidized ferredoxin	-	?

Organism

Organism	UniProt	Comment	Textmining
uncultured actinobacterium	-	-	-
uncultured marine phage	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant GST-tagged enzyme, cleavage fo the tag by PreScission Protease and tag removal	uncultured marine phage
recombinant GST-tagged enzymes from Escherichia coli strain BL21(DE3) by glutathione affinity chromatography, ultrafiltration, and gel filtration	uncultured actinobacterium

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
biliverdin IXalpha + 2 reduced ferredoxin	-	uncultured actinobacterium	(3Z)-phycoerythrobilin + 2 oxidized ferredoxin	-	?
biliverdin IXalpha + 2 reduced ferredoxin	-	uncultured marine phage	(3Z)-phycoerythrobilin + 2 oxidized ferredoxin	-	?
biliverdin IXalpha + 2 reduced ferredoxin	importance of the correct electron donor for the activity of the PcyX-like FDBRs	uncultured actinobacterium	(3Z)-phycoerythrobilin + 2 oxidized ferredoxin	-	?
biliverdin IXalpha + 2 reduced ferredoxin	importance of the correct electron donor for the activity of the PcyX-like FDBRs	uncultured marine phage	(3Z)-phycoerythrobilin + 2 oxidized ferredoxin	-	?
additional information	recombinant metagenomically derived enzymes PcyX-EBQ and PcyX-ECK are functional FDBRs catalysing the reduction of BV via 5,16-dihydrobiliverdin (DHBV) to phycoerythrobilin (PEB). The rate determining step in the PcyX-catalysed reaction is the conversion of the intermediate DHBV to the final product PEB. Enzyme PcyX_ECK forms 3(E/Z)-PPhiB as side products	uncultured actinobacterium	?	-	-

Synonyms

Synonyms	Comment	Organism
EBK42635	-	uncultured marine phage
FDBR	-	uncultured actinobacterium
FDBR	-	uncultured marine phage
ferredoxin-dependent bilin reductase	-	uncultured actinobacterium
ferredoxin-dependent bilin reductase	-	uncultured marine phage
PcyX	-	uncultured actinobacterium
PcyX	-	uncultured marine phage
PebS	-	uncultured actinobacterium
PhiPcyX	-	uncultured marine phage

Cofactor

Cofactor	Comment	Organism
Ferredoxin	[2Fe-2S]-ferredoxin, the type of ferredoxin and its concentration has a drastic effect on the formation of PEB	uncultured actinobacterium
Ferredoxin	[2Fe-2S]-ferredoxin, the type of ferredoxin and its concentration has a drastic effect on the formation of PEB	uncultured marine phage
[2Fe-2S]-center	-	uncultured actinobacterium
[2Fe-2S]-center	-	uncultured marine phage

General Information

General Information	Comment	Organism
evolution	ferredoxin-dependent bilin reductases (FDBRs) are a class of enzymes reducing the heme metabolite biliverdin IXa (BV) to form open-chain tetrapyrroles used for light-perception and light-harvesting in photosynthetic organisms. Evolution and molecular mechanism of four-electron reducing ferredoxin-dependent bilin reductases from oceanic phages, overview. PcyX is originally identified from metagenomics data derived from phage. PcyA (EC 1.3.7.2) is the closest relative catalysing the reduction of biliverdin (BV) to phycocyanobilin (PEB). But PcyX converts the same substrate to phycoerythrobilin, resembling the reaction catalysed by cyanophage PebS	uncultured marine phage
evolution	ferredoxin-dependent bilin reductases (FDBRs) are a class of enzymes reducing the heme metabolite biliverdin IXa (BV) to form open-chain tetrapyrroles used for light-perception and light-harvesting in photosynthetic organisms. Evolution and molecular mechanism of four-electron reducing ferredoxin-dependent bilin reductases from oceanic phages, overview. PcyX is originally identified from metagenomics data derived from phage. PcyA (EC 1.3.7.2) is the closest relative catalysing the reduction of biliverdin (BV) to phycocyanobilin (PEB). But PcyX converts the same substrate to phycoerythrobilin, resembling the reaction catalysed by cyanophage PebS. The change in regiospecificity from PcyA to PcyX is not only caused by individual catalytic amino acid residues. Rather the combination of the architecture of the active site with the positioning of the substrate triggers specific proton transfer yielding the individual phycobilin products. Phylogenetic analysis and tree suggest PcyX sequences forming a distinct clade	uncultured actinobacterium
additional information	a conserved aspartate-histidine pair is critical for activity of PcyX. Strutcure comparisons of FDBRs, PcyA and PcyX, overview. Ile86 in PcyA is replaced by Met67, whereas Val90 is substituted by Cys71 in PcyX. Both are strictly conserved in all PcyX sequences, but small hydrophobic residues in all other FDBR. Due to the disorder on the distal side of the binding pocket, residues corresponding to Asn219 in PcyA or to Asp206 in PebS are not visible in our PcyX structure. Modelling of the substrate into the active site. His69 and Asp86 are catalytic important residues, the Asp86/His69 pair of PcyX is critical for catalysis. Also Met67 is crucial for the activity of PcyX, Asn198 is essential for the correct binding of the substrate	uncultured actinobacterium
additional information	the X-ray structure of PhiPcyX (EBK42635) shows the typical alpha/beta/alpha-sandwich fold, with a central antiparallel beta-sheet, flanked by alpha-helices, as described before for other FDBRs. Analysis of the substrate binding pocket structure of PcyX, structure comparisons, overview	uncultured marine phage