Any feedback?
Please rate this page
(literature.php)
(0/150)

BRENDA support

Literature summary extracted from

  • Miyanaga, A.; Fushinobu, S.; Ito, K.; Shoun, H.; Wakagi, T.
    Mutational and structural analysis of cobalt-containing nitrile hydratase on substrate and metal binding (2004), Eur. J. Biochem., 271, 429-438.
    View publication on PubMed

Activating Compound

EC Number Activating Compound Comment Organism Structure
4.2.1.84 nitrile hydratase activator protein residue aTyr114 may be involved in the interaction with the nitrile hydratase activator protein of Pseudomonas thermophila Pseudonocardia thermophila

Cloned(Commentary)

EC Number Cloned (Comment) Organism
4.2.1.84 expression of wild-type and mutant enzymes in Escherichia coli strain JM109 Pseudonocardia thermophila

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
4.2.1.84 purified recombinant apoenzyme and mutant enzymes, sitting drop vapor diffusion method, 8 mg/ml protein, with a reservoir solution containing 1.4 M tri-sodium citrate, and 0.1 M HEPES-NaOH, pH 7.5, 5°C, crystals of a complex with n-butyric acid are prepared by soaking a native crystal in a reservoir solution containing 15 mM n-butyric acid for 3 h, X-ray diffraction structure determination and analysis at 1.63-2.0 A resolution, molecular replacement and modeling Pseudonocardia thermophila

Protein Variants

EC Number Protein Variants Comment Organism
4.2.1.84 alphaT109S site-directed mutagenesis, the mutant shows similar characteristics compared to the wild-type enzyme Pseudonocardia thermophila
4.2.1.84 alphaY114T site-directed mutagenesis, the mutant shows a very low cobalt content and catalytic activity compared to the wild-type enzyme, and oxidative modifications of aCys111 and aCys113 residues are not observed Pseudonocardia thermophila
4.2.1.84 betaY68F site-directed mutagenesis, the mutant shows an elevated Km value and a significantly decreased kcat value compared to the wild-type enzyme Pseudonocardia thermophila
4.2.1.84 T109S similar characteristics to the wild-type enzyme Pseudonocardia thermophila
4.2.1.84 Y114T very low cobalt content and catalytic activity compared to the wild-type enzyme Pseudonocardia thermophila

Inhibitors

EC Number Inhibitors Comment Organism Structure
4.2.1.84 benzoic acid competitive inhibitor; competitive inhibitor Pseudonocardia thermophila
4.2.1.84 n-butyric acid competitive inhibitor, the hydroxyl group of enzyme residue betaTyr68 forms hydrogen bonds with both n-butyric acid and residue alphaSer112, which is located in the active center. Butyric acid acts as a stabilizer of Fe-type NHase, Co-type NHase is more stable; competitive inhibitor, the hydroxyl group of enzyme residue betaTyr68 forms hydrogen bonds with both n-butyric acid and residue alphaSer112, which is located in the active center. Butyric acid acts as a stabilizer of Fe-type NHase, Co-type NHase is more stable Pseudonocardia thermophila
4.2.1.84 propionic acid competitive inhibitor; competitive inhibitor Pseudonocardia thermophila

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
4.2.1.84 additional information
-
 additional information the wild-type enzyme shows significantly lower Km values for aromatic substrates than for aliphatic ones Pseudonocardia thermophila
4.2.1.84 0.02
-
 Benzonitrile pH 7.6, 25°C, recombinant wild-type enzyme Pseudonocardia thermophila
4.2.1.84 0.025
-
 Benzonitrile pH 7.6, 25°C, recombinant mutant alphaT109S Pseudonocardia thermophila
4.2.1.84 0.079
-
 4-cyanopyridine pH 7.6, 25°C, recombinant wild-type enzyme Pseudonocardia thermophila
4.2.1.84 0.11
-
 Benzonitrile pH 7.6, 25°C, recombinant mutant alphaY114T Pseudonocardia thermophila
4.2.1.84 0.12
-
 3-Cyanopyridine pH 7.6, 25°C, recombinant wild-type enzyme Pseudonocardia thermophila
4.2.1.84 0.23
-
 Benzonitrile pH 7.6, 25°C, recombinant mutant betaY68F Pseudonocardia thermophila
4.2.1.84 0.49
-
 Methacrylonitrile pH 7.6, 25°C, recombinant wild-type enzyme Pseudonocardia thermophila
4.2.1.84 2.3
-
 Methacrylonitrile pH 7.6, 25°C, recombinant mutant alphaY114T Pseudonocardia thermophila
4.2.1.84 2.6
-
 Methacrylonitrile pH 7.6, 25°C, recombinant mutant betaY68F Pseudonocardia thermophila
4.2.1.84 3.6
-
 acrylonitrile pH 7.6, 25°C, recombinant wild-type enzyme Pseudonocardia thermophila
4.2.1.84 4.7
-
 Methacrylonitrile pH 7.6, 25°C, recombinant mutant alphaT109S Pseudonocardia thermophila
4.2.1.84 9.9
-
 acrylonitrile pH 7.6, 25°C, recombinant mutant alphaY114T Pseudonocardia thermophila
4.2.1.84 58
-
 acrylonitrile pH 7.6, 25°C, recombinant mutant betaY68F Pseudonocardia thermophila
4.2.1.84 107
-
 acrylonitrile pH 7.6, 25°C, recombinant mutant alphaT109S Pseudonocardia thermophila

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
4.2.1.84 Co2+ cobalt-containing nitrile hydratase Pseudonocardia thermophila
4.2.1.84 additional information the apoenzyme shows no detectable activity, a disulfide bond between highly conserved Co-binding residues alphaCys108 and alphaCys113 is formed in the apoenzyme structure Pseudonocardia thermophila

Organism

EC Number Organism UniProt Comment Textmining
4.2.1.84 Pseudonocardia thermophila
-
-
-
4.2.1.84 Pseudonocardia thermophila Q7SID2 alpha-subunit
-
4.2.1.84 Pseudonocardia thermophila Q7SID3 beta-subunit
-
4.2.1.84 Pseudonocardia thermophila JCM 3095
-
-
-
4.2.1.84 Pseudonocardia thermophila JCM 3095 Q7SID2 alpha-subunit
-
4.2.1.84 Pseudonocardia thermophila JCM 3095 Q7SID3 beta-subunit
-

Purification (Commentary)

EC Number Purification (Comment) Organism
4.2.1.84 recombinant soluble wild-type and mutant enzymes from Escherichia coli strain JM109 by ammonium sulfate fractionation and two different steps of anion echange chromatography Pseudonocardia thermophila

Storage Stability

EC Number Storage Stability Organism
4.2.1.84 4°C, purified recombinant wild-type enzyme, retains complete activity for 1 year, and the catalytic center does not change as confimed by analysis of the crystal structure Pseudonocardia thermophila

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4.2.1.84 3-cyanopyridine + H2O substrate of recombinant wild-type enzyme, not of mutant enzymes Pseudonocardia thermophila nicotinamide
-
 ?
4.2.1.84 3-cyanopyridine + H2O substrate of recombinant wild-type enzyme, not of mutant enzymes Pseudonocardia thermophila JCM 3095 nicotinamide
-
 ?
4.2.1.84 4-cyanopyridine + H2O substrate of recombinant wild-type enzyme, not of mutant enzymes Pseudonocardia thermophila isonicotinamide
-
 ?
4.2.1.84 4-cyanopyridine + H2O substrate of recombinant wild-type enzyme, not of mutant enzymes Pseudonocardia thermophila JCM 3095 isonicotinamide
-
 ?
4.2.1.84 acrylonitrile + H2O substrate of recombinant wild-type and mutant enzymes Pseudonocardia thermophila acrylamide
-
 ?
4.2.1.84 acrylonitrile + H2O substrate of recombinant wild-type and mutant enzymes Pseudonocardia thermophila JCM 3095 acrylamide
-
 ?
4.2.1.84 benzonitrile + H2O substrate of recombinant wild-type and mutant enzymes Pseudonocardia thermophila benzamide
-
 ?
4.2.1.84 benzonitrile + H2O substrate of recombinant wild-type and mutant enzymes Pseudonocardia thermophila JCM 3095 benzamide
-
 ?
4.2.1.84 methacrylonitrile + H2O substrate of recombinant wild-type and mutant enzymes Pseudonocardia thermophila methacrylamide
-
 ?
4.2.1.84 methacrylonitrile + H2O substrate of recombinant wild-type and mutant enzymes Pseudonocardia thermophila JCM 3095 methacrylamide
-
 ?

Synonyms

EC Number Synonyms Comment Organism
4.2.1.84 cobalt-containing nitrile hydratase
-
 Pseudonocardia thermophila
4.2.1.84 NHase
-
 Pseudonocardia thermophila

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
4.2.1.84 25
-
 assay at Pseudonocardia thermophila

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
4.2.1.84 4.9
-
 Benzonitrile pH 7.6, 25°C, recombinant mutant alphaY114T Pseudonocardia thermophila
4.2.1.84 7.4
-
 Benzonitrile pH 7.6, 25°C, recombinant mutant betaY68F Pseudonocardia thermophila
4.2.1.84 14.1
-
 Methacrylonitrile pH 7.6, 25°C, recombinant mutant betaY68F Pseudonocardia thermophila
4.2.1.84 15.2
-
 acrylonitrile pH 7.6, 25°C, recombinant mutant betaY68F Pseudonocardia thermophila
4.2.1.84 33.9
-
 acrylonitrile pH 7.6, 25°C, recombinant mutant alphaY114T Pseudonocardia thermophila
4.2.1.84 34.6
-
 Methacrylonitrile pH 7.6, 25°C, recombinant mutant alphaY114T Pseudonocardia thermophila
4.2.1.84 90
-
 4-cyanopyridine pH 7.6, 25°C, recombinant wild-type enzyme Pseudonocardia thermophila
4.2.1.84 123
-
 Benzonitrile pH 7.6, 25°C, recombinant wild-type enzyme Pseudonocardia thermophila
4.2.1.84 131
-
 3-Cyanopyridine pH 7.6, 25°C, recombinant wild-type enzyme Pseudonocardia thermophila
4.2.1.84 132
-
 Benzonitrile pH 7.6, 25°C, recombinant mutant alphaT109S Pseudonocardia thermophila
4.2.1.84 482
-
 Methacrylonitrile pH 7.6, 25°C, recombinant mutant alphaT109S Pseudonocardia thermophila
4.2.1.84 621
-
 acrylonitrile pH 7.6, 25°C, recombinant mutant alphaT109S Pseudonocardia thermophila
4.2.1.84 1000
-
 Methacrylonitrile pH 7.6, 25°C, recombinant wild-type enzyme Pseudonocardia thermophila
4.2.1.84 1910
-
 acrylonitrile pH 7.6, 25°C, recombinant wild-type enzyme Pseudonocardia thermophila

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
4.2.1.84 7.6
-
 assay at Pseudonocardia thermophila

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
4.2.1.84 additional information
-
 additional information the wild-type enzyme shows significantly lower Ki values for aromatic inhibitors than for aliphatic ones Pseudonocardia thermophila
4.2.1.84 0.033
-
 benzoic acid pH 7.6, 25°C, recombinant wild-type enzyme Pseudonocardia thermophila
4.2.1.84 1.3
-
 n-butyric acid pH 7.6, 25°C, recombinant wild-type enzyme Pseudonocardia thermophila
4.2.1.84 9.9
-
 propionic acid pH 7.6, 25°C, recombinant wild-type enzyme Pseudonocardia thermophila

General Information

EC Number General Information Comment Organism
4.2.1.84 additional information in Co-type NHase, the side-chain of alphaThr109, located iin the cysteine cluster region, undergoes a hydrophobic interaction with the side-chain of alphaVal136. The hydroxyl group of residue alphaTyr114, located near the cysteine cluster region, forms hydrogen bonds with the main-chain oxygen atoms of alphaLeu119 and alphaLeu121, via a water molecule Pseudonocardia thermophila

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
4.2.1.84 0.26
-
 acrylonitrile pH 7.6, 25°C, recombinant mutant betaY68F Pseudonocardia thermophila
4.2.1.84 3.4
-
 acrylonitrile pH 7.6, 25°C, recombinant mutant alphaY114T Pseudonocardia thermophila
4.2.1.84 5.4
-
 Methacrylonitrile pH 7.6, 25°C, recombinant mutant betaY68F Pseudonocardia thermophila
4.2.1.84 5.8
-
 acrylonitrile pH 7.6, 25°C, recombinant mutant alphaT109S Pseudonocardia thermophila
4.2.1.84 15
-
 Methacrylonitrile pH 7.6, 25°C, recombinant mutant alphaY114T Pseudonocardia thermophila
4.2.1.84 32
-
 Benzonitrile pH 7.6, 25°C, recombinant mutant betaY68F Pseudonocardia thermophila
4.2.1.84 45
-
 Benzonitrile pH 7.6, 25°C, recombinant mutant alphaY114T Pseudonocardia thermophila
4.2.1.84 103
-
 Methacrylonitrile pH 7.6, 25°C, recombinant mutant alphaT109S Pseudonocardia thermophila
4.2.1.84 537
-
 acrylonitrile pH 7.6, 25°C, recombinant wild-type enzyme Pseudonocardia thermophila
4.2.1.84 1090
-
 3-Cyanopyridine pH 7.6, 25°C, recombinant wild-type enzyme Pseudonocardia thermophila
4.2.1.84 1140
-
 4-cyanopyridine pH 7.6, 25°C, recombinant wild-type enzyme Pseudonocardia thermophila
4.2.1.84 2040
-
 Methacrylonitrile pH 7.6, 25°C, recombinant wild-type enzyme Pseudonocardia thermophila
4.2.1.84 5290
-
 Benzonitrile pH 7.6, 25°C, recombinant mutant alphaT109S Pseudonocardia thermophila
4.2.1.84 6150
-
 Benzonitrile pH 7.6, 25°C, recombinant wild-type enzyme Pseudonocardia thermophila