Information on EC 1.3.1.33 - protochlorophyllide reductase

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

EC NUMBER
COMMENTARY hide
1.3.1.33
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RECOMMENDED NAME
GeneOntology No.
protochlorophyllide reductase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
chlorophyllide a + NADP+ = protochlorophyllide + NADPH + H+
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
redox reaction
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-
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reduction
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
3,8-divinyl-chlorophyllide a biosynthesis I (aerobic, light-dependent)
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Biosynthesis of secondary metabolites
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chlorophyll metabolism
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Metabolic pathways
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Porphyrin and chlorophyll metabolism
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SYSTEMATIC NAME
IUBMB Comments
chlorophyllide-a:NADP+ 7,8-oxidoreductase
The enzyme catalyses a light-dependent trans-reduction of the D-ring of protochlorophyllide; the product has the (7S,8S)-configuration.
CAS REGISTRY NUMBER
COMMENTARY hide
68518-04-7
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
PCC 7120
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-
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
strain CS-41, enzyme components protein L, protein N and protein B are all transcribed constitutively independent of illumination and presence of glucose. Steady-state amounts of proteins in the light-grown cells are two- to threefold greater than in dark-grown cells. Approximately the same amount of protein is present in cultures grown mixtrophically or heterotrophically both containing glucose. Much less protein is present in photoautotrophic cells. Therefore light-independent enzyme activity depends on post-transcriptional and post-translational regulation and switches on both in light and dark
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Manually annotated by BRENDA team
strain CS-41, enzyme components protein L, protein N and protein B are all transcribed constitutively independent of illumination and presence of glucose. Steady-state amounts of proteins in the light-grown cells are two- to threefold greater than in dark-grown cells. Approximately the same amount of protein is present in cultures grown mixtrophically or heterotrophically both containing glucose. Much less protein is present in photoautotrophic cells. Therefore light-independent enzyme activity depends on post-transcriptional and post-translational regulation and switches on both in light and dark
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Manually annotated by BRENDA team
strain ATCC 49652
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Manually annotated by BRENDA team
subunit L, fragment
UniProt
Manually annotated by BRENDA team
subunit B, subunit L, subunit N, fragments
B0F835 and B0F841 and B0F842
UniProt
Manually annotated by BRENDA team
squash
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-
Manually annotated by BRENDA team
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SwissProt
Manually annotated by BRENDA team
subunit B, subunit L, subunit N, fragments
B0F833 and B0F836 and B0F837
UniProt
Manually annotated by BRENDA team
subunit L, subunit N, fragments
B0F838 and B0F839
UniProt
Manually annotated by BRENDA team
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-
Manually annotated by BRENDA team
cress
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-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
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SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
8-ethyl-chlorophyll a + NADPH + H+
? + NADP+
show the reaction diagram
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-
-
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?
8-ethyl-chlorophyll b + NADPH + H+
? + NADP+
show the reaction diagram
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-
-
-
?
8-vinyl-chlorophyll a + NADPH + H+
? + NADP+
show the reaction diagram
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-
-
-
?
8-vinyl-chlorophyll b + NADPH + H+
? + NADP+
show the reaction diagram
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-
-
-
?
C8-ethyl-C13(2)-(r)-protochlorophyllide + NADPH
? + NADP+
show the reaction diagram
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stereoisomer of the substrate
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-
?
chlorophyllide a + NADP+
protochlorophyllide + NADPH + H+
show the reaction diagram
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POR catalyzes the NADPH-dependent reduction of the C17-C18 double bond of protochlorophyllide to form chlorophyllide
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r
divinyl protochlorophyllide a + NADPH
divinyl chlorophyllide a + NADP+
show the reaction diagram
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-
-
-
?
monovinyl protochlorophyllide a + NADPH
monovinyl chlorophyllide a + NADP+
show the reaction diagram
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-
-
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?
protochlorophyllide + dithionite
chlorophyllide + SO2
show the reaction diagram
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-
-
-
?
protochlorophyllide + dithiothreitol
chlorophyllide + oxidized dithiothreitol
show the reaction diagram
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-
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?
protochlorophyllide + NADPH
chlorophyllide + NADP+
show the reaction diagram
protochlorophyllide + NADPH + H+
chlorophyllide + NADP+
show the reaction diagram
protochlorophyllide + NADPH + H+
chlorophyllide a + NADP+
show the reaction diagram
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-
-
-
?
protochlorophyllide + reduced ferredoxin
chlorophyllide + oxidized ferredoxin
show the reaction diagram
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ferredoxin is the natural electron donor
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?
protochlorophyllide a + NADPH
chlorophyllide a
show the reaction diagram
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-
-
?
protochlorophyllide a + NADPH
chlorophyllide a + NADP+
show the reaction diagram
protochlorophyllide a + NADPH + H+
chlorophyllide a + NADP+
show the reaction diagram
protochlorophyllide b + NADPH + H+
chlorophyllide b + NADP+
show the reaction diagram
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-
-
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?
Zn-protopheophorbide a + NADPH + H+
? + NADP+
show the reaction diagram
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efficient substrate
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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
chlorophyllide a + NADP+
protochlorophyllide + NADPH + H+
show the reaction diagram
-
POR catalyzes the NADPH-dependent reduction of the C17-C18 double bond of protochlorophyllide to form chlorophyllide
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r
divinyl protochlorophyllide a + NADPH
divinyl chlorophyllide a + NADP+
show the reaction diagram
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-
-
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?
monovinyl protochlorophyllide a + NADPH
monovinyl chlorophyllide a + NADP+
show the reaction diagram
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-
-
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?
protochlorophyllide + NADPH
chlorophyllide + NADP+
show the reaction diagram
protochlorophyllide + NADPH + H+
chlorophyllide + NADP+
show the reaction diagram
protochlorophyllide + reduced ferredoxin
chlorophyllide + oxidized ferredoxin
show the reaction diagram
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ferredoxin is the natural electron donor
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-
?
protochlorophyllide a + NADPH
chlorophyllide a + NADP+
show the reaction diagram
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?
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4Fe-4S center
Ferredoxin
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
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specifically stimulated by Ca2+
Fe2+
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contains two 4Fe-4S clusters
Iron
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enzyme component L-protein contains an oxygen-sensitive 4Fe-4S cluster similar to nitrogenase Fe protein. Activity quickly disappears upon exposure to air with a half-life of 20 s
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2',5'-ADP
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competitive inhibition of NADPH binding
5,5'-dithiobis-(2-nitrobenzoic acid)
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inhibits the regeneration of the enzyme-substrates complex, but has no effect on the photoconversion of the preformed complex
chlorophyll c1
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competitive
EGTA
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the enzyme activity is abolished in the presence of 10 mM EGTA
N-ethylmaleimide
additional information
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the reaction is completely insensitive to illumination (5-20 w/m2 tungsten light)
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ATP
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the enzyme uses ATP to drive the production of chlorophyllide a
light
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light-activated enzyme, activation is possible at cryogenic temperatures
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protochlorophyllide
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stringent requirement
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0527
Dithionite
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in 100 mM HEPES-NaOH (pH 7.5), 2 mM ATP, 5 mM MgCl2
0.00083
divinyl protochlorophyllide a
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pH 7.5
0.00136
monovinyl protochlorophyllide a
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pH 7.5
0.000012 - 0.0595
NADPH
0.00015 - 0.035
protochlorophyllide
0.00027 - 0.0086
protochlorophyllide a
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.011 - 0.165
NADPH
0.003 - 0.165
protochlorophyllide
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00302
2',5'-ADP
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calculated competitive inhibition constant
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.00315
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in 100 mM HEPES-NaOH (pH 7.5), 2 mM ATP, 5 mM MgCl2
0.5
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etioplast membranes
0.7
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prolamellar body
0.8
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sonicated membranes
3.3
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prothylakoid
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 9
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6 - 8.5
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10 - 60
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linear drop of the relative concentration of the enzyme protein up to 30C
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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translocon constituent toc33 is indispensable for the import of isoform PorB
Manually annotated by BRENDA team
low expression, light-response during greening, expression in mature cells
Manually annotated by BRENDA team
additional information
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photosynthetic active tissue
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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PORA precursor, pPORA
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
32000
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gel filtration
37800
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amino acid composition
40000
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determined by SDS-PAGE
41000
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identified with monospecific polyclonal antibody
41200
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predicted from cDNA
42000
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determined by SDS-PAGE
60000
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purified ChlL subunit, gel filtration
78800
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recombinant fusion protein, gel filtration
112000
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substrate-enzyme complex, gel filtration
210000
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purified ChlNB complex, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterotetramer
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2 * 46199 + 2 * 58729, purified ChlNB complex, deduced from amino acid sequence
homodimer
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2 * 32395, purified ChlL subunit, deduced from amino acid sequence
monomer
octamer
heterooctameric complex: subunits N and B are structurally homologous, generating a pseudo-2fold symmetry axis that is colinear with the molecular twofold axis of L2. Both [4Fe-4S] clusters are centered around this extended axis: the L2 cluster is symmetrically ligated by four cysteinyl ligands between the two subunits, whereas the NB cluster is asymmetrically ligated by three cysteine residues from N and one aspartate residue from B
oligomer
additional information
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recombinant enzyme component L-protein forms a dimer, recombinant components NB-protein form a heterotetramer, gel filtration
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
proteolytic modification
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x * 46000 Da, precursor protein, x * 38000 Da, mature protein
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
the X-ray crystallographic structure of the substrate-bound, ADP-aluminium fluoride-stabilized (ADP-AlF3-stabilized) transition state complex between the DPOR components L2 and (NB)2 from the marine cyanobacterium Prochlorococcus marinus is reported
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
90
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the enzyme activity is abolished after heating of the membranes (90C, 5 min)
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
active enzyme is protected by NADPH, protochlorophyllide and glycerol
protochlorophyllide and NADPH enhances the stability of the enzyme
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rapid breakdown of the enzyme protein in vitro, caused by a membrane-bound proteolytic activity
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rapid loss of activity during attempts to purify
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unstable membrane-bound protein
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
0C, can be kept in elution buffer for at least 3h without any detectable loss of activity
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4C, stored in elution buffer retains full activity for many weeks
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enzyme in crude extracts stable for more than 6 months when maintained anaerobically at 4C, with no significant loss of activity
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
both activities of the components protein L and proteins NB are rapidly lost during purification procedures, such as affinity chromatography with S-protein agarose. Attempts to purify the active components have so far been unsuccessful
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by Ni-NTA agarose chromatography
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etioplasts are isolated
glutathione Sepharose column chromatography
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glutathione-Sepharose column chromatography
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isolated from pea leaves
NB-protein component from Pchlide oxidoreductase
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PORb heterologously overexpressed in Escherichia coli
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recombinant fusion protein
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using a chitin bead column, the chitin-intein tag is removed; using a chitin bead column, the chitin-intein tag is removed
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using His-Bind resin
wild-type and recombinant pea enzyme, heterologously expressed in Escherichia coli
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
a genomic library is constructed, the vector pZERO is used; a genomic library is constructed, the vector pZERO is used
A9UGZ2, C6KHP5 and Q6H056 and Q6H058
barley POR expressed in Escherichia coli as a beta-galactosidase fusion protein
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cDNA library constructed in pBR322, full-length cDNA cloned, cDNA inserts subcloned in the M13 phage and expressed in Escherichia coli
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cDNAs and/or gens encoding POR, also referred to as LPCR or PCR in some studies isolated; full-length cDNA, coding for PCR
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cloned and overproduced in Escherichia coli with a hexahistidine tag at the N-terminus
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cloning of the gene encoding protochlorophyllide reductase
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cloning of the nuclear gene
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expressed in Escherichia coli
expressed in Escherichia coli BL21(DE3) Codon Plus RIL cells
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expressed in Escherichia coli strain SG13009
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expressed in Escherichia coli; expression in Escherichia coli cells
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expressed in Rhodobacter capsulatus SB1003, JDA, JDB and ZY5
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expression in Escherichia coli BL21
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expression in Escherichia coli cells
expression in Rhodobacter capsulatus mutant deficient in Bchl biosynthesis. The NADPH:protochlorophyllide oxidoreductase is integrated in the porphyrin biosynthesis network and its activity leads to the formation of photosynthetic chlorophyll proteins
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for sequencing
full-length cDNA, coding for PCR
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full-length cDNA, coding for PCR; POR C cloned and expressed in Escherichia coli
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into the vector pQE30
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mature pea POR cloned into the pAlter-1 mutagenesis vector, expressed in Escherichia coli JM109
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overexpressed as a fusion with maltose-binding protein in Escherichia coli using expression plasmid pKK233-2
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overexpressed in Escherichia coli as a fusion protein with the maltose-binding protein
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overexpression in Escherichia coli
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overexpression in Escherichia coli as a fusion protein with maltose-binding protein
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overexpression in Escherichia coli as a His6-tagged protein
yes into the vector pTYB for expression in Escherichia coli BL21DE3 cells; yes into the vector pTYB for expression in Escherichia coli BL21DE3 cells, for the construction of transgenic plants pDONR/ZeO and pGWB2 vectors are used
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
down-regulated in green light and in the dark; down-regulated in the dark
A9UGZ2, C6KHP5 and Q6H056 and Q6H058
OsPORA is decreased dramatically in fully mature leaves
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OsPORA is expressed at high levels in developing leaves; OsPORB expression is rapidly upregulated by high light treatment
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OsPORB expression is regulated in a circadian rhythm in short-day conditions
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PORA represents the negatively light-regulated POR enzyme
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up-regulated in green light
A9UGZ2, C6KHP5 and Q6H056 and Q6H058
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C103S
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the BchN subunit variant shows 0.5% residual activity and is essentially inactive
C21S
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the BchN subunit variant shows 0.5% residual activity and is essentially inactive
C46S
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the BchN subunit variant shows 0.5% residual activity and is essentially inactive
C104A
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no detectable effect on the import of protein to plastid and processing in darkness
C166A
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no detectable effect on the import of protein to plastid and processing in darkness
C195A
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mutant, constructed for the identification of the protochlorophyllide binding site
C222A
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mutant, constructed for the identification of the protochlorophyllide binding site
C276A
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decrease in mature enzyme protein present in plastid and decrease in the amount of protochlorophyllide bound to enzyme. C276 constitutes the protochlorophyllide binding site in the active centre of enzyme
C303A
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decrease in mature enzyme protein present in plastid and decrease in the amount of protochlorophyllide bound to enzyme. C303 constitutes a low affinity protochlorophyllide binding site involved in assembly and stabilization of imported enzyme inside etioplasts
C33A
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mutant, constructed for the identification of the protochlorophyllide binding site
C85A
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mutant, constructed for the identification of the protochlorophyllide binding site
H394A
mutant retains only a moderate activity which points to a critical role of this residue in the specific protonation at C-18, probably by positioning a water molecule at a distance of 3.2 A from C-18 above the ring
Y189F
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mutant, the putative proton donor, Tyr 189, is replaced by a phenylalanine residue
C199/C226S
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mutant, mutation of the absolutely conserved cysteine residues
C199S
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kcat comparable to wild-type, relative activity comparable to wild-type, Kd (NADPH) comparable to wild-type, Kd (protochlorophyllide) comparable to wild-type; mutant, Cys199 has a relatively minor role in catalysis
C199S/C226S
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kcat more that 10fold decreased to wild-type, relative activity highly decreased compared to wild-type, Kd (NADPH) 2fold increased compared to wild-type, Kd (protochlorophyllide) 6fold increased compared to wild-type
C226S
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kcat more that 10fold decreased to wild-type, relative activity highly decreased compared to wild-type, Kd (NADPH) comparable to wild-type, Kd (protochlorophyllide) 4fold increased compared to wild-type; mutant, mutation causes a remarkable change in the mechansim of the hydrogen transfer reactions
C37S
-
kcat comparable to wild-type, relative activity comparable to wild-type, Kd (NADPH) increased compared to wild-type, Kd (protochlorophyllide) equal to wild-type; mutant, Cys37 has a relatively minor role in catalysis
C37S/C199S
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kcat comparable to wild-type, relative activity comparable to wild-type, Kd (NADPH) 10fold increased compared to wild-type, Kd (protochlorophyllide) comparable to wild-type; mutant, mutation of the absolutely conserved cysteine residues
C37S/C199S/C226S
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kcat more that 10fold decreased to wild-type, relative activity highly decreased compared to wild-type, Kd (NADPH) 10fold increased compared to wild-type, Kd (protochlorophyllide) 6fold increased compared to wild-type; mutant, mutation of the absolutely conserved cysteine residues
C37S/C226S
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kcat more that 10fold decreased to wild-type, relative activity highly decreased compared to wild-type, Kd (NADPH) 10fold increased compared to wild-type, Kd (protochlorophyllide) 6fold increased compared to wild-type; mutant, mutation of the absolutely conserved cysteine residues
K197A
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mutant, constructed for analysing the role of the conserved active site lysine
K197Q
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mutant, constructed for analysing the role of the conserved active site lysine
K197R
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mutant, constructed for analysing the role of the conserved active site lysine
Y193A
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mutant, constructed for analysing the role of the conserved active site tyrosine
Y193F
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mutant, constructed for analysing the role of the conserved active site tyrosine
Y193S
-
mutant, constructed for analysing the role of the conserved active site tyrosine
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
-
POR is an ideal model for studying catalysis near the solvent glass transition
Show AA Sequence (317 entries)
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