Information on EC 1.3.7.5 - phycocyanobilin:ferredoxin oxidoreductase

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

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EC NUMBER
COMMENTARY hide
1.3.7.5
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RECOMMENDED NAME
GeneOntology No.
phycocyanobilin:ferredoxin oxidoreductase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(3Z)-phycocyanobilin + 4 oxidized ferredoxin = biliverdin IXalpha + 4 reduced ferredoxin
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
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-
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redox reaction
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-
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reduction
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-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
phycocyanobilin biosynthesis
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phycoviolobilin biosynthesis
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Porphyrin and chlorophyll metabolism
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Biosynthesis of secondary metabolites
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SYSTEMATIC NAME
IUBMB Comments
(3Z)-phycocyanobilin:ferredoxin oxidoreductase
Catalyses the four-electron reduction of biliverdin IXalpha (2-electron reduction at both the A and D rings). Reaction proceeds via an isolatable 2-electron intermediate, 181,182-dihydrobiliverdin. Flavodoxins can be used instead of ferredoxin. The direct conversion of biliverdin IXalpha (BV) to (3Z)-phycocyanolbilin (PCB) in the cyanobacteria Synechocystis sp. PCC 6803, Anabaena sp. PCC7120 and Nostoc punctiforme is in contrast to the proposed pathways of PCB biosynthesis in the red alga Cyanidium caldarium, which involves (3Z)-phycoerythrobilin (PEB) as an intermediate [2] and in the green alga Mesotaenium caldariorum, in which PCB is an isolable intermediate.
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CAS REGISTRY NUMBER
COMMENTARY hide
347401-12-1
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
green alga
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Manually annotated by BRENDA team
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Uniprot
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
Prochlorococcus sp. CCMP1378 / MED4
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Manually annotated by BRENDA team
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
metabolism
physiological function
additional information
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SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(3Z)-phycocyanobilin + oxidized ferredoxin
biliverdin IXa + reduced ferredoxin
show the reaction diagram
biliverdin IXa + reduced ferredoxin
(3Z)-phycocyanobilin + oxidized ferredoxin
show the reaction diagram
biliverdin IXa diamide + reduced ferredoxin
? + oxidized ferredoxin
show the reaction diagram
biliverdin IXalpha + reduced ferredoxin
(3E)-phycocyanobilin + oxidized ferredoxin
show the reaction diagram
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Synechococcus ferredoxin
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-
?
biliverdin Ixalpha + reduced ferredoxin
(3Z)-phycocyanobilin + oxidized ferredoxin
show the reaction diagram
biliverdin IXalpha + reduced ferredoxin
phycocyanobilin + oxidized ferredoxin
show the reaction diagram
biliverdin IXalpha 12-monoamide + reduced ferredoxin
phycocyanobilin 12-monoamide + oxidized ferredoxin
show the reaction diagram
biliverdin IXalpha 8-monoamide + reduced ferredoxin
phycocyanobilin 8-monoamide + oxidized ferredoxin
show the reaction diagram
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products are corresponding phycocyanobilin monoamides with a mix of the 3E and 3Z forms
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?
biliverdin XIII + reduced ferredoxin
phycocyanobilin + oxidized ferredoxin
show the reaction diagram
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biliverdin XIII contains two endovinyl groups, so PcyA can convert either (or both) to an ethylidene
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?
biliverdin XIIIalpha + reduced ferredoxin
(3E)-isophytochromobilin + oxidized ferredoxin
show the reaction diagram
biliverdin XIIIalpha + reduced ferredoxin
(3Z)-isophytochromobilin + oxidized ferredoxin
show the reaction diagram
biliverdin XIIIalpha + reduced ferredoxin
? + oxidized ferredoxin
show the reaction diagram
biliverdin XIIIalpha monoamide + reduced ferredoxin
? + oxidized ferredoxin
show the reaction diagram
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mixture of products
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?
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
biliverdin Ixalpha + reduced ferredoxin
(3Z)-phycocyanobilin + oxidized ferredoxin
show the reaction diagram
biliverdin IXalpha + reduced ferredoxin
phycocyanobilin + oxidized ferredoxin
show the reaction diagram
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Ferredoxin
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Diethylpyrocarbonate
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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
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6
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assay with substrate biliverdin diamide
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
16
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assay at
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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PDB
SCOP
CATH
UNIPROT
ORGANISM
Q93TN0
Nostoc sp. (strain PCC 7120 / SAG 25.82 / UTEX 2576);
Synechocystis sp. (strain PCC 6803 / Kazusa);
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
27000
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x * 27000, recombinant enzyme, SDS-PAGE
28700
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SDS-PAGE
28726
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1 * 28726, deduced from nucleotide sequence
28900
SDS-PAGE
30400
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gel filtration, glycerol gradient sedimentation
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 27000, recombinant enzyme, SDS-PAGE
monomer
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1 * 28726, deduced from nucleotide sequence
additional information
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Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
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
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
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PcyA–biliverdin 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
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, 20°C, a few days, X-ray diffraction structure determination and analysis at 1.9 A resolution
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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, 20°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
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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, 21°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
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purified recombinant wild-type PcyA and PcyA-E76Q mutant in complex with 18EtBV or biloverdin IXalpha and biliverdin XIIIalpha, hanging drop vapor diffusion method, 20°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
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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
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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
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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
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-30°C, purified recombinant enzyme, 50 mM HEPES-NaOH, pH 7.5, containing 100 mM NaCl, 6 months, full activity is maintained
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-80°C
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
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
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloned into plasmid containing the sequence coding for HO1 protein
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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
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expression in Arabidopsis thaliana
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expression in Escherichia coli
expression of mutant enzymes in Escherichia coli strain BL21(DE3)
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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)
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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recombinant functional expression of PcyA in Escherichia coli with production of phycocyanobilin, functional co-expression with cyanobacterial heme oxygenase, and the phycocyanin alpha-subunit, CpcA, from Synechocystis sp. PCC 6803 or Synechococcus sp. PCC 7002, and with the phycocyanin alpha-subunit phycocyanobilin lyase, CpcE/CpcF, or the phycoerythrocyanin alpha-subunit phycocyanobilin isomerizing lyase, PecE/PecF, from Noctoc sp. PCC 7120. Production levels of fluorescent pigments and chromophore analysis, overview
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subcloned into pKT210, co-expression with heme oxygenase in Escherichia coli cells
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wild-type and mutants, mutant plasmid constructs expressed in Escherichia coli strain DH5alpha
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C210A
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retains 90% relative to wild type
C86A
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retains 90% activity relative to wild type
D102N
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retains 11% activity relative to wild type
D116N
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retains 11% activity relative to wild type
D217N
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retains 87% relative to wild type
H71A
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retains less than 1% activity relative to wild type
H71D
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retains 28% activity relative to wild type
H71E
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retains 50% activity relative to wild type
H71N
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retains 46% activity relative to wild type
H71Q
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retains 65% activity relative to wild type
H85A
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retains less than 1% activity relative to wild type
H85D
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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
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retains 5% activity relative to wild type
K218E
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retains 20% activity relative to wild type
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
E76Q
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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
H74A
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site-directed mutagenesis, inactive mutant
H74E
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site-directed mutagenesis, the mutant retains reasonable activity
H74Q
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site-directed mutagenesis, the mutant retains reasonable activity
V225D
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site-directed mutagenesis,substrate binding structure, overview
I86D
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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+
additional information
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
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construction of a plasmid containing genes of apo-allophycocyanin alpha-subunit without chromophore and chromophore synthetases HO1, i.e. ferredoxin-dependent heme oxygenase, and PcyA, i.e. phycocyanobilin:ferredoxin oxidoreductase, and expression in Escherichia coli. Holo-allophycocyanin, i.e. allophycocyanin alpha-subunit with chromophore, can be synthesized by autocatalysis in Escherichia coli. Recombinant holo-allophycocyanin alpha-subunit shows the same spectral and fluorescent properties as phycocyanin and serves as a good substitute for native phycocyanin for fluorescent tagging. Recombinant allophycocyanin alpha-subunit can inhibit hydroxyl and peroxyl radicals more strongly than holo-allophycocyanin alpha-subunit and native allophycocyanin
additional information
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Show AA Sequence (334 entries)
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LINKS TO OTHER DATABASES (specific for EC-Number 1.3.7.5)
ExplorEnz
ExPASy
KEGG
MetaCyc
SABIO-RK
NCBI: PubMed, Protein, Nucleotide, Structure, Genome, OMIM
IUBMB Enzyme Nomenclature
PROSITE Database of protein families and domains
InterPro (database of protein families, domains and functional sites)