Literature summary for 1.3.7.12 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.

Cloned(Commentary)

Cloned (Comment)	Organism
expression of GST-tagged wild-type and F218V mutant RCCR lacking the chloroplast transit peptide, Met1 to Gln39	Arabidopsis thaliana
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)

Crystallization (Comment)	Organism
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
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

Crystallization (Comment)

Organism

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

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

Protein Variants	Comment	Organism
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
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
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

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

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
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
red chlorophyll catabolite + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+	Arabidopsis thaliana	-	primary fluorescent chlorophyll catabolite + 2 oxidized ferredoxin [iron-sulfur] cluster	-	?

Organism

Organism	UniProt	Comment	Textmining
Arabidopsis thaliana	Q8LDU4	-	-

Purification (Commentary)

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

Source Tissue

Source Tissue	Comment	Organism	Textmining
leaf	-	Arabidopsis thaliana	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
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
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
red chlorophyll catabolite + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+	-	Arabidopsis thaliana	primary fluorescent chlorophyll catabolite + 2 oxidized ferredoxin [iron-sulfur] cluster	-	?
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

Subunits	Comment	Organism
homodimer	RCCR folds into a characteristic alphabetaalpha sandwich	Arabidopsis thaliana
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

Synonyms	Comment	Organism
RCCR	-	Arabidopsis thaliana
red chlorophyll catabolite reductase	-	Arabidopsis thaliana

Cofactor

Cofactor	Comment	Organism
Ferredoxin	dependent on	Arabidopsis thaliana
NADP+	-	Arabidopsis thaliana
NADPH	-	Arabidopsis thaliana

General Information

General Information	Comment	Organism
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
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
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