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Literature summary for 1.3.7.7 extracted from

  • Muraki, N.; Nomata, J.; Ebata, K.; Mizoguchi, T.; Shiba, T.; Tamiaki, H.; Kurisu, G.; Fujita, Y.
    X-ray crystal structure of the light-independent protochlorophyllide reductase (2010), Nature, 465, 110-114.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli JM105 cells Rhodobacter capsulatus
protochlorophyllide-bound and protochlorophyllide-free forms of NB-protein are overexpressed in Rhodobacter capsulatus strain DB176 and Escherichia coli strain JM105 Rhodobacter capsulatus

Crystallization (Commentary)

Crystallization (Comment) Organism
hanging drop vapor diffusion method, the protochlorophyllide-bound form of NB-protein is crystallized using 200 mM sodium/potassium phosphate buffer (pH 5.0) containing 5 mM dithiothreitol and 10% (w/v) ethylene glycol at 4°C, to which 16% (w/v) and 14% (w/v) PEG4K are added in aerobic and anaerobic conditions, respectively, as precipitants. Protochlorophyllide -free and selenomethionine-substituted recombinant NB-proteins are crystallized at 20°C using 20% (w/v) PEG3350 containing 200 mM ammonium chloride and 5mM dithiothreitol. D36C and D36A variants are crystallized at 4°C using 20% (w/v) PEG3350 containing 200 mM sodium chloride, 100 mM MOPS/NaOH (pH 7.0) and 5 mM dithiothreitol as a precipitant Rhodobacter capsulatus
purified recombinant protochlorophyllide-bound and protochlorophyllide-free forms of NB-protein of DPOR, and purified recombinant selenomethionine-substituted protochlorophyllide-free forms of mutants D36A and D36C, hanging-drop vapour diffusion method., X-ray diffraction structure determination and analysis at 2.3-2.9 A resolution Rhodobacter capsulatus

Protein Variants

Protein Variants Comment Organism
C112A inactive Rhodobacter capsulatus
C26A inactive Rhodobacter capsulatus
C51A the mutation almost abolishes the activity of the enzyme (less than 5% compared to the wild type enzyme) Rhodobacter capsulatus
C95A inactive Rhodobacter capsulatus
D36A site-directed mutagenesis, mutation in BchB, mutant NB-cluster structure compered to the wild-type enzyme Rhodobacter capsulatus
D36A the mutant exhibits low activity (13% compared to the wild type enzyme) Rhodobacter capsulatus
D36C site-directed mutagenesis, mutation in BchB, mutant NB-cluster structure compered to the wild-type enzyme Rhodobacter capsulatus
D36C the mutation almost abolishes the activity of the enzyme (less than 5% compared to the wild type enzyme) Rhodobacter capsulatus
D36S site-directed mutagenesis, mutation in BchB, mutant NB-cluster structure compered to the wild-type enzyme Rhodobacter capsulatus
D36S the mutation almost abolishes the activity of the enzyme (less than 5% compared to the wild type enzyme) Rhodobacter capsulatus

Inhibitors

Inhibitors Comment Organism Structure
chlorophyll c competitive inhibitor Rhodobacter capsulatus

Metals/Ions

Metals/Ions Comment Organism Structure
Fe2+ iron-sulfur cluster in the NB protein Rhodobacter capsulatus
Mg2+
-
Rhodobacter capsulatus

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
chlorophyllide a + reduced ferredoxin + 2 ATP + 2 H2O Rhodobacter capsulatus
-
protochlorophyllide + oxidized ferredoxin + 2 ADP + 2 phosphate + 2 H+
-
?
chlorophyllide a + reduced ferredoxin + 2 ATP + 2 H2O Rhodobacter capsulatus DB176
-
protochlorophyllide + oxidized ferredoxin + 2 ADP + 2 phosphate + 2 H+
-
?

Organism

Organism UniProt Comment Textmining
Rhodobacter capsulatus
-
-
-
Rhodobacter capsulatus DB176
-
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant protochlorophyllide-bound and protochlorophyllide-free forms of NB-protein from Rhodobacter capsulatus strain DB176 and Escherichia coli strain JM105 by affinity chromatography and gel filtration Rhodobacter capsulatus
Strep-tactin column chromatography and Superdex 200 gel filtration Rhodobacter capsulatus

Reaction

Reaction Comment Organism Reaction ID
chlorophyllide a + oxidized ferredoxin + 2 ADP + 2 phosphate = protochlorophyllide a + reduced ferredoxin + 2 ATP + 2 H2O reaction mechanism for trans-specific reduction, overview Rhodobacter capsulatus

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
0.026
-
purified recombinant protochlorophyllide-bound NB-protein, recovered from crystals, pH not specified in the publication, temperature not specified in the publication Rhodobacter capsulatus
0.05
-
purified recombinant protochlorophyllide-free NB-protein, recovered from crystals, pH not specified in the publication, temperature not specified in the publication Rhodobacter capsulatus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
chlorophyllide a + reduced ferredoxin + 2 ATP + 2 H2O
-
Rhodobacter capsulatus protochlorophyllide + oxidized ferredoxin + 2 ADP + 2 phosphate + 2 H+
-
?
chlorophyllide a + reduced ferredoxin + 2 ATP + 2 H2O
-
Rhodobacter capsulatus DB176 protochlorophyllide + oxidized ferredoxin + 2 ADP + 2 phosphate + 2 H+
-
?
chlorophyllide a + reduced ferredoxin + ATP
-
Rhodobacter capsulatus protochlorophyllide + oxidized ferredoxin + ADP + phosphate
-
?
chlorophyllide a + reduced ferredoxin + ATP
-
Rhodobacter capsulatus DB176 protochlorophyllide + oxidized ferredoxin + ADP + phosphate
-
?
additional information DPOR is a nitrogenase-like enzyme consisting of two components, L-protein (a BchL dimer) and NB-protein (a BchN-BchB heterotetramer) Rhodobacter capsulatus ?
-
?
additional information each catalytic BchN-BchB unit contains one protochlorophyllidee and one iron-sulfur NB-cluster coordinated uniquely by one aspartate and three cysteines. Unique aspartate ligation is not necessarily needed for the cluster assembly but is essential for the catalytic activity. Specific protochlorophyllide-binding accompanies the partial unwinding of an alpha-helix that belongs to the next catalytic BchN-BchB unit, unique trans-specific reduction mechanism in which the distorted C17-propionate of protochlorophyllid and an aspartate from BchB serve as proton donors for C18 and C17 of protochlorophyllide, respectively, overview Rhodobacter capsulatus ?
-
?
additional information the C175C18 double bond of chlorophyll c is not reduced by DPOR Rhodobacter capsulatus ?
-
?
additional information DPOR is a nitrogenase-like enzyme consisting of two components, L-protein (a BchL dimer) and NB-protein (a BchN-BchB heterotetramer) Rhodobacter capsulatus DB176 ?
-
?
additional information each catalytic BchN-BchB unit contains one protochlorophyllidee and one iron-sulfur NB-cluster coordinated uniquely by one aspartate and three cysteines. Unique aspartate ligation is not necessarily needed for the cluster assembly but is essential for the catalytic activity. Specific protochlorophyllide-binding accompanies the partial unwinding of an alpha-helix that belongs to the next catalytic BchN-BchB unit, unique trans-specific reduction mechanism in which the distorted C17-propionate of protochlorophyllid and an aspartate from BchB serve as proton donors for C18 and C17 of protochlorophyllide, respectively, overview Rhodobacter capsulatus DB176 ?
-
?
additional information the C175C18 double bond of chlorophyll c is not reduced by DPOR Rhodobacter capsulatus DB176 ?
-
?

Subunits

Subunits Comment Organism
heterooctamer (alpha2)2(betagamma)4, DPOR is a nitrogenase-like enzyme consisting of two components, L-protein, a BchL dimer, and NB-protein, a BchN-BchB heterotetramer, which are structurally related to nitrogenase Fe protein and MoFe protein, respectively Rhodobacter capsulatus
More each catalytic BchN-BchB unit contains one protochlorophyllide and one iron-sulfur NB-cluster coordinated uniquely by one aspartate and three cysteines. Unique aspartate ligation is not necessarily needed for the cluster assembly but is essential for the catalytic activity. Specific protochlorophyllide-binding accompanies the partial unwinding of an alpha-helix that belongs to the next catalytic BchN-BchB unit, unique trans-specific reduction mechanism in which the distorted C17-propionate of protochlorophyllide and an aspartate from BchB serve as proton donors for C18 and C17 of protochlorophyllide, respectively, overview Rhodobacter capsulatus

Synonyms

Synonyms Comment Organism
dark-operative Pchlide oxidoreductase
-
Rhodobacter capsulatus
DPOR
-
Rhodobacter capsulatus
light-independent (dark-operative) Pchlide oxidoreductase
-
Rhodobacter capsulatus
light-independent protochlorophyllide reductase
-
Rhodobacter capsulatus

Cofactor

Cofactor Comment Organism Structure
4Fe-4S-center
-
Rhodobacter capsulatus
ATP
-
Rhodobacter capsulatus
Ferredoxin each catalytic BchN-BchB unit contains one protochlorophyllidee and one iron-sulfur NB-cluster coordinated uniquely by one aspartate and three cysteines. Unique aspartate ligation is not necessarily needed for the cluster assembly but is essential for the catalytic activity, overview Rhodobacter capsulatus

General Information

General Information Comment Organism
physiological function DPOR performs reduction of the C17-C18 double bond of protochlorophyllide to form chlorophyllide a, the direct precursor of chlorophyll a in a light-independent, dark-operative way of action Rhodobacter capsulatus