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Information on EC 1.3.3.4 - protoporphyrinogen oxidase and Organism(s) Bacillus subtilis and UniProt Accession P32397

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     1 Oxidoreductases
         1.3 Acting on the CH-CH group of donors
             1.3.3 With oxygen as acceptor
                1.3.3.4 protoporphyrinogen oxidase
IUBMB Comments
This is the last common enzyme in the biosynthesis of chlorophylls and heme . Two isoenzymes exist in plants: one in plastids and the other in mitochondria. This is the target enzyme of phthalimide-type and diphenylether-type herbicides . The enzyme from oxygen-dependent species contains FAD . Also slowly oxidizes mesoporphyrinogen IX.
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Bacillus subtilis
UNIPROT: P32397
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Word Map
The taxonomic range for the selected organisms is: Bacillus subtilis
The enzyme appears in selected viruses and cellular organisms
Synonyms
protoporphyrinogen oxidase, ppox, protox, protoporphyrinogen ix oxidase, mtppo, protox enzyme, protoporphyrinogen oxidase ix, ppo1-1, ppox i, protein yfex, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
protoporphyrinogen IX oxidase
-
Protox enzyme
-
protoporphyrinogen IX oxidase
-
-
-
-
protoporphyrinogen oxidase
-
-
-
-
protoporphyrinogenase
-
-
-
-
protox
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
redox reaction
-
-
-
-
oxidation
-
-
-
-
reduction
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
protoporphyrinogen-IX:oxygen oxidoreductase
This is the last common enzyme in the biosynthesis of chlorophylls and heme [8]. Two isoenzymes exist in plants: one in plastids and the other in mitochondria. This is the target enzyme of phthalimide-type and diphenylether-type herbicides [8]. The enzyme from oxygen-dependent species contains FAD [9]. Also slowly oxidizes mesoporphyrinogen IX.
CAS REGISTRY NUMBER
COMMENTARY hide
53986-32-6
-
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
protoporphyrinogen IX + 3 O2
protoporphyrin + 3 H2O2
show the reaction diagram
protoporphyrinogen IX + 3 O2
protoporphyrin IX + 3 H2O2
show the reaction diagram
-
-
-
?
protoporphyrinogen IX + O2
protoporphyrin IX + H2O
show the reaction diagram
20fold lower activity with protoporphyrinogen IX compared to coproporphyrinogen III
-
-
?
protoporphyrinogen-IX + 3 O2
protoporphyrin-IX + 3 H2O2
show the reaction diagram
-
-
-
?
coproporphyrinogen-III + 3 O2
coproporphyrin-III + 3 H2O2
show the reaction diagram
-
-
-
-
?
mesoporphyrinogen-IX + 3 O2
mesoporphyrin-IX + 3 H2O2
show the reaction diagram
-
-
-
-
?
protoporphyrinogen IX + 3 O2
protoporphyrin + 3 H2O2
show the reaction diagram
protoporphyrinogen-IX + 3 O2
protoporphyrin-IX + 3 H2O2
show the reaction diagram
additional information
?
-
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
protoporphyrinogen IX + 3 O2
protoporphyrin + 3 H2O2
show the reaction diagram
-
-
-
?
protoporphyrinogen IX + 3 O2
protoporphyrin IX + 3 H2O2
show the reaction diagram
-
-
-
?
protoporphyrinogen IX + O2
protoporphyrin IX + H2O
show the reaction diagram
20fold lower activity with protoporphyrinogen IX compared to coproporphyrinogen III
-
-
?
protoporphyrinogen-IX + 3 O2
protoporphyrin-IX + 3 H2O2
show the reaction diagram
-
-
-
?
protoporphyrinogen IX + 3 O2
protoporphyrin + 3 H2O2
show the reaction diagram
-
-
-
-
?
protoporphyrinogen-IX + 3 O2
protoporphyrin-IX + 3 H2O2
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
acifluorfen
NaCl
inhibits recombinant wild-type enzyme and mutant V311M
oxyfluorfen
a diphenyl ether herbicide, competitive, the mutant V311M is slightly less sensitive to inhibition than the wild-type enzyme, inhibition kinetics
acifluorfen
acifluorfen-methyl
-
weak
bilirubin
-
competitive
Biliverdin
-
competitive
carfentrazone-ethyl
-
22% less inhibition is found in T3 transgenic lines
hemin
-
competitive
oxyfluorfen
-
26% less inhibition is found in T3 transgenic lines
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
imidazole
activates recombinant wild-type enzyme and mutant V311M
Tween 20
activates recombinant wild-type enzyme and mutant V311M
n-octylglucopyranoside
-
1 mM results in maximal activity
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00067 - 0.00354
protoporphyrinogen IX
0.0032
protoporphyrinogen-IX
recombinant wild-type enzyme and recombinant mutant V311M, pH 7.4, 25°C
0.0053
Coproporphyrinogen-III
-
-
0.0049
Mesoporphyrinogen-IX
-
-
0.007
protoporphyrinogen IX
-
pH 7.5, 25°C
0.001 - 0.0063
protoporphyrinogen-IX
additional information
additional information
-
steady-state kinetics, overview
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00015 - 0.0181
protoporphyrinogen IX
12.9 - 14.1
protoporphyrinogen-IX
0.000833
Coproporphyrinogen-III
-
-
0.0448
Mesoporphyrinogen-IX
-
-
0.00000783 - 0.00317
protoporphyrinogen-IX
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.016 - 0.784
protoporphyrinogen IX
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.029 - 0.154
acifluorfen
0.011 - 0.0177
oxyfluorfen
4.4
bilirubin
-
-
4
Biliverdin
-
-
2.3
hemin
-
-
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.133 - 0.617
acifluorfen
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.4
recombinant wild-type enzyme
7.5
-
assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22
assay at room temperature
25
-
assay at
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
Bacillus subtilis PPO is unique among PPO family members in that it is a soluble monomer
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
malfunction
-
when PPO is inhibited, protoporphyrin IX accumulates to cause light-dependent membrane damage
metabolism
-
protoporphyrinogen oxidase acts in the tetrapyrrole biosynthetic pathway that leads to the formation of both heme and chlorophylls
physiological function
-
protoporphyrinogen oxidase is one of the most important action targets of commercial herbicides
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
53000
x * 53000, SDS-PAGE
56000
x * 56000, recombinant wild-type enzyme and mutant V311M, SDS-PAGE
54000
-
gel filtration
56000
-
SDS-PAGE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
Bacillus subtilis PPO is unique among PPO family members in that it is a soluble monomer
monomer
-
gel filtration, SDS-PAGE
additional information
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified native and SeMet-labeled enzyme in complex with inhibitor acifluorfen, sitting-drop, vapor-diffusion method, 10 mg/ml wild-type bsPPO protein in 50 mM TrisHCl, pH 7.5, 200 mM sodium chloride, 5 mM DTT, 1 mM EDTA, and acifluorfen in a 1:5 ratio, equilibrating against a reservoir solution of 2.4 M ammonium phosphate dibasic, 0.1 M Tris-HCl, pH 7.5, 1 week, 20°C, X-ray diffraction structure determination and analysis at 2.8-3.5 A resolution
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
F227R
the mutant shows reduced activity compared to the wild-type enzyme
I176A
the mutant shows reduced activity compared to the wild-type enzyme
K71A
the mutant shows reduced activity compared to the wild-type enzyme
P64A
the mutant shows reduced activity compared to the wild-type enzyme
V311M
site-directed mutagenesis, mutant shows similar kinetics compared to the wild-type enzyme, but slightly decreased activity dependent on pH, and NaCl, Tween 20, and imidazole concentrations
Y366A
site-directed mutagenesis, the mutant enzyme shows 10% of wild-type enzyme activity
Y366E
site-directed mutagenesis, the mutant enzyme shows 1% of wild-type enzyme activity
Y366H
site-directed mutagenesis, the mutant enzyme shows 10% of wild-type enzyme activity
additional information
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
Ni2+ affinity column chromatography
recombinant His-tagged wild-type and mutant V311M from Escherichia coli by nickel affinity chromatography
recombinant wild-type and mutant enzymes from Escherichia coli strain XL-1 Blue inclusion bodies
cloned enzyme
-
recombinant His-tagged enzyme from Escherichia coli by nickel affinity chromatography
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression of wild-type and mutant enzymes in Escherichia coli strain XL-1 Blue in inclusion bodies
expression of wild-type and mutant V311M as His-tagged enzymes in Escherichia coli strain BL21(DE3)
the enzyme gene is fused in its 3'-end to a polynucleotide encoding six histidine residues and expressed in both Escherichia coli BL21(DE3) cells and Bacillus subtilis 168A cells
expression of His-tagged enzyme in Escherichia coli
-
expression of the enzyme in transgenic rice plants using Agrobacterium tumefaciens transformation system
-
in Escherichia coli, His-tag inserted for purification
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
-
protoporphyrinogen oxidase is one of the most important action targets of commercial herbicides
additional information
-
transgenic rice lines are resistant to the herbicides carfentrazone-ethyl and oxyfluorfen
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Corrigall, A.V.; Siziba, K.B.; Maneli, M.H.; Shephard, E.G.; Ziman, M.; Dailey, T.A.; Dailey, H.A.; Kirsch, R.E.; Meissner, P.N.
Purification of and kinetic studies on a cloned protoporphyrinogen oxidase from the aerobic bacterium Bacillus subtilis
Arch. Biochem. Biophys.
358
251-256
1998
Bacillus subtilis
Manually annotated by BRENDA team
Jeong, E.; Han, O.
Purification and characterization of Bacillus subtilis protoporphyrinogen oxidase and pre-equilibrium behavior during oxidation of protoporphyrinogen IX
J. Biochem. Mol. Biol.
34
39-42
2001
Bacillus subtilis
-
Manually annotated by BRENDA team
Jeong, E.; Houn, T.; Kuk, Y.; Kim, E.S.; Chandru, H.K.; Baik, M.; Back, K.; Guh, J.O.; Han, O.
A point mutation of valine-311 to methionine in Bacillus subtilis protoporphyrinogen oxidase does not greatly increase resistance to the diphenyl ether herbicide oxyfluorfen
Bioorg. Chem.
31
389-397
2003
Bacillus subtilis (P32397), Bacillus subtilis
Manually annotated by BRENDA team
Jung, S.; Chung, J.S.; Chon, S.U.; Kuk, Y.I.; Lee, H.J.; Guh, J.O.; Back, K.
Expression of recombinant protoporphyrinogen oxidase influences growth and morphological characteristics in transgenic rice
Plant Growth Regul.
42
283-288
2004
Bacillus subtilis
-
Manually annotated by BRENDA team
Kuk, Y.I.; Lee, H.J.; Chung, J.S.; Kim, K.M.; Lee, S.B.; Ha, S.B.; Back, K.; Guh, J.O.
Expression of a Bacillus subtilis protoporphyrinogen oxidase gene in rice plants reduces sensitivity to peroxidizing herbicides
Biol. Plant.
49
577-583
2005
Bacillus subtilis, Oryza sativa
-
Manually annotated by BRENDA team
Sun, L.; Wen, X.; Tan, Y.; Li, H.; Yang, X.; Zhao, Y.; Wang, B.; Cao, Q.; Niu, C.; Xi, Z.
Site-directed mutagenesis and computational study of the Y366 active site in Bacillus subtilis protoporphyrinogen oxidase
Amino Acids
37
523-530
2009
Bacillus subtilis (P32397), Bacillus subtilis
Manually annotated by BRENDA team
Tan, Y.; Sun, L.; Xi, Z.; Yang, G.F.; Jiang, D.Q.; Yan, X.P.; Yang, X.; Li, H.Y.
A capillary electrophoresis assay for recombinant Bacillus subtilis protoporphyrinogen oxidase
Anal. Biochem.
383
200-204
2008
Bacillus subtilis
Manually annotated by BRENDA team
Qin, X.; Sun, L.; Wen, X.; Yang, X.; Tan, Y.; Jin, H.; Cao, Q.; Zhou, W.; Xi, Z.; Shen, Y.
Structural insight into unique properties of protoporphyrinogen oxidase from Bacillus subtilis
J. Struct. Biol.
170
76-82
2010
Bacillus subtilis (P32397), Bacillus subtilis
Manually annotated by BRENDA team
Hansson, M.; Gustafsson, M.C.; Kannangara, C.G.; Hederstedt, L.
Isolated Bacillus subtilis HemY has coproporphyrinogen III to coproporphyrin III oxidase activity
Biochim. Biophys. Acta
1340
97-104
1997
Bacillus subtilis (P32397), Bacillus subtilis 168 (P32397)
Manually annotated by BRENDA team