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Literature summary 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 .
    View publication on PubMedView publication on EuropePMC

Activating Compound

EC Number Activating Compound Comment Organism Structure
1.13.12.19 ascorbate stimulates Pseudomonas savastanoi pv. phaseolicola
1.13.12.19 DTT stimulates Pseudomonas savastanoi pv. phaseolicola
1.14.20.7 ascorbate
-
 Pseudomonas savastanoi pv. phaseolicola
1.14.20.7 dithiothreitol
-
 Pseudomonas savastanoi pv. phaseolicola

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.13.12.19 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
1.14.20.7 expressed in Escherichia coli BL21(DE3) cells Pseudomonas savastanoi pv. phaseolicola

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.13.12.19 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
1.14.20.7 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 Pseudomonas savastanoi pv. phaseolicola

Protein Variants

EC Number Protein Variants Comment Organism
1.13.12.19 E84D site-directed mutagenesis, the mutant does not produce ethylene Pseudomonas savastanoi pv. phaseolicola
1.13.12.19 E84Q site-directed mutagenesis, the mutant does not produce ethylene Pseudomonas savastanoi pv. phaseolicola
1.13.12.19 R171A site-directed mutagenesis, the mutant does not produce ethylene Pseudomonas savastanoi pv. phaseolicola
1.13.12.19 R171K site-directed mutagenesis, the mutant does not produce ethylene Pseudomonas savastanoi pv. phaseolicola
1.13.12.19 R316A site-directed mutagenesis, the mutant shows reduced ethylene production compared to the wild-type enzyme Pseudomonas savastanoi pv. phaseolicola
1.13.12.19 R316K site-directed mutagenesis, the mutant shows reduced ethylene production compared to the wild-type enzyme Pseudomonas savastanoi pv. phaseolicola
1.13.12.19 Y172F site-directed mutagenesis, the mutant shows reduced ethylene production compared to the wild-type enzyme Pseudomonas savastanoi pv. phaseolicola
1.14.20.7 C317A the mutant shows about 34% of wild type activity Pseudomonas savastanoi pv. phaseolicola
1.14.20.7 C317S the mutant shows about 21% of wild type activity Pseudomonas savastanoi pv. phaseolicola
1.14.20.7 E84D inactive Pseudomonas savastanoi pv. phaseolicola
1.14.20.7 E84Q inactive Pseudomonas savastanoi pv. phaseolicola
1.14.20.7 F175Y the mutant shows about 18% of wild type activity Pseudomonas savastanoi pv. phaseolicola
1.14.20.7 R171A inactive Pseudomonas savastanoi pv. phaseolicola
1.14.20.7 R171K inactive Pseudomonas savastanoi pv. phaseolicola
1.14.20.7 R316A the mutant shows about 3.7% of wild type activity Pseudomonas savastanoi pv. phaseolicola
1.14.20.7 R316K the mutant shows about 13% of wild type activity Pseudomonas savastanoi pv. phaseolicola
1.14.20.7 T86S the mutant shows about 31% of wild type activity Pseudomonas savastanoi pv. phaseolicola
1.14.20.7 T86V inactive Pseudomonas savastanoi pv. phaseolicola
1.14.20.7 V270T the mutant shows about 4.3 % of wild type activity Pseudomonas savastanoi pv. phaseolicola
1.14.20.7 Y192F the mutant shows about 5.6% of wild type activity Pseudomonas savastanoi pv. phaseolicola
1.14.20.7 Y318F the mutant shows about 65% of wild type activity Pseudomonas savastanoi pv. phaseolicola

Inhibitors

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

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.13.12.19 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
1.14.20.7 Fe2+ required Pseudomonas savastanoi pv. phaseolicola

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.13.12.19 2-oxoglutarate + O2 Pseudomonas savastanoi pv. phaseolicola
-
 ethylene + 3 CO2 + H2O
-
 ?
1.14.20.7 2-oxoglutarate + L-arginine + O2 Pseudomonas savastanoi pv. phaseolicola
-
 succinate + CO2 + guanidine + (S)-1-pyrroline-5-carboxylate + H2O
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.13.12.19 Pseudomonas savastanoi pv. phaseolicola P32021
-
-
1.14.20.7 Pseudomonas savastanoi pv. phaseolicola P32021
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.13.12.19 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
1.14.20.7 His-Trap column chromatography Pseudomonas savastanoi pv. phaseolicola

Reaction

EC Number Reaction Comment Organism Reaction ID
1.13.12.19 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)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.13.12.19 2-oxoglutarate + O2
-
 Pseudomonas savastanoi pv. phaseolicola ethylene + 3 CO2 + H2O
-
 ?
1.13.12.19 2-oxoglutarate + O2 the enzyme is dependent on 2-oxoglutarate Pseudomonas savastanoi pv. phaseolicola ethylene + 3 CO2 + H2O
-
 ?
1.13.12.19 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 ?
-
 ?
1.14.20.7 2-oxoglutarate + L-arginine + O2
-
 Pseudomonas savastanoi pv. phaseolicola succinate + CO2 + guanidine + (S)-1-pyrroline-5-carboxylate + H2O
-
 ?

Subunits

EC Number Subunits Comment Organism
1.13.12.19 monomer by SDS-PAGE and gel filtration Pseudomonas savastanoi pv. phaseolicola
1.13.12.19 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

EC Number Synonyms Comment Organism
1.13.12.19 PsEFE
-
 Pseudomonas savastanoi pv. phaseolicola
1.14.20.7 EFE
-
 Pseudomonas savastanoi pv. phaseolicola
1.14.20.7 ethylene-forming enzyme
-
 Pseudomonas savastanoi pv. phaseolicola

General Information

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