Literature summary for 4.2.1.84 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.

Cloned(Commentary)

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

Crystallization (Commentary)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

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

KM Value [mM]

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

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
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

Organism	UniProt	Comment	Textmining
Pseudomonas putida	-	-	-
Pseudomonas putida NRRL-18668	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
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)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
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	?	-	?
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	?	-	?
n-valeronitrile + H2O	-	Pseudomonas putida	n-valeramide	-	?
n-valeronitrile + H2O	-	Pseudomonas putida NRRL-18668	n-valeramide	-	?

Subunits

Subunits	Comment	Organism
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

Synonyms	Comment	Organism
Co-type nitrile hydratase	-	Pseudomonas putida
ppNHase	-	Pseudomonas putida

Temperature Optimum [°C]

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

Turnover Number [1/s]

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

pH Optimum

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

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.0002	-	n-valeronitrile	pH 6.7, 0°C, recombinant mutant betaE56Q	Pseudomonas putida
0.00045	-	n-valeronitrile	pH 6.7, 0°C, recombinant mutant betaH147N	Pseudomonas putida
0.00121	-	n-valeronitrile	pH 6.7, 0°C, recombinant mutant betaH71F	Pseudomonas putida
0.00122	-	n-valeronitrile	pH 6.7, 0°C, recombinant mutant betaH71N	Pseudomonas putida
0.002	-	n-valeronitrile	pH 6.7, 0°C, recombinant mutant betaH71L	Pseudomonas putida
0.0025	-	n-valeronitrile	pH 6.7, 0°C, recombinant mutant alphaD164N	Pseudomonas putida
0.014	-	n-valeronitrile	pH 6.7, 0°C, recombinant mutant alphaE168Q	Pseudomonas putida
0.0152	-	n-valeronitrile	pH 6.7, 0°C, recombinant mutant betaY215F	Pseudomonas putida
0.023	-	n-valeronitrile	pH 6.7, 0°C, recombinant mutant alphaY171F	Pseudomonas putida
0.032	-	n-valeronitrile	pH 6.7, 0°C, recombinant mutant alphaR170N	Pseudomonas putida
0.05	-	n-valeronitrile	pH 6.7, 0°C, recombinant wild-type enzyme	Pseudomonas putida