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Literature summary 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 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.3.7.2 gene pcyA or BRADO1265, CAL75167, phylogenetic analysis and tree, recombinant expression of GST-tagged enzyme in Escherichia coli strain BL21(DE3), coexpression of the chaperone pair GroES/GroEL Bradyrhizobium sp. ORS 278
1.3.7.6 gene pcyX, phylogenetic analysis and tree, recombinant overexpression of synthetic construct of GST-tagged enzyme uncultured marine phage
1.3.7.6 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)

EC Number Crystallization (Comment) Organism
1.3.7.6 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

EC Number Protein Variants Comment Organism
1.3.7.2 D105N site-directed mutagenesis, altered substrate biliverdin binding compared to wild-type, the mutant shows 89% reduced activity compared to wild-type Bradyrhizobium sp. ORS 278
1.3.7.2 E76Q site-directed mutagenesis, the mutant shows 80% reduced activity compared to wild-type Bradyrhizobium sp. ORS 278
1.3.7.2 H88Q site-directed mutagenesis, altered substrate biliverdin binding compared to wild-type, the mutant shows 95% reduced activity compared to wild-type Bradyrhizobium sp. ORS 278
1.3.7.2 I86D site-directed mutagenesis, inactive mutant Bradyrhizobium sp. ORS 278
1.3.7.6 C71A site-directed mutagenesis, mutant shows reduced activity compared to wild-type uncultured actinobacterium
1.3.7.6 D55N site-directed mutagenesis uncultured actinobacterium
1.3.7.6 D86N site-directed mutagenesis, inactive mutant uncultured actinobacterium
1.3.7.6 H200Q site-directed mutagenesis, the PcyX mutant shows a faster turnover compared with to wild-type enzyme uncultured actinobacterium
1.3.7.6 H69Q site-directed mutagenesis, altered substrate biliverdin binding compared to wild-type, the mutant shows highly reduced activity compared to wild-type uncultured actinobacterium
1.3.7.6 M67I site-directed mutagenesis, mutant shows highly reduced activity compared to wild-type uncultured actinobacterium
1.3.7.6 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
1.3.7.6 N198D site-directed mutagenesis, inactive mutant uncultured actinobacterium

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.3.7.2 Fe2+ in cofactor ferredoxin Bradyrhizobium sp. ORS 278
1.3.7.6 Fe2+ in cofactor ferredoxin, [2Fe-2S]-center uncultured actinobacterium
1.3.7.6 Fe2+ in cofactor ferredoxin, [2Fe-2S]-center uncultured marine phage

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.3.7.2 biliverdin IXalpha + reduced ferredoxin Bradyrhizobium sp. ORS 278
-
 15,16-dihydrobiliverdin + oxidized ferredoxin
-
 ?
1.3.7.6 biliverdin IXalpha + 2 reduced ferredoxin uncultured actinobacterium
-
 (3Z)-phycoerythrobilin + 2 oxidized ferredoxin
-
 ?
1.3.7.6 biliverdin IXalpha + 2 reduced ferredoxin uncultured marine phage
-
 (3Z)-phycoerythrobilin + 2 oxidized ferredoxin
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.3.7.2 Bradyrhizobium sp. ORS 278
-
-
-
1.3.7.6 uncultured actinobacterium
-
-
-
1.3.7.6 uncultured marine phage
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.3.7.2 recombinant GST-tagged enzyme from Escherichia coli strain BL21(DE3) by glutathione affinity chromatography, ultrafiltration, and gel filtration Bradyrhizobium sp. ORS 278
1.3.7.6 recombinant GST-tagged enzyme, cleavage fo the tag by PreScission Protease and tag removal uncultured marine phage
1.3.7.6 recombinant GST-tagged enzymes from Escherichia coli strain BL21(DE3) by glutathione affinity chromatography, ultrafiltration, and gel filtration uncultured actinobacterium

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.3.7.2 biliverdin IXalpha + reduced ferredoxin
-
 Bradyrhizobium sp. ORS 278 15,16-dihydrobiliverdin + oxidized ferredoxin
-
 ?
1.3.7.6 biliverdin IXalpha + 2 reduced ferredoxin
-
 uncultured actinobacterium (3Z)-phycoerythrobilin + 2 oxidized ferredoxin
-
 ?
1.3.7.6 biliverdin IXalpha + 2 reduced ferredoxin
-
 uncultured marine phage (3Z)-phycoerythrobilin + 2 oxidized ferredoxin
-
 ?
1.3.7.6 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
-
 ?
1.3.7.6 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
-
 ?
1.3.7.6 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

EC Number Synonyms Comment Organism
1.3.7.2 BRADO1265
-
 Bradyrhizobium sp. ORS 278
1.3.7.2 FDBR
-
 Bradyrhizobium sp. ORS 278
1.3.7.2 ferredoxin-dependent bilin reductase
-
 Bradyrhizobium sp. ORS 278
1.3.7.2 PcyA
-
 Bradyrhizobium sp. ORS 278
1.3.7.6 EBK42635
-
 uncultured marine phage
1.3.7.6 FDBR
-
 uncultured actinobacterium
1.3.7.6 FDBR
-
 uncultured marine phage
1.3.7.6 ferredoxin-dependent bilin reductase
-
 uncultured actinobacterium
1.3.7.6 ferredoxin-dependent bilin reductase
-
 uncultured marine phage
1.3.7.6 PcyX
-
 uncultured actinobacterium
1.3.7.6 PcyX
-
 uncultured marine phage
1.3.7.6 PebS
-
 uncultured actinobacterium
1.3.7.6 PhiPcyX
-
 uncultured marine phage

Cofactor

EC Number Cofactor Comment Organism Structure
1.3.7.2 Ferredoxin
-
 Bradyrhizobium sp. ORS 278
1.3.7.6 Ferredoxin [2Fe-2S]-ferredoxin, the type of ferredoxin and its concentration has a drastic effect on the formation of PEB uncultured actinobacterium
1.3.7.6 Ferredoxin [2Fe-2S]-ferredoxin, the type of ferredoxin and its concentration has a drastic effect on the formation of PEB uncultured marine phage
1.3.7.6 [2Fe-2S]-center
-
 uncultured actinobacterium
1.3.7.6 [2Fe-2S]-center
-
 uncultured marine phage

General Information

EC Number General Information Comment Organism
1.3.7.2 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. But PcyX converts the same substrate to phycoerythrobilin, resembling the reaction catalysed by cyanophage PebS. But 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 Bradyrhizobium sp. ORS 278
1.3.7.2 additional information a conserved aspartate-histidine pair is critical for activity of PcyA. PcyA contains the catalytic Asp-His-Glu triad. 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. Glu76 of PcyA is central for exovinyl-reduction Bradyrhizobium sp. ORS 278
1.3.7.6 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
1.3.7.6 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
1.3.7.6 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
1.3.7.6 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