Information on EC 1.3.7.5 - phycocyanobilin:ferredoxin oxidoreductase

biliverdin IXalpha + reduced ferredoxin

(3E)-phycocyanobilin + oxidized ferredoxin

-

Synechococcus ferredoxin

655796

-

?

biliverdin Ixalpha + reduced ferredoxin

(3Z)-phycocyanobilin + oxidized ferredoxin

15 entries

biliverdin IXalpha + reduced ferredoxin

phycocyanobilin + oxidized ferredoxin

5 entries

biliverdin IXalpha 12-monoamide + reduced ferredoxin

phycocyanobilin 12-monoamide + oxidized ferredoxin

biliverdin IXalpha 8-monoamide + reduced ferredoxin

phycocyanobilin 8-monoamide + oxidized ferredoxin

-

products are corresponding phycocyanobilin monoamides with a mix of the 3E and 3Z forms

-

?

biliverdin XIII + reduced ferredoxin

phycocyanobilin + oxidized ferredoxin

-

biliverdin XIII contains two endovinyl groups, so PcyA can convert either (or both) to an ethylidene

-

?

biliverdin XIIIalpha + reduced ferredoxin

(3E)-isophytochromobilin + oxidized ferredoxin

biliverdin XIIIalpha + reduced ferredoxin

(3Z)-isophytochromobilin + oxidized ferredoxin

biliverdin XIIIalpha + reduced ferredoxin

? + oxidized ferredoxin

biliverdin XIIIalpha monoamide + reduced ferredoxin

? + oxidized ferredoxin

-

mixture of products

-

?

additional information

?

-

biliverdin Ixalpha + reduced ferredoxin

(3Z)-phycocyanobilin + oxidized ferredoxin

9 entries

biliverdin IXalpha + reduced ferredoxin

phycocyanobilin + oxidized ferredoxin

Ferredoxin

5 entries

-

diethylpyrocarbonate

-

reduces PcyA activity to less than 20% of mock-treated enzyme within 10 min, diethylpyrocarbonate-inactivation can be prevented by the presence of biliverdin IXa substrate

6

-

assay with substrate biliverdin diamide

7.5

-

assay at

8.5

3 entries

16

-

assay at

25

-

assay at

3 entries

Anabaena sp. PCC 7120

-

674771, 724287

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Mesotaenium caldariorum

green alga

-

-

Uniprot

-

-

-

-

Prochlorococcus sp.

-

-

Prochlorococcus sp. CCMP1378 / MED4

-

-

Synechococcus sp.

-

437673, 673636, 724317, 725252

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Synechococcus sp. 7002

-

724317

-

Synechococcus sp. W8020

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673636

-

Synechococcus sp. WH8102

-

-

9 entries

-

742318, 742524

UniProt

-

-

chloroplast

Mesotaenium caldariorum

-

soluble

evolution

malfunction

metabolism

physiological function

4 entries

additional information

Synechocystis sp. PCC 6803 substr. Kazusa

4 entries

27000

-

x * 27000, recombinant enzyme, SDS-PAGE

28700

-

SDS-PAGE

28726

-

1 * 28726, deduced from nucleotide sequence

28900

SDS-PAGE

30400

-

gel filtration, glycerol gradient sedimentation

656197

?

-

x * 27000, recombinant enzyme, SDS-PAGE

monomer

-

1 * 28726, deduced from nucleotide sequence

656197

additional information

hanging drop vapour diffusion method with 18% PEG-8000, 0.025 M MES-NaOH at pH 6.5, 0.05 M Ca(OAc)2 at pH 6.5, and 5% dioxane (or 5% ethanol)

high-field electron paramagnetic resonance spectroscopy of frozen solutions and single crystals of the one-electron reduced protein-substrate complex of mutant D102N. Spectra reveal a biliverdin radical with a very narrow g tensor. This g tensor is consistent with a biliverdin radical where the carbonyl oxygen atoms on both the A and the D pyrrole rings are protonated

698489

high-field electron paramagnetic resonance spectroscopy of frozen solutions and single crystals of the one-electron reduced protein-substrate complex of mutant D105N. Spectra reveal a biliverdin radical with a very narrow g tensor with principal values 2.00359(5), 2.00341(5), and 2.00218(5). This g tensor is consistent with a biliverdin radical where the carbonyl oxygen atoms on both the A and the D pyrrole rings are protonated

698489

PcyAâbiliverdin IXalpha complex by the hanging-drop vapor-diffusion method, PcyA is folded in a three-layer alpha/beta/alpha sandwich structure, in which biliverdin IXalpha in a cyclic conformation is positioned between the beta-sheet and C-terminal alpha-helices

676847

purified recombinant enzyme mutant V225D in complex with substrates biliverdin IXalpha or biliverdin XIIIalpha, mixing of 0.0009 ml of protein solution, containing 11.5 mg/ml protein and biliverdin, with 0.0009 ml of reservoir solution containing 0.8-1.0 M NaH2PO4, 1.0-1.2 M K2HPO4, and 100 mM sodium acetate, pH 4.0, 20Â°C, a few days, X-ray diffraction structure determination and analysis at 1.9 A resolution

711117

purified recombinant mutant enzymes, hanging drop vapour diffusion method, dithionite-treated reduced D105N PcyA crystals from 1.45-1.8 M ammonium sulfate, 0.15-0.4 M NaCl, and 0.1 M HEPES, pH 7.0, dithionite-treated reduced H88Q PcyA crystals from 1.7-2.2 M ammonium sulfate, 0.26-0.32 M NaCl, and 0.1 M sodium cacodylate, pH 7.0, 20Â°C in the dark, cryoprotectant solution is consisting of 30% v/v ethylene glycol in mother liquor, X-ray diffraction structure determination and analysis at 1.5 A resolution

-

724288

purified recombinant wild-type and mutant enzymes, hanging drop vapor diffusion method, mixing of 0.002 ml of protein solution containing 15 mg/mL protein and 0.67 mM biliverdin IXalpha, with 0.002 ml of reservoir solution containing 1-1.25 M sodium citrate, 0.1-0.4 M NaCl, and 0.1 M Tris HCl, pH 7.0, 21Â°C, 1-2 weeks. Crystal trials are set up under green safelight and stored in the dark, X-ray diffraction structure determination and analysis at 1.18-1.49 A resolution

-

purified recombinant wild-type PcyA and PcyA-E76Q mutant in complex with 18EtBV or biloverdin IXalpha and biliverdin XIIIalpha, hanging drop vapor diffusion method, 20Â°C, method optimization, protein solution containing wild-type PcyA or mutant E76Q and bilin is mixed with reservoir solutions containing 0.85 M sodium citrate, 0.1 M sodium cacodylate, pH 7.0, for the PcyA-18EtBV complex and 2.0 M ammonium sulfate, 0.2 M NaCl, and 0.1M sodium cacodylate, pH7.0, for the PcyA-BV13 complex, for the mutant a reservoir solution containing 1.7 M ammonium sulfate, 2% PEG 400, and 0.1 M HEPES, pH 7.0, is used, X-ray diffraction structure determination and analysis at 1.04-1.48 A resolution

712411

substrate-free form of PcyA by the hanging-drop vapor-diffusion method, at 2.5 A resolution, the side-chain of Asp105 is located at a site that would be underneath the biliverdin IXa A-ring in the PcyA-biliverdin IXa complex and hydrogen-bonded with His88, biliverdin IXa may be protonated by a mechanism involving conformational changes of these two residues before reduction

-

673657

quantum mechanical approaches show that the propensity of biliverdin to bind PcyA is dominated by electrostatic interactions, especially related to residues Arg149 and Lys221, while H-bonds are formed with His88 and Ser114. The antioxidant activity is dependent on the intramolecular noncovalent bond interactions. The surrounding residues increase the antioxidant character of biliverdin by 2 eV

742318

structure of the I86D-BVH+ complex and the protonation states of residues Asp105 and Glu76 in PcyA. Asp105 adopts a fixed conformation in the I86D mutant, but has dual conformations in wild-type PcyA which reflects the protonation states of biliverdin

742524

C210A

-

retains 90% relative to wild type

C86A

-

retains 90% activity relative to wild type

D102N

-

retains 11% activity relative to wild type

D116N

-

retains 11% activity relative to wild type

D217N

-

retains 87% relative to wild type

H71A

-

retains less than 1% activity relative to wild type

H71D

-

retains 28% activity relative to wild type

H71E

-

retains 50% activity relative to wild type

H71N

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retains 46% activity relative to wild type

H71Q

-

retains 65% activity relative to wild type

H85A

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retains less than 1% activity relative to wild type

H85D

-

retains less than 1% activity relative to wild type

H85E

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retains 5% activity relative to wild type

H85N

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retains less than 1% activity relative to wild type

H85Q

-

retains 5% activity relative to wild type

K218E

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retains 20% activity relative to wild type

C86A

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retains 90% activity relative to wild type

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D217N

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retains 87% relative to wild type

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H71A

-

retains less than 1% activity relative to wild type

-

H71N

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retains 46% activity relative to wild type

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H71Q

-

retains 65% activity relative to wild type

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D102N

E73Q

exhibits 20% of wild type activity

H71Q

exhibits 65% of wild type activity

H85Q

exhibits 5% of wild type activity

K218E

exhibits 20% of wild type activity

D105N

E76Q

site-directed mutagenesis, substrate-binding structure compared to the wild-type enzyme. Overall folds and the binding sites of the U-shaped substrates of all three complexes are similar with wild-type PcyABV, the orientation of the Glu76 side chain, which is in close contact with the exo-vinyl group in PcyA-biliverdin IXalpha, is rotated away from the bilin D-ring. The local structures around the A-rings in the three complexes, which all retain the ability to reduce the A-ring of their bound pigments, are nearly identical with that of wild-type PcyA-biliverdin IXalpha

712411

H74A

-

site-directed mutagenesis, inactive mutant

H74E

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site-directed mutagenesis, the mutant retains reasonable activity

H74Q

-

site-directed mutagenesis, the mutant retains reasonable activity

H88Q

V225D

site-directed mutagenesis,substrate binding structure, overview

711117

I86D

biliverdin bound to the I86D mutant is fully protonated (BVH+) and can accept an electron, but I86D is unable to donate protons for the reduction. Compared to the wild-type PcyA, the I86D mutant stabilizes BVH+

742524

additional information

3 entries

-30Â°C, purified recombinant enzyme, 50 mM HEPES-NaOH, pH 7.5, containing 100 mM NaCl, 6 months, full activity is maintained

-

-80Â°C

-

-

by gel filtration

recombinant enzyme from Escherichia coli strain BL21(DE3) by anion-exchange chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, and gel filtration

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recombinant GST-tagged PcyA from Escherichia coli by glutathione affinity chromatography

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recombinant mutant enzymes from Escherichia coli strain BL21(DE3) by cyanide affinity chromatography

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recombinant PcyA

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recombinant wild-type and mutant enzymes from Escherichia coli strain Bl21(DE3)

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Superdex G-200 gel filtration

wild-type and mutants, histidine mutants purified to homogeneity

-

-

6 entries

cloned into plasmid containing the sequence coding for HO1 protein

-

437764

coexpression in Escherichia coli BL21(DE3) with cpcB(C155I) or pecB(C155I) and with cpeS, ho1 and pcyA, resulting in biosynthesis of the respective beta-subunits singly chromophorylated at cysteine-beta84

-

674771

expression in Arabidopsis thaliana

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657164

expression in Escherichia coli

expression of mutant enzymes in Escherichia coli strain BL21(DE3)

-

724288

gene pycA, expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)

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into vector pET-21a and expressed in Escherichia coli C41(DE3)

676847

PcyA expression in Escherichia coli strain BL21(DE3)

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recombinant expression of GST-tagged PcyA in Escherichia coli

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