Literature summary for 1.13.12.19 extracted from

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 (2017), Proc. Natl. Acad. Sci. USA, 114, 4667-4672 .

Search for more literature for 1.13.12.19

Activating Compound

Activating Compound	Comment	Organism	Structure
ascorbate	stimulates	Pseudomonas savastanoi pv. phaseolicola
DTT	stimulates	Pseudomonas savastanoi pv. phaseolicola

Cloned(Commentary)

Cloned (Comment)	Organism
gene efe, recombinant expression of N-terminally His-tagged or N-terminally His-SUMO-tagged enzyme in Escherichia coli strain BL21(DE3)	Pseudomonas savastanoi pv. phaseolicola

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant enzyme in three structures: of enzyme in complex with manganese and 2-oxoglutarate, of enzyme in complex with with manganese and bis-Tris-propane buffer, and enzyme in complex with iron, L-Arg, and N-oxalylglycine, hanging drop vapour diffusion method, mixing of 0.003 ml of 12-20 mg/ml protein solution with 0.003 ml of reservoir solution containing 17.5-25% w/v PEG 3350 and PEG 6000, 0.1 M bis-Tris-propane, pH 7.0, 0.04 M sodium/potassium phosphate, 3-5 mM MnCl2, and with or without 10 mM 2-oxoglutarate, and equilibration against 0.5 ml reservoir solution, at 20°, microseeding, method optimization, X-ray diffraction structure determination and analysis at 1.08-1.55 A resolution, single-wavelength anomalous diffraction data obtained from selenomethionine-derivatized PsEFE:Mn:bis-Tris-propane crystals, structure modeling	Pseudomonas savastanoi pv. phaseolicola

Crystallization (Comment)

Organism

purified recombinant enzyme in three structures: of enzyme in complex with manganese and 2-oxoglutarate, of enzyme in complex with with manganese and bis-Tris-propane buffer, and enzyme in complex with iron, L-Arg, and N-oxalylglycine, hanging drop vapour diffusion method, mixing of 0.003 ml of 12-20 mg/ml protein solution with 0.003 ml of reservoir solution containing 17.5-25% w/v PEG 3350 and PEG 6000, 0.1 M bis-Tris-propane, pH 7.0, 0.04 M sodium/potassium phosphate, 3-5 mM MnCl2, and with or without 10 mM 2-oxoglutarate, and equilibration against 0.5 ml reservoir solution, at 20°, microseeding, method optimization, X-ray diffraction structure determination and analysis at 1.08-1.55 A resolution, single-wavelength anomalous diffraction data obtained from selenomethionine-derivatized PsEFE:Mn:bis-Tris-propane crystals, structure modeling

Pseudomonas savastanoi pv. phaseolicola

Protein Variants

Protein Variants	Comment	Organism
E84D	site-directed mutagenesis, the mutant does not produce ethylene	Pseudomonas savastanoi pv. phaseolicola
E84Q	site-directed mutagenesis, the mutant does not produce ethylene	Pseudomonas savastanoi pv. phaseolicola
R171A	site-directed mutagenesis, the mutant does not produce ethylene	Pseudomonas savastanoi pv. phaseolicola
R171K	site-directed mutagenesis, the mutant does not produce ethylene	Pseudomonas savastanoi pv. phaseolicola
R316A	site-directed mutagenesis, the mutant shows reduced ethylene production compared to the wild-type enzyme	Pseudomonas savastanoi pv. phaseolicola
R316K	site-directed mutagenesis, the mutant shows reduced ethylene production compared to the wild-type enzyme	Pseudomonas savastanoi pv. phaseolicola
Y172F	site-directed mutagenesis, the mutant shows reduced ethylene production compared to the wild-type enzyme	Pseudomonas savastanoi pv. phaseolicola

Inhibitors

Inhibitors	Comment	Organism	Structure
N-oxalylglycine	an unreactive 2-oxoglutarate analogue, enzyme binding structure analysis, overview	Pseudomonas savastanoi pv. phaseolicola

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Fe2+	dependent on, required for catalysis. The overall metal-binding mode of PsEFE is relatively typical for 2OG oxygenases, with the metal coordinated by His189 (C terminus of DSBH II), Asp191 (loop linking DSBH II and III), and His268 (N-terminus of DSBH VII)	Pseudomonas savastanoi pv. phaseolicola

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
2-oxoglutarate + O2	Pseudomonas savastanoi pv. phaseolicola	-	ethylene + 3 CO2 + H2O	-	?

Organism

Organism	UniProt	Comment	Textmining
Pseudomonas savastanoi pv. phaseolicola	P32021	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant enzyme from Escherichia coli strain BL21(DE3) to near homogeneity by affinityy chromatography, desalting gel filtration, tag cleavage by human SenP2 protease, followed by another step of affinityy chromatography, and gel filtration	Pseudomonas savastanoi pv. phaseolicola

Reaction

Reaction	Comment	Organism	Reaction ID
2-oxoglutarate + O2 = ethene + 3 CO2 + H2O	Pseudomonas syringae ethylene-forming enzyme reveal a branched mechanism. In one branch, an apparently typical 2-oxoglutarate oxygenase reaction to give succinate, carbon dioxide, and sometimes pyrroline-5-carboxylate occurs, reaction of EC 1.13.11.34. Alternatively, Grob-type oxidative fragmentation of a 2-oxoglutarate-derived intermediate occurs to give ethylene and carbon dioxide, EC 1.13.12.19. Fragmentation to give ethylene is promoted by binding of L-arginine in a nonoxidized conformation and of 2-oxoglutarate in an unprecedented high-energy conformation that favors ethylene, relative to succinate formation. Induced fit reaction mechanism, detailed overview	Pseudomonas savastanoi pv. phaseolicola	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
2-oxoglutarate + O2	-	Pseudomonas savastanoi pv. phaseolicola	ethylene + 3 CO2 + H2O	-	?
2-oxoglutarate + O2	the enzyme is dependent on 2-oxoglutarate	Pseudomonas savastanoi pv. phaseolicola	ethylene + 3 CO2 + H2O	-	?
additional information	Pseudomonas syringae ethylene-forming enzyme reveal a branched mechanism. In one branch, an apparently typical 2-oxoglutarate oxygenase reaction to give succinate, carbon dioxide, and sometimes pyrroline-5-carboxylate occurs, reaction of EC 1.13.11.34. Alternatively, Grob-type oxidative fragmentation of a 2-oxoglutarate-derived intermediate occurs to give ethylene and carbon dioxide, EC 1.13.12.19. Fragmentation to give ethylene is promoted by binding of L-arginine in a nonoxidized conformation and of 2-oxoglutarate in an unprecedented high-energy conformation that favors ethylene, relative to succinate formation. Role for Tyr192 in catalysis, substrate binding structures, structure-function analysis, overview	Pseudomonas savastanoi pv. phaseolicola	?	-	?

Subunits

Subunits	Comment	Organism
monomer	by SDS-PAGE and gel filtration	Pseudomonas savastanoi pv. phaseolicola
More	the overall PsEFE fold comprises 10 alpha-helices and 14 beta-strands, of which eight beta-strands (I-VIII) form the major and minor beta-sheets of the conserved distorted double-stranded beta helix (DSBH): the 2-oxoglutarate oxygenase characteristic fold. beta-Strands beta1 and beta2 at the N-terminus extend the major beta-sheet at the end of the DSBH away from the active site, beta-strands beta3 and beta6 extend the other end of the major beta-sheet close to the active site. alpha-Helices alpha2 and alpha5 bind across the surface of the major beta-sheet and likely stabilize it. A loop region (residues 80-93), located between beta3 and beta6, which harbors beta4 and beta5, acts as a lid partially covering the active site and provides residues that bind the L-Arg cofactor/substrate. Three alpha-helices (alpha8, alpha9, alpha10) at the C-terminus also contribute to the active site	Pseudomonas savastanoi pv. phaseolicola

Synonyms

Synonyms	Comment	Organism
PsEFE	-	Pseudomonas savastanoi pv. phaseolicola

General Information

General Information	Comment	Organism
evolution	emzyme PsEFE should be regarded as a hybrid of subgroups I and II, in terms of its classification	Pseudomonas savastanoi pv. phaseolicola