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Literature summary extracted from

  • Sugishima, M.; Okamoto, Y.; Noguchi, M.; Kohchi, T.; Tamiaki, H.; Fukuyama, K.
    Crystal structures of the substrate-bound forms of red chlorophyll catabolite reductase: implications for site-specific and stereospecific reaction (2010), J. Mol. Biol., 402, 879-891.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.3.1.8 expression of GST-tagged wild-type and F218V mutant RCCR lacking the chloroplast transit peptide, Met1 to Gln39 Arabidopsis thaliana
1.3.7.12 expression of GST-tagged wild-type and F218V mutant RCCR lacking the chloroplast transit peptide, Met1 to Gln39 Arabidopsis thaliana
1.3.7.12 expression of wild-type and mutant enzymes lacking the chloroplast transit peptide (Met1 to Gln39) and N-termial residues 40-48 as GST-tagged enzymes Arabidopsis thaliana

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.3.1.8 purified recombinant red chlorophyll catabolite-bound RCCRDELTA49, and red chlorophyll catabolite-bound or substrate-free F218V RCCRDELTA49, sitting drop vapor diffusion method, 20°C, protein solution is mixed with an equal volume of reservoir solution and equilibrated against reservoir solution containing 30% w/v PEG 2000 monomethyl ether, 0.1 M ammonium acetate, 3% v/v dioxane, and 0.1 M 4-morpholineethanesulfonic acid–NaOH, pH 6.5, 1 day, X-ray diffraction structure determination and analysis at 2.0-2.6 A resolution Arabidopsis thaliana
1.3.7.12 purified recombinant RCC-bound wild-type enzyme AtRCCRDELTA49, RCC-bound and substrate-free enzyme mutant F218V AtRCCRDELTA49, sitting-drop vapor diffusion method, mixing of 10 mg/ml substrate-free and substrate-bound proteins in 20 mM Tris-HCl, pH 7.4, and 200 mM NaCl, with an equal volume of reservoir solution containing 30% or 35%, respectively, w/v PEG 2000 monomethyl ether, 0.1 M ammonium acetate, 3% v/v dioxane, and 0.1 M 4-morpholineethanesulfonic acid-NaOH, pH 6.5, equilibration gainst reservoir solution, 20°C, 1 day, X-ray diffraction structure determination and analysis at 2.0-2.6 A resolution Arabidopsis thaliana
1.3.7.12 purified recombinant red chlorophyll catabolite-bound RCCRDELTA49, and red chlorophyll catabolite-bound or substrate-free F218V RCCRDELTA49, sitting drop vapor diffusion method, 20°C, protein solution is mixed with an equal volume of reservoir solution and equilibrated against reservoir solution containing 30% w/v PEG 2000 monomethyl ether, 0.1 M ammonium acetate, 3% v/v dioxane, and 0.1 M 4-morpholineethanesulfonic acid-NaOH, pH 6.5, 1 day, X-ray diffraction structure determination and analysis at 2.0-2.6 A resolution Arabidopsis thaliana

Protein Variants

EC Number Protein Variants Comment Organism
1.3.1.8 F218V a mutant protein that produces the stereoisomer of primary fluorescent chlorophyll catabolites at the C1 position, the F218V mutation changes the stereospecificity in RCCR. Construction of wild-type and F218V mutant RCCR lacking the chloroplast transit peptide, Met1 to Gln39, i.e. RCCRDELTA49 Arabidopsis thaliana
1.3.7.12 F218V a mutant protein that produces the stereoisomer of primary fluorescent chlorophyll catabolites at the C1 position, the F218V mutation changes the stereospecificity in RCCR. Construction of wild-type and F218V mutant RCCR lacking the chloroplast transit peptide, Met1 to Gln39, i.e. RCCRDELTA49 Arabidopsis thaliana
1.3.7.12 F218V the AtRCCR mutant protein produces the stereoisomer of primary fluorescent chlorophyll catabolites at the C1 position. The RCC in F218V AtRCCR rotates slightly compared with that in wild-type to fill in the space generated by the substitution of Phe218 with valine. Concomitantly, the two carboxy groups of Glu154 and Asp291 move slightly away from the C20/C1 double bond. Analysis of substrate-free and substrate-bound enzyme crystal structures, and comparison to wild-type structures, overview Arabidopsis thaliana
1.3.7.12 additional information construction of N-terminally truncated variants of wild-type enzyme and enzyme mutant F218V, i.e. AtRCCRDELTA49 and F218V AtRCCRDELTA49 Arabidopsis thaliana

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
1.3.7.12 chloroplast stroma Arabidopsis thaliana 9507
-

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.3.1.8 primary fluorescent chlorophyll catabolite + NADP+ Arabidopsis thaliana stereospecific reaction. RCCR catalyzes the ferredoxin-dependent and site-specific reduction of the C20/C1 double bond of red chlorophyll catabolite, RCC, the catabolic intermediate produced in chlorophyll degradation red chlorophyll catabolite + NADPH + H+
-
 r
1.3.7.12 primary fluorescent chlorophyll catabolite + NADP+ Arabidopsis thaliana stereospecific reaction. RCCR catalyzes the ferredoxin-dependent and site-specific reduction of the C20/C1 double bond of red chlorophyll catabolite, RCC, the catabolic intermediate produced in chlorophyll degradation red chlorophyll catabolite + NADPH + H+
-
 r
1.3.7.12 red chlorophyll catabolite + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ Arabidopsis thaliana
-
 primary fluorescent chlorophyll catabolite + 2 oxidized ferredoxin [iron-sulfur] cluster
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.3.1.8 Arabidopsis thaliana
-
-
-
1.3.7.12 Arabidopsis thaliana Q8LDU4
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.3.1.8 recombinant GST-tagged wild-type and F218V mutant RCCR lacking the chloroplast transit peptide, Met1 to Gln39, i.e. RCCRDELTA49 Arabidopsis thaliana
1.3.7.12 recombinant GST-tagged wild-type and F218V mutant RCCR lacking the chloroplast transit peptide, Met1 to Gln39, i.e. RCCRDELTA49 Arabidopsis thaliana
1.3.7.12 recombinant truncated wild-type and mutant enzymes AtRCCRDELTA49 and F218V AtRCCRDELTA49 Arabidopsis thaliana

Source Tissue

EC Number Source Tissue Comment Organism Textmining
1.3.7.12 leaf
-
 Arabidopsis thaliana
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.3.1.8 primary fluorescent chlorophyll catabolite + NADP+ stereospecific reaction Arabidopsis thaliana red chlorophyll catabolite + NADPH + H+ red chlorophyll catabolite, RCC, binding does not drastically change the RCCR structure, binding structure and mechanism analysis, overview. Comparison of the RCC-binding pockets of wild-type RCCRDELTA49 and F218V RCCRDELTA49, overview r
1.3.1.8 primary fluorescent chlorophyll catabolite + NADP+ stereospecific reaction. RCCR catalyzes the ferredoxin-dependent and site-specific reduction of the C20/C1 double bond of red chlorophyll catabolite, RCC, the catabolic intermediate produced in chlorophyll degradation Arabidopsis thaliana red chlorophyll catabolite + NADPH + H+
-
 r
1.3.7.12 primary fluorescent chlorophyll catabolite + NADP+ stereospecific reaction Arabidopsis thaliana red chlorophyll catabolite + NADPH + H+ red chlorophyll catabolite, RCC, binding does not drastically change the RCCR structure, binding structure and mechanism analysis, overview. Comparison of the RCC-binding pockets of wild-type RCCRDELTA49 and F218V RCCRDELTA49, overview r
1.3.7.12 primary fluorescent chlorophyll catabolite + NADP+ stereospecific reaction. RCCR catalyzes the ferredoxin-dependent and site-specific reduction of the C20/C1 double bond of red chlorophyll catabolite, RCC, the catabolic intermediate produced in chlorophyll degradation Arabidopsis thaliana red chlorophyll catabolite + NADPH + H+
-
 r
1.3.7.12 red chlorophyll catabolite + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+
-
 Arabidopsis thaliana primary fluorescent chlorophyll catabolite + 2 oxidized ferredoxin [iron-sulfur] cluster
-
 ?
1.3.7.12 red chlorophyll catabolite + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ loose substrate binding allows for conformation change during the reaction, stereospcific reaction, mechanism, overview Arabidopsis thaliana primary fluorescent chlorophyll catabolite + 2 oxidized ferredoxin [iron-sulfur] cluster formation of a stereospecific product, overview ?

Subunits

EC Number Subunits Comment Organism
1.3.1.8 More RCCR folds into a characteristic alpha/beta/alpha sandwich, similar to that observed in the ferredoxin-dependent bilin reductase family, structure comparisosns, overview Arabidopsis thaliana
1.3.7.12 homodimer RCCR folds into a characteristic alphabetaalpha sandwich Arabidopsis thaliana
1.3.7.12 More RCCR folds into a characteristic alpha/beta/alpha sandwich, similar to that observed in the ferredoxin-dependent bilin reductase family, structure comparisosns, overview Arabidopsis thaliana

Synonyms

EC Number Synonyms Comment Organism
1.3.1.8 RCCR
-
 Arabidopsis thaliana
1.3.7.12 RCCR
-
 Arabidopsis thaliana
1.3.7.12 red chlorophyll catabolite reductase
-
 Arabidopsis thaliana

Cofactor

EC Number Cofactor Comment Organism Structure
1.3.1.8 NADP+
-
 Arabidopsis thaliana
1.3.1.8 NADPH
-
 Arabidopsis thaliana
1.3.7.12 Ferredoxin dependent on Arabidopsis thaliana
1.3.7.12 NADP+
-
 Arabidopsis thaliana
1.3.7.12 NADPH
-
 Arabidopsis thaliana

General Information

EC Number General Information Comment Organism
1.3.7.12 evolution the enzyme belongs to the ferredoxin-dependent bilin reductase (FDBR) family. RCC is bound to the pocket between the beta-sheet and the C-terminal alpha-helices, as seen in substrate-bound FDBRs, but RCC binding to RCCR is much looser than substrate binding to FDBRs Arabidopsis thaliana
1.3.7.12 additional information the substrate red chlorophyll catabolite is bound to the pocket between the beta-sheet and the C-terminal alpha-helices. Substrate RCC binds quiet lossely to the enzyme. The loose binding seems beneficial to the large conformational change in red chlorophyll catabolite upon reduction. Two conserved acidic residues, Glu154 and Asp291, sandwich the C20/C1 double bond of RCC, suggesting that these two residues are involved in site-specific reduction. The geometrical arrangement of RCC and the carboxy groups of Glu154 and Asp291 in RCCR is essential for the stereospecificity of the RCCR reaction, substrate binding mechanism, overview. Analysis of substrate-free and substrate-bound enzyme crystal structures, and comparison to F218V enzyme mutant structures, overview Arabidopsis thaliana
1.3.7.12 physiological function red chlorophyll catabolite reductase (RCCR) catalyzes the ferredoxin-dependent reduction of the C20/C1 double bond of red chlorophyll catabolite (RCC), the catabolic intermediate produced in chlorophyll degradation Arabidopsis thaliana