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Literature summary extracted from

  • Martinez, S.; Wu, R.; Krzywda, K.; Opalka, V.; Chan, H.; Liu, D.; Holz, R.C.
    Analyzing the catalytic role of active site residues in the Fe-type nitrile hydratase from Comamonas testosteroni Ni1 (2015), J. Biol. Inorg. Chem., 20, 885-894 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
4.2.1.84 expression in Escherichia coli BL21(DE3) Comamonas testosteroni

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
4.2.1.84 hanging drop vapor diffusion method, X-ray crystal structures of the alphaH80A/alphaH81A, alphaH80W/alphaH81W, and alphaR157A mutant CtNHase enzymes are solved to 2.0, 2.8, and 2.5 A resolutions, respectively Comamonas testosteroni

Protein Variants

EC Number Protein Variants Comment Organism
4.2.1.84 H80A mutant of alpha-subunit, activity (kcat = 220/s) accounts for about 20% of the wild-type activity (kcat = 1100/s) while the Km value is slightly reduced (187 mM) Comamonas testosteroni
4.2.1.84 H80A/H81A mutant of alpha-subunit, activity (kcat = 132/s) accounts for 12% of the wild-type activity (kcat = 1100/s) while the Km value is nearly unchanged at 205 mM. Hydrogen-bonding interactions crucial for the catalytic function of the alphaCys104-SOH ligand are disrupted. Disruption of these hydrogen bonding interactions likely alters the nucleophilicity of the sulfenic acid oxygen and the Lewis acidity of the active site Fe(III) ion Comamonas testosteroni
4.2.1.84 H80W/H81W mutant of alpha-subunit, disruption of these hydrogen bonding interactions likely alters the nucleophilicity of the sulfenic acid oxygen and the Lewis acidity of the active site Fe(III) ion Comamonas testosteroni
4.2.1.84 H81A mutant of alpha-subunit, activity (kcat = 77/s) accounts for 4% of the wild-type activity (kcat = 1100/s) while the Km value is slightly reduced (179 mM) Comamonas testosteroni
4.2.1.84 R157A mutant of alpha-subunit, activity (kcat = 10/s) accounts for less than 1% of the wild-type activity (kcat = 1100/s) while the Km value is nearly unchanged at 205 mM Comamonas testosteroni
4.2.1.84 R157A mutant of alpha-subunit, disruption of these hydrogen bonding interactions likely alters the nucleophilicity of the sulfenic acid oxygen and the Lewis acidity of the active site Fe(III) ion Comamonas testosteroni

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
4.2.1.84 179
-
 acrylonitrile pH 7.0, 25°C, alpha-subunit mutant enzyme H81A Comamonas testosteroni
4.2.1.84 187
-
 acrylonitrile pH 7.0, 25°C, alpha-subunit mutant enzyme H80A Comamonas testosteroni
4.2.1.84 204
-
 acrylonitrile pH 7.0, 25°C, alpha-subunit mutant enzyme R157A Comamonas testosteroni
4.2.1.84 213
-
 acrylonitrile pH 7.0, 25°C, alpha-subunit mutant enzyme H80A/H81A Comamonas testosteroni
4.2.1.84 232
-
 acrylonitrile pH 7.0, 25°C, alpha-subunit mutant enzyme H80W/H81W Comamonas testosteroni
4.2.1.84 239
-
 acrylonitrile pH 7.0, 25°C, alpha-subunit mutant enzyme R157K Comamonas testosteroni
4.2.1.84 250
-
 acrylonitrile pH 7.0, 25°C, wild-type enzyme Comamonas testosteroni

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
4.2.1.84 Fe3+ Fe-type nitrile hydratase. Iron content of wild-type enzyme is 1.4 equivalents of iron per (alphabeta)2 heterotetramer. Mutating the active site alphaH80, alphaHs81, and alphaR157 residues have little or no effect on the incorporation of iron into the active site. In the alpha-subunit mutant enzyme (H80W/H81W, H80A/H81A and R157A), hydrogen-bonding interactions crucial for the catalytic function of the alphaCys104-SOH ligand are disrupted. Disruption of these hydrogen bonding interactions likely alters the nucleophilicity of the sulfenic acid oxygen and the Lewis acidity of the active site Fe(III) ion Comamonas testosteroni

Organism

EC Number Organism UniProt Comment Textmining
4.2.1.84 Comamonas testosteroni J9PBS0 AND J9PBS1 J9PBS0: alpha-subunit, J9PBS1: beta-subunit
-
4.2.1.84 Comamonas testosteroni Ni1 J9PBS0 AND J9PBS1 J9PBS0: alpha-subunit, J9PBS1: beta-subunit
-

Purification (Commentary)

EC Number Purification (Comment) Organism
4.2.1.84
-
 Comamonas testosteroni

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4.2.1.84 acrylonitrile + H2O
-
 Comamonas testosteroni ?
-
 ?
4.2.1.84 acrylonitrile + H2O
-
 Comamonas testosteroni Ni1 ?
-
 ?

Subunits

EC Number Subunits Comment Organism
4.2.1.84 heterotetramer
-
 Comamonas testosteroni

Synonyms

EC Number Synonyms Comment Organism
4.2.1.84 CtNHase
-
 Comamonas testosteroni

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
4.2.1.84 10
-
 acrylonitrile pH 7.0, 25°C, alpha-subunit mutant enzyme R157A Comamonas testosteroni
4.2.1.84 32
-
 acrylonitrile pH 7.0, 25°C, alpha-subunit mutant enzyme R157K Comamonas testosteroni
4.2.1.84 77
-
 acrylonitrile pH 7.0, 25°C, alpha-subunit mutant enzyme H81A Comamonas testosteroni
4.2.1.84 79
-
 acrylonitrile pH 7.0, 25°C, alpha-subunit mutant enzyme H80W/H81W Comamonas testosteroni
4.2.1.84 132
-
 acrylonitrile pH 7.0, 25°C, alpha-subunit mutant enzyme H80A/H81A Comamonas testosteroni
4.2.1.84 220
-
 acrylonitrile pH 7.0, 25°C, alpha-subunit mutant enzyme H80A Comamonas testosteroni
4.2.1.84 1100
-
 acrylonitrile pH 7.0, 25°C, wild-type enzyme Comamonas testosteroni