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Literature summary extracted from

  • de Geus, D.C.; Thomassen, E.A.; Hagedoorn, P.L.; Pannu, N.S.; van Duijn, E.; Abrahamass spectrometry, J.P.
    Crystal structure of chlorite dismutase, a detoxifying enzyme producing molecular oxygen (2009), J. Mol. Biol., 387, 192-206.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.13.11.49 enzyme gene chemically synthesized without signal sequence, cloned into vector pET28a with histidine tag and thrombin cleavage site (removed before crystallization), and overexpressed Azospira oryzae

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.13.11.49 thiocyanate inhibited enzyme with incorporated heme, crystals grown from well solution containing 100 mM Mes buffer (pH 5.5), 25% polyethylene glycol monomethyl ether 2000, 0.3 M KSCN, 5% glycerol, 160-260 mM (NH4)2SO4, before data collection crystals are soaked in a solution of the mother liquor including 16% glycerol, crystal is flash-frozen and kept at -173°C (100 K) for multiple anomalous dispersion (MAD) with synchrotron radiation Azospira oryzae

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.13.11.49 Fe2+ activating compound Azospira oryzae

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.13.11.49 30000
-
 4 * 30000 Da, elution position on a gel-filtration column using Stokes radius to determine molecular weight points to globular tetramer, collisional activation of the ions ejects one monomer of the pentamer, so that low-charged tetramer counter complex ions are also observed with native mass spectrometry Azospira oryzae
1.13.11.49 30000
-
 5 * 30000 Da, tandem mass spectrometry, native mass spectrometry, active state in solution Azospira oryzae
1.13.11.49 30000
-
 6 * 30000, multiple anomalous dispersion crystal structure, one heme per monomer (histidine 170 axial heme ligand), arginine 183 probably with essential role in substrate positioning and activation, activity of the hexamer not measurable because of the presence of the thiocyanate inhibitor, each monomer appears to be acting on its own, therefore, different quaternary states may be biologically relevant depending on protein concentration, pH, or other factors Azospira oryzae
1.13.11.49 30420
-
 monomer without heme, native mass spectrometry, enzyme buffer-exchanged to 50 mM ammonium acetate, pH 6.8, with centrifugal filter units with 30 kDa cutoff Azospira oryzae
1.13.11.49 31030
-
 monomer with heme, native mass spectrometry, enzyme buffer-exchanged to 50 mM ammonium acetate, pH 6.8, with centrifugal filter units with 30 kDa cutoff Azospira oryzae
1.13.11.49 31032
-
 1 * 30420 without heme, 1 * 31032 with heme Azospira oryzae
1.13.11.49 155000
-
 pentamer, native mass spectrometry, native mass spectrometry, enzyme buffer-exchanged to 50 mM ammonium acetate, pH 6.8, with centrifugal filter units with 30 kDa cutoff Azospira oryzae

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.13.11.49 chlorite Azospira oryzae catalytic mechanism via ferryl species and ClO--anion chloride + O2
-
 ?
1.13.11.49 chlorite Azospira oryzae GR-1 catalytic mechanism via ferryl species and ClO--anion chloride + O2
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.13.11.49 Azospira oryzae E2DI02
-
-
1.13.11.49 Azospira oryzae GR-1 E2DI02
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.13.11.49 HiLoad 16/60 Superdex 200 prep-grade column, equilibrated with 20 mM Tris-HCl (pH 7.5) and 135 mM NaCl to determin molecular Stokes radius Azospira oryzae

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.13.11.49 1500
-
 100 mM K3PO4 (pH 7.0), 25°C, oxygen removal by bubbling with high-purity argon, measurement with modified Clarke electrode Azospira oryzae

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.13.11.49 chlorite catalytic mechanism via ferryl species and ClO--anion Azospira oryzae chloride + O2
-
 ?
1.13.11.49 chlorite catalytic mechanism via ferryl species and ClO--anion Azospira oryzae GR-1 chloride + O2
-
 ?

Subunits

EC Number Subunits Comment Organism
1.13.11.49 hexamer 6 * 30000, multiple anomalous dispersion crystal structure, one heme per monomer (histidine 170 axial heme ligand), arginine 183 probably with essential role in substrate positioning and activation, activity of the hexamer not measurable because of the presence of the thiocyanate inhibitor, each monomer appears to be acting on its own, therefore, different quaternary states may be biologically relevant depending on protein concentration, pH, or other factors Azospira oryzae
1.13.11.49 monomer 1 * 30420 without heme, 1 * 31032 with heme Azospira oryzae
1.13.11.49 pentamer 5 * 30000 Da, tandem mass spectrometry, native mass spectrometry, active state in solution Azospira oryzae
1.13.11.49 tetramer 4 * 30000 Da, elution position on a gel-filtration column using Stokes radius to determine molecular weight points to globular tetramer, collisional activation of the ions ejects one monomer of the pentamer, so that low-charged tetramer counter complex ions are also observed with native mass spectrometry Azospira oryzae

Cofactor

EC Number Cofactor Comment Organism Structure
1.13.11.49 heme
-
 Azospira oryzae