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Literature summary extracted from

  • Brodkin, H.R.; Novak, W.R.; Milne, A.C.; DAquino, J.A.; Karabacak, N.M.; Goldberg, I.G.; Agar, J.N.; Payne, M.S.; Petsko, G.A.; Ondrechen, M.J.; Ringe, D.
    Evidence of the participation of remote residues in the catalytic activity of Co-type nitrile hydratase from Pseudomonas putida (2011), Biochemistry, 50, 4923-4935.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
4.2.1.84 expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Pseudomonas putida

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
4.2.1.84 purified recombinant wild-type and mutant enzymes, hanging drop vapour diffusion method, 25°C, from 20 mg/mL ppNHase and a reservoir containing 22% polyacrylic acid sodium salt 5100, 100 mM HEPES, pH 7.5, 20 mM magnesium chloride, and 4% acetone, single crystals are transferred to 17.6% polyacrylic acid sodium salt 5100 and 20% glycerol in 100 mM HEPES, pH 7.5, as cryoprotectant, X-ray diffraction structure determination and analysis Pseudomonas putida

Protein Variants

EC Number Protein Variants Comment Organism
4.2.1.84 alphaD164N site-directed mutagenesis Pseudomonas putida
4.2.1.84 alphaE168Q site-directed mutagenesis Pseudomonas putida
4.2.1.84 alphaR170Q site-directed mutagenesis Pseudomonas putida
4.2.1.84 betaE56Q site-directed mutagenesis Pseudomonas putida
4.2.1.84 betaH71F site-directed mutagenesis Pseudomonas putida
4.2.1.84 betaH71L site-directed mutagenesis Pseudomonas putida
4.2.1.84 betaH71N site-directed mutagenesis Pseudomonas putida
4.2.1.84 betaY215F site-directed mutagenesis Pseudomonas putida

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
4.2.1.84 additional information
-
 additional information recombinant wild-type and mutant enzymes, kinetics analysis, overview Pseudomonas putida
4.2.1.84 0.32
-
 n-valeronitrile pH 6.7, 0°C, recombinant mutant alphaR170N Pseudomonas putida
4.2.1.84 1.8
-
 n-valeronitrile pH 6.7, 0°C, recombinant mutant alphaD164N Pseudomonas putida
4.2.1.84 2.7
-
 n-valeronitrile pH 6.7, 0°C, recombinant mutant betaY215F Pseudomonas putida
4.2.1.84 4.5
-
 n-valeronitrile pH 6.7, 0°C, recombinant mutant alphaE168Q Pseudomonas putida
4.2.1.84 6.6
-
 n-valeronitrile pH 6.7, 0°C, recombinant wild-type enzyme Pseudomonas putida
4.2.1.84 9.3
-
 n-valeronitrile pH 6.7, 0°C, recombinant mutant alphaY171F Pseudomonas putida
4.2.1.84 10
-
 n-valeronitrile pH 6.7, 0°C, recombinant mutant betaH71L Pseudomonas putida
4.2.1.84 15.3
-
 n-valeronitrile pH 6.7, 0°C, recombinant mutant betaE56Q Pseudomonas putida
4.2.1.84 20.1
-
 n-valeronitrile pH 6.7, 0°C, recombinant mutant betaH71N Pseudomonas putida
4.2.1.84 21.2
-
 n-valeronitrile pH 6.7, 0°C, recombinant mutant betaH71F Pseudomonas putida
4.2.1.84 26.4
-
 n-valeronitrile pH 6.7, 0°C, recombinant mutant betaH147N Pseudomonas putida

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
4.2.1.84 23000
-
 1 * 23000, about, alpha-subunit + 1 x 23000, about, beta-subunit, Co-type NHases are bacterial heterodimers, consisting of nonhomologous alpha- and beta-subunits Pseudomonas putida

Organism

EC Number Organism UniProt Comment Textmining
4.2.1.84 Pseudomonas putida
-
-
-
4.2.1.84 Pseudomonas putida NRRL-18668
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
4.2.1.84 recombinant soluble wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by anion exchange chromatography, ammonium sulfate fractionation, and hydrophobic interaction chromatography, followed by ultrafiltration and another step of anion exchange chromatography Pseudomonas putida

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4.2.1.84 additional information roles of second- and third-shell residues of the active site structure in catalysis, overview. Three of the predicted second-shell residues, alpha-Asp164, beta-Glu56, and beta-His147, and one predicted third-shell residue, beta-His71, have significant effects on the catalytic efficiency of the enzyme, while one of the predicted residues, alpha-Glu168, and the three residues not predicted, alpha-Arg170, alpha-Tyr171, and beta-Tyr215, do not have any significant effects on the catalytic efficiency of the enzyme Pseudomonas putida ?
-
 ?
4.2.1.84 additional information roles of second- and third-shell residues of the active site structure in catalysis, overview. Three of the predicted second-shell residues, alpha-Asp164, beta-Glu56, and beta-His147, and one predicted third-shell residue, beta-His71, have significant effects on the catalytic efficiency of the enzyme, while one of the predicted residues, alpha-Glu168, and the three residues not predicted, alpha-Arg170, alpha-Tyr171, and beta-Tyr215, do not have any significant effects on the catalytic efficiency of the enzyme Pseudomonas putida NRRL-18668 ?
-
 ?
4.2.1.84 n-valeronitrile + H2O
-
 Pseudomonas putida n-valeramide
-
 ?
4.2.1.84 n-valeronitrile + H2O
-
 Pseudomonas putida NRRL-18668 n-valeramide
-
 ?

Subunits

EC Number Subunits Comment Organism
4.2.1.84 heterodimer 1 * 23000, about, alpha-subunit + 1 x 23000, about, beta-subunit, Co-type NHases are bacterial heterodimers, consisting of nonhomologous alpha- and beta-subunits Pseudomonas putida

Synonyms

EC Number Synonyms Comment Organism
4.2.1.84 Co-type nitrile hydratase
-
 Pseudomonas putida
4.2.1.84 ppNHase
-
 Pseudomonas putida

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
4.2.1.84
-
-
 assay at Pseudomonas putida

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
4.2.1.84 0.0035
-
 n-valeronitrile pH 6.7, 0°C, recombinant mutant betaE56Q Pseudomonas putida
4.2.1.84 0.0045
-
 n-valeronitrile pH 6.7, 0°C, recombinant mutant alphaD164N Pseudomonas putida
4.2.1.84 0.01
-
 n-valeronitrile pH 6.7, 0°C, recombinant mutant alphaR170N Pseudomonas putida
4.2.1.84 0.012
-
 n-valeronitrile pH 6.7, 0°C, recombinant mutant betaH147N Pseudomonas putida
4.2.1.84 0.02
-
 n-valeronitrile pH 6.7, 0°C, recombinant mutant betaH71L Pseudomonas putida
4.2.1.84 0.025
-
 n-valeronitrile pH 6.7, 0°C, recombinant mutant betaH71N Pseudomonas putida
4.2.1.84 0.027
-
 n-valeronitrile pH 6.7, 0°C, recombinant mutant betaH71F Pseudomonas putida
4.2.1.84 0.04
-
 n-valeronitrile pH 6.7, 0°C, recombinant mutant betaY215F Pseudomonas putida
4.2.1.84 0.0617
-
 n-valeronitrile pH 6.7, 0°C, recombinant mutant alphaE168Q Pseudomonas putida
4.2.1.84 0.217
-
 n-valeronitrile pH 6.7, 0°C, recombinant mutant alphaY171F Pseudomonas putida
4.2.1.84 0.327
-
 n-valeronitrile pH 6.7, 0°C, recombinant wild-type enzyme Pseudomonas putida

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
4.2.1.84 6.7
-
 assay at Pseudomonas putida

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 0.0002
-
 n-valeronitrile pH 6.7, 0°C, recombinant mutant betaE56Q Pseudomonas putida
4.2.1.84 0.00045
-
 n-valeronitrile pH 6.7, 0°C, recombinant mutant betaH147N Pseudomonas putida
4.2.1.84 0.00121
-
 n-valeronitrile pH 6.7, 0°C, recombinant mutant betaH71F Pseudomonas putida
4.2.1.84 0.00122
-
 n-valeronitrile pH 6.7, 0°C, recombinant mutant betaH71N Pseudomonas putida
4.2.1.84 0.002
-
 n-valeronitrile pH 6.7, 0°C, recombinant mutant betaH71L Pseudomonas putida
4.2.1.84 0.0025
-
 n-valeronitrile pH 6.7, 0°C, recombinant mutant alphaD164N Pseudomonas putida
4.2.1.84 0.014
-
 n-valeronitrile pH 6.7, 0°C, recombinant mutant alphaE168Q Pseudomonas putida
4.2.1.84 0.0152
-
 n-valeronitrile pH 6.7, 0°C, recombinant mutant betaY215F Pseudomonas putida
4.2.1.84 0.023
-
 n-valeronitrile pH 6.7, 0°C, recombinant mutant alphaY171F Pseudomonas putida
4.2.1.84 0.032
-
 n-valeronitrile pH 6.7, 0°C, recombinant mutant alphaR170N Pseudomonas putida
4.2.1.84 0.05
-
 n-valeronitrile pH 6.7, 0°C, recombinant wild-type enzyme Pseudomonas putida