Literature summary for 1.3.7.15 extracted from

Moser, J.; Jasper, J.; Ramos, J.; Sowa, S.; Layer, G.
Expression, purification, and activity analysis of chlorophyllide oxidoreductase and Ni2+-sirohydrochlorin a,c-diamide reductase (2019), Methods Mol. Biol., 1876, 125-140 .

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant overexpression of enzymatically active subunit (BchY/BchZ)2 and BchX2 subcomplexes in Escherichia coli strain BL21(DE3), expression of N-terminally His6-tagged BchY, N-terminally thioredoxin/His/S-tagged BchX2, and untagged BchZ under the control of individual promoters. Supplementation with Fe(III)citrate and L-cysteine as sources of iron and sulfur, respectively, as media constituents has significant influence on the in vivo assembly of [4Fe-4S] clusters, growth temperature is 17°C	Roseobacter denitrificans

Cloned (Comment)

Organism

recombinant overexpression of enzymatically active subunit (BchY/BchZ)2 and BchX2 subcomplexes in Escherichia coli strain BL21(DE3), expression of N-terminally His6-tagged BchY, N-terminally thioredoxin/His/S-tagged BchX2, and untagged BchZ under the control of individual promoters. Supplementation with Fe(III)citrate and L-cysteine as sources of iron and sulfur, respectively, as media constituents has significant influence on the in vivo assembly of [4Fe-4S] clusters, growth temperature is 17°C

Roseobacter denitrificans

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Fe2+	in cofactors	Roseobacter denitrificans
Mg2+	required	Roseobacter denitrificans

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
3-acetyl-3-devinylchlorophyllide a + 2 reduced ferredoxin [iron-sulfur] cluster + ATP + H2O + 2 H+	Roseobacter denitrificans	-	bacteriochlorophyllide a + 2 oxidized ferredoxin [iron-sulfur] cluster + ADP + phosphate	-	?
3-acetyl-3-devinylchlorophyllide a + 2 reduced ferredoxin [iron-sulfur] cluster + ATP + H2O + 2 H+	Roseobacter denitrificans ATCC 33942	-	bacteriochlorophyllide a + 2 oxidized ferredoxin [iron-sulfur] cluster + ADP + phosphate	-	?
8-vinyl chlorophyllide a + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+	Roseobacter denitrificans	-	chlorophyllide a + 2 oxidized ferredoxin [iron-sulfur] cluster	-	?
8-vinyl chlorophyllide a + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+	Roseobacter denitrificans ATCC 33942	-	chlorophyllide a + 2 oxidized ferredoxin [iron-sulfur] cluster	-	?

Organism

Organism	UniProt	Comment	Textmining
Roseobacter denitrificans	P26277 AND Q16DU8 AND Q16DU7	genes bchX, bchY, and bchZ; Erythrobacter sp. strain OCh 114	-
Roseobacter denitrificans ATCC 33942	P26277 AND Q16DU8 AND Q16DU7	genes bchX, bchY, and bchZ; Erythrobacter sp. strain OCh 114	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant N-terminally His6-tagged BchY and untagged BchZ as a subcomplex, and N-terminally thioredoxin/His/S-tagged BchX2, from Escherichia coli strain BL21(DE3) by nickel affinity chromatography under strict anaerobic conditions (i.e. in an anaerobic chamber), which is essential for the preservation of enzymatic activities. Method evaluation, overview	Roseobacter denitrificans

Reaction

Reaction	Comment	Organism	Reaction ID
3-deacetyl-3-vinylbacteriochlorophyllide a + 2 oxidized ferredoxin [iron-sulfur] cluster + ADP + phosphate = chlorophyllide a + 2 reduced ferredoxin [iron-sulfur] cluster + ATP + H2O + 2 H+	binding of the substrate to (BchY/BchZ)2 is the initial step of COR catalysis, which is followed by formation of the ternary complex between (BchY/BchZ)2 and (BchX)2	Roseobacter denitrificans	a

Renatured (Commentary)

Renatured (Comment)	Organism
robust reconstitution of recombinant COR activity is achieved by coupling it to another enzymatic assay using the green pigment, protochlorophyllide, as a substrate of the nitrogenase-like enzyme, dark-operative protochlorophyllide oxidoreductase (DPOR). The DPOR reaction results in the formation of chlorophyllide (Chlide), which then acts as a direct substrate of COR. Reconstitution of DPOR and COR activities requires high concentrations of ATP, as well as a suitable electron donor	Roseobacter denitrificans

Renatured (Comment)

Organism

robust reconstitution of recombinant COR activity is achieved by coupling it to another enzymatic assay using the green pigment, protochlorophyllide, as a substrate of the nitrogenase-like enzyme, dark-operative protochlorophyllide oxidoreductase (DPOR). The DPOR reaction results in the formation of chlorophyllide (Chlide), which then acts as a direct substrate of COR. Reconstitution of DPOR and COR activities requires high concentrations of ATP, as well as a suitable electron donor

Roseobacter denitrificans

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
3-acetyl-3-devinylchlorophyllide a + 2 reduced ferredoxin [iron-sulfur] cluster + ATP + H2O + 2 H+	-	Roseobacter denitrificans	bacteriochlorophyllide a + 2 oxidized ferredoxin [iron-sulfur] cluster + ADP + phosphate	-	?
3-acetyl-3-devinylchlorophyllide a + 2 reduced ferredoxin [iron-sulfur] cluster + ATP + H2O + 2 H+	-	Roseobacter denitrificans ATCC 33942	bacteriochlorophyllide a + 2 oxidized ferredoxin [iron-sulfur] cluster + ADP + phosphate	-	?
8-vinyl chlorophyllide a + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+	-	Roseobacter denitrificans	chlorophyllide a + 2 oxidized ferredoxin [iron-sulfur] cluster	-	?
8-vinyl chlorophyllide a + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+	the substrate is purified from the Rhodobacter capsulatus strain CB1200	Roseobacter denitrificans	chlorophyllide a + 2 oxidized ferredoxin [iron-sulfur] cluster	-	?
8-vinyl chlorophyllide a + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+	-	Roseobacter denitrificans ATCC 33942	chlorophyllide a + 2 oxidized ferredoxin [iron-sulfur] cluster	-	?
8-vinyl chlorophyllide a + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+	the substrate is purified from the Rhodobacter capsulatus strain CB1200	Roseobacter denitrificans ATCC 33942	chlorophyllide a + 2 oxidized ferredoxin [iron-sulfur] cluster	-	?
additional information	for the standard DPOR/COR activity assay, a cell-free Escherichia coli extract containing overproduced Chlorobaculum tepidum DPOR subunits, BchN, BchB and BchL, is used in combination with purified recombinant COR subcomplexes, BchX2 and (BchY/BchZ)2. When using the chemical reducing agent, dithionite, instead of the natural electron donor (which might be a ferredoxin), it is essential to conduct control reactions in the absence of BchX2 and (BchY/BchZ)2 to ensure the dependence of the observed COR activity on both protein subcomplexes. The maximum COR activity is only achieved using an optimal ratio between BchX2 and (BchY/BchZ)2. Protein-protein interaction of BchX2 and (BchY/BchZ)2 is investigated in the presence of an analogue of the cosubstrate ATP, MgADP-AlF4-. Assay method evaluation, overview	Roseobacter denitrificans	?	-	-
additional information	for the standard DPOR/COR activity assay, a cell-free Escherichia coli extract containing overproduced Chlorobaculum tepidum DPOR subunits, BchN, BchB and BchL, is used in combination with purified recombinant COR subcomplexes, BchX2 and (BchY/BchZ)2. When using the chemical reducing agent, dithionite, instead of the natural electron donor (which might be a ferredoxin), it is essential to conduct control reactions in the absence of BchX2 and (BchY/BchZ)2 to ensure the dependence of the observed COR activity on both protein subcomplexes. The maximum COR activity is only achieved using an optimal ratio between BchX2 and (BchY/BchZ)2. Protein-protein interaction of BchX2 and (BchY/BchZ)2 is investigated in the presence of an analogue of the cosubstrate ATP, MgADP-AlF4-. Assay method evaluation, overview	Roseobacter denitrificans ATCC 33942	?	-	-

Subunits

Subunits	Comment	Organism
oligomer	the COR components (BchY/BchZ)2 and BchX2 are organized in a ternary COR complex	Roseobacter denitrificans

Synonyms

Synonyms	Comment	Organism
BchX	-	Roseobacter denitrificans
BchY	-	Roseobacter denitrificans
BchZ	-	Roseobacter denitrificans
chlorophyllide oxidoreductase	-	Roseobacter denitrificans
COR	-	Roseobacter denitrificans

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
34	-	assay at	Roseobacter denitrificans

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Roseobacter denitrificans

Cofactor

Cofactor	Comment	Organism
ATP	-	Roseobacter denitrificans
Fe-S center	[4Fe-4S] clusters	Roseobacter denitrificans
Ferredoxin	putatively the natural electron donor	Roseobacter denitrificans
additional information	sodium dithionite can substitute for ferredoxin as electron donor	Roseobacter denitrificans

General Information

General Information	Comment	Organism
metabolism	the biosynthesis of all bacteriochlorophylls involves the two-electron reduction of the C7-C8 double bond of the green pigment chlorophyllide, which is catalyzed by the nitrogenase-like two-component metalloenzyme chlorophyllide oxidoreductase (COR), whereas in all methanogenic microbes, another nitrogenase-like system, CfbC/D, is responsible for the sophisticated six-electron reduction of Ni2+-sirohydrochlorin a,c-diamide in the course of coenzyme F430 biosynthesis	Roseobacter denitrificans
physiological function	the biosynthesis of all bacteriochlorophylls involves the two-electron reduction of the C7-C8 double bond of the green pigment chlorophyllide, which is catalyzed by the nitrogenase-like two-component metalloenzyme chlorophyllide oxidoreductase (COR), whereas in all methanogenic microbes, another nitrogenase-like system, CfbC/D, is responsible for the sophisticated six-electron reduction of Ni2+-sirohydrochlorin a,c-diamide in the course of coenzyme F430 biosynthesis	Roseobacter denitrificans