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Information on EC 1.14.20.7 - 2-oxoglutarate/L-arginine monooxygenase/decarboxylase (succinate-forming) and Organism(s) Pseudomonas savastanoi pv. phaseolicola and UniProt Accession P32021

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IUBMB Comments
This is one of two simultaneous reactions catalysed by the enzyme, which is responsible for ethylene production in bacteria of the Pseudomonas syringae group. In the other reaction [EC 1.13.12.19, 2-oxoglutarate dioxygenase (ethene-forming)] the enzyme catalyses the dioxygenation of 2-oxoglutarate forming ethene and three molecules of carbon dioxide.The enzyme catalyses two cycles of the ethene-forming reaction for each cycle of the succinate-forming reaction, so that the stoichiometry of the products ethene and succinate is 2:1.
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Pseudomonas savastanoi pv. phaseolicola
UNIPROT: P32021
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The taxonomic range for the selected organisms is: Pseudomonas savastanoi pv. phaseolicola
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
Reaction Schemes
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L-arginine
+
2-oxoglutarate
+
O2
=
succinate
+
CO2
+
guanidine
+
(S)-1-pyrroline-5-carboxylate
+
H2O
+
+
=
+
+
+
+
Synonyms
2-oxoglutarate-dependent oxygenase-type ethylene-forming-enzyme, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2-oxoglutarate-Fe(II) oxygenase
-
additional information
cf. EC 1.13.12.19
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
L-arginine + 2-oxoglutarate + O2 = succinate + CO2 + guanidine + (S)-1-pyrroline-5-carboxylate + H2O
show the reaction diagram
PATHWAY SOURCE
PATHWAYS
-
-
SYSTEMATIC NAME
IUBMB Comments
L-arginine,2-oxoglutarate:oxygen oxidoreductase (succinate-forming)
This is one of two simultaneous reactions catalysed by the enzyme, which is responsible for ethylene production in bacteria of the Pseudomonas syringae group. In the other reaction [EC 1.13.12.19, 2-oxoglutarate dioxygenase (ethene-forming)] the enzyme catalyses the dioxygenation of 2-oxoglutarate forming ethene and three molecules of carbon dioxide.The enzyme catalyses two cycles of the ethene-forming reaction for each cycle of the succinate-forming reaction, so that the stoichiometry of the products ethene and succinate is 2:1.
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2-oxoadipate + L-arginine + O2
glutarate + CO2 + guanidine + (S)-1-pyrroline-5-carboxylate + H2O
show the reaction diagram
-
-
-
?
2-oxoglutarate + L-arginine + O2
succinate + CO2 + 5-hydroxy-L-arginine
show the reaction diagram
-
-
-
?
2-oxoglutarate + L-arginine + O2
succinate + CO2 + guanidine + (S)-1-pyrroline-5-carboxylate + H2O
show the reaction diagram
-
-
-
?
2-oxoglutarate + O2
ethylene + CO2
show the reaction diagram
-
-
-
?
3 2-oxoglutarate + L-arginine + 3 O2
2 C2H4 + succinate + 7 CO2 + 3 H2O + guanidine + L-DELTA1-pyrroline-5-carboxylate
show the reaction diagram
cf. EC 1.14.11.34, reaction, mechanism of the two reaction catalyzed at the same time, overview. Enzyme EFE converts 2-oxoglutarate into ethylene plus three CO2 molecules while also catalyzing the C5 hydroxylation of L-arginine driven by the oxidative decarboxylation of 2-oxoglutarate to form succinate and CO2
-
-
?
L-arginine + 2-oxoglutarate + O2
succinate + CO2 + guanidine + (S)-1-pyrroline-5-carboxylate + H2O
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
2-oxoglutarate + L-arginine + O2
succinate + CO2 + 5-hydroxy-L-arginine
show the reaction diagram
-
-
-
?
2-oxoglutarate + L-arginine + O2
succinate + CO2 + guanidine + (S)-1-pyrroline-5-carboxylate + H2O
show the reaction diagram
-
-
-
?
2-oxoglutarate + O2
ethylene + CO2
show the reaction diagram
-
-
-
?
L-arginine + 2-oxoglutarate + O2
succinate + CO2 + guanidine + (S)-1-pyrroline-5-carboxylate + H2O
show the reaction diagram
-
-
-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ascorbate
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.031
2-oxoadipate
pH 7.5, 25°C, recombinant enzyme
0.057
2-oxoglutarate
pH 7.5, 25°C, recombinant enzyme
0.05 - 0.071
L-arginine
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0042
2-oxoadipate
pH 7.5, 25°C, recombinant enzyme
2.067
2-oxoglutarate
pH 7.5, 25°C, recombinant enzyme
0.0045 - 0.048
L-arginine
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.134
2-oxoadipate
pH 7.5, 25°C, recombinant enzyme
36.26
2-oxoglutarate
pH 7.5, 25°C, recombinant enzyme
0.97
L-arginine
at pH 7.5 and 25°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 9
over 50% of maximal activity within this range
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
physiological function
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION?
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
39370
recombinant detagged enzyme, gel filtration
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
1 * 40000, recombinant detagged enzyme, SDS-PAGE, 1 * 39670, recombinant detagged enzyme, mass spectrometry
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
analysis of crystal structure of the enzyme in complex with several ligands, detailed overview
in complex with manganese and 2-oxoglutarate, with manganese and the buffer bis-Tris-propane, and in complex with iron, L-Arg, and N-oxalylglycine, sitting drop vapor diffusion method, using 17.5-25% (w/v) polyethylene glycol (PEG) 3350 and 6000
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A198V
A199G
the mutant shows reduced activity compared to the wild type enzyme
A218V
the mutant shows about 3% of wild type activity
A281V
site-directed mutagenesis, the mutant produces low levels of products in comparison to the wild-type enzyme
C317A
the mutant shows about 34% of wild type activity
C317S
the mutant shows about 21% of wild type activity
D191E
the D191E variant degrades L-Arg and 2-oxoglutarateto pyrroline-5-carboxylate (again detected after reduction to proline and Fmoc derivatization) and succinate nearly stoichiometrically, with only about 5% of the cosubstrate being fragmented to ethylene
E213A
the mutant shows about 90% of wild type activity
E213A/E215A
the mutant shows about 5% of wild type activity
E215A
the mutant shows about 5% of wild type activity
E235D
the mutant shows about wild type activity
E285A
the mutant shows about 10% of wild type activity
E285Q
the mutant shows about 25% of wild type activity
F175Y
the mutant shows about 18% of wild type activity
F278Y
the mutant shows about wild type activity
F283A
F283R
F283V
F283W
the mutant shows about 20% of wild type activity
F283Y
F310R
the mutant shows about 3% of wild type activity
F310W
the mutant shows about 30% of wild type activity
H116Q
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
H169Q
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
H233Q
site-directed mutagenesis, inactive mutant
H284Q
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
H309Q
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
I254M
the mutant shows about wild type activity
I304N
the mutant shows reduced activity compared to the wild type enzyme
I322V
the mutant shows about wild type activity
L22M
the mutant shows about wild type activity
L73K
the mutant shows about 60% of wild type activity
L73R
the mutant shows about 40% of wild type activity
R171A
R184A
the mutant shows about 75% of wild type activity
R277A
site-directed mutagenesis, the mutant is expressed in inclusion bodies
R316A
the mutant shows about 3.7% of wild type activity
R316K
the mutant shows about 13% of wild type activity
S81R
the mutant shows about 5% of wild type activity
S81Y
the mutant shows about 5% of wild type activity
T86S
the mutant shows about 31% of wild type activity
V172T
the mutant shows about wild type activity
V196F
V212Y/E213S
the mutant shows reduced activity compared to the wild type enzyme
V270T
the mutant shows about 4.3 % of wild type activity
Y192F
the mutant shows about 5.6% of wild type activity
Y306A
the mutant shows about 3% of wild type activity
Y306F
the mutant shows about 5% of wild type activity
Y318F
the mutant shows about 65% of wild type activity
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
His-Trap column chromatography
recombinant His-tagged enzyme from Escherichia coli strain BL21 Gold (DE3) by nickel affinity chromatography, tag cleavage by a TEV protease mutant, and another step of nickel affinity chromatography, and dialysis of the flow through
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) cells
gene efe, large scale expression of His6-tagged enzyme in Escherichia coli strain BL21 Gold (DE3), method optimization and evaluation
recombinant expression of wild-type and mutant enzymes
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Martinez, S.; Hausinger, R.P.
Biochemical and spectroscopic characterization of the non-heme Fe(II)- and 2-oxoglutarate-dependent ethylene-forming enzyme from Pseudomonas syringae pv. phaseolicola PK2
Biochemistry
55
5989-5999
2016
Pseudomonas savastanoi pv. phaseolicola (P32021), Pseudomonas savastanoi pv. phaseolicola PK2 (P32021)
Manually annotated by BRENDA team
Johansson, N.; Persson, K.O.; Larsson, C.; Norbeck, J.
Comparative sequence analysis and mutagenesis of ethylene forming enzyme (EFE) 2-oxoglutarate/Fe(II)-dependent dioxygenase homologs
BMC Biochem.
15
22
2014
no activity in Penicillium chrysogenum, Penicillium digitatum (K9GDR0), Pseudomonas savastanoi pv. phaseolicola (P32021)
Manually annotated by BRENDA team
Martinez, S.; Fellner, M.; Herr, C.Q.; Ritchie, A.; Hu, J.; Hausinger, R.P.
Structures and mechanisms of the non-heme Fe(II)- and 2-oxoglutarate-dependent ethylene-forming enzyme Substrate binding creates a twist
J. Am. Chem. Soc.
139
11980-11988
2017
Pseudomonas savastanoi pv. phaseolicola (P32021), Pseudomonas savastanoi pv. phaseolicola PK2 (P32021)
Manually annotated by BRENDA team
Zhang, Z.; Smart, T.J.; Choi, H.; Hardy, F.; Lohans, C.T.; Abboud, M.I.; Richardson, M.S.W.; Paton, R.S.; McDonough, M.A.; Schofield, C.J.
Structural and stereoelectronic insights into oxygenase-catalyzed formation of ethylene from 2-oxoglutarate
Proc. Natl. Acad. Sci. USA
114
4667-4672
2017
Pseudomonas savastanoi pv. phaseolicola (P32021)
Manually annotated by BRENDA team
Copeland, R.A.; Davis, K.M.; Shoda, T.K.C.; Blaesi, E.J.; Boal, A.K.; Krebs, C.; Bollinger, J.M.
An iron(IV)-oxo intermediate initiating L-arginine oxidation but not ethylene production by the 2-oxoglutarate-dependent oxygenase, ethylene-forming enzyme
J. Am. Chem. Soc.
143
2293-2303
2021
Pseudomonas savastanoi pv. phaseolicola (P32021)
Manually annotated by BRENDA team
Xue, J.; Lu, J.; Lai, W.
Mechanistic insights into a non-heme 2-oxoglutarate-dependent ethylene-forming enzyme selectivity of ethylene-formation versusl-Arg hydroxylation
Phys. Chem. Chem. Phys.
21
9957-9968
2019
Pseudomonas savastanoi pv. phaseolicola (P32021)
Manually annotated by BRENDA team