Literature summary for 4.2.1.84 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.

Activating Compound

Activating Compound	Comment	Organism	Structure
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)

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

Crystallization (Commentary)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

Protein Variants	Comment	Organism
alphaT109S	site-directed mutagenesis, the mutant shows similar characteristics compared to the wild-type enzyme	Pseudonocardia thermophila
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
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
T109S	similar characteristics to the wild-type enzyme	Pseudonocardia thermophila
Y114T	very low cobalt content and catalytic activity compared to the wild-type enzyme	Pseudonocardia thermophila

Inhibitors

Inhibitors	Comment	Organism
benzoic acid	competitive inhibitor; competitive inhibitor	Pseudonocardia thermophila
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
propionic acid	competitive inhibitor; competitive inhibitor	Pseudonocardia thermophila

KM Value [mM]

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

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Co2+	cobalt-containing nitrile hydratase	Pseudonocardia thermophila
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

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

Purification (Commentary)

Purification (Comment)	Organism
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

Storage Stability	Organism
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)

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

Synonyms

Synonyms	Comment	Organism
cobalt-containing nitrile hydratase	-	Pseudonocardia thermophila
NHase	-	Pseudonocardia thermophila

Temperature Optimum [°C]

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

Turnover Number [1/s]

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

pH Optimum

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

Ki Value [mM]

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

General Information

General Information	Comment	Organism
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]

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