Literature summary extracted from

Baum, S.; Williamson, D.S.; Sewell, T.; Stolz, A.
Conversion of sterically demanding alpha,alpha-disubstituted phenylacetonitriles by the arylacetonitrilase from Pseudomonas fluorescens EBC191 (2012), Appl. Environ. Microbiol., 78, 48-57.

Application

EC Number	Application	Comment	Organism
3.5.5.1	synthesis	recombinant cells expressing the enzyme are promising catalysts for the synthesis of stable chiral quaternary carbon centers from ketones	Alcaligenes faecalis
3.5.5.1	synthesis	recombinant cells expressing the enzyme are promising catalysts for the synthesis of stable chiral quaternary carbon centers from ketones	Rhodococcus sp.
3.5.5.1	synthesis	recombinant cells expressing the enzyme are promising catalysts for the synthesis of stable chiral quaternary carbon centers from ketones	Synechocystis sp.
3.5.5.1	synthesis	recombinant cells expressing the enzyme are promising catalysts for the synthesis of stable chiral quaternary carbon centers from ketones	Pseudomonas fluorescens
3.5.5.5	synthesis	recombinant cells expressing the enzyme are promising catalysts for the synthesis of stable chiral quaternary carbon centers from ketones	Pseudomonas fluorescens

Cloned(Commentary)

EC Number	Cloned (Comment)	Organism
3.5.5.1	expression under the control of a rhamnose-inducible promoter in Escherichia coli strain JM109	Alcaligenes faecalis
3.5.5.1	expression under the control of a rhamnose-inducible promoter in Escherichia coli strain JM109	Rhodococcus sp.
3.5.5.1	expression under the control of a rhamnose-inducible promoter in Escherichia coli strain JM109	Synechocystis sp.
3.5.5.1	expression under the control of a rhamnose-inducible promoter in Escherichia coli strain JM109	Pseudomonas fluorescens
3.5.5.5	expression under the control of a rhamnose-inducible promoter in Escherichia coli strain JM109	Pseudomonas fluorescens

Protein Variants

EC Number	Protein Variants	Comment	Organism
3.5.5.1	Y54A	site-directed mutagenesis, the mutant enzyme converts 2-hydroxy-2-phenylpropionitrile with about the same activity as that of the wild-type enzyme, but forms significantly reduced amounts of amides from mandelonitrile and acetophenone cyanohydrin, it shows different kinetics of acetophenone cyanohydrin conversion and product formation compared to the wild-type	Pseudomonas fluorescens
3.5.5.1	Y54F	site-directed mutagenesis, altered substrate specificity and enantioselectivity compared to the wild-type enzyme, overview	Pseudomonas fluorescens
3.5.5.1	Y54M	site-directed mutagenesis, altered substrate specificity and enantioselectivity compared to the wild-type enzyme, overview	Pseudomonas fluorescens
3.5.5.1	Y54P	site-directed mutagenesis, altered substrate specificity and enantioselectivity compared to the wild-type enzyme, overview	Pseudomonas fluorescens
3.5.5.1	Y54V	site-directed mutagenesis, altered substrate specificity and enantioselectivity compared to the wild-type enzyme, overview	Pseudomonas fluorescens
3.5.5.5	Tyr54C	the mutant shows wild type activity but forms significantly decreased relative amounts of atrolactamide from 2-hydroxy-2-phenylpropionitrile	Pseudomonas fluorescens
3.5.5.5	Tyr54P	the mutant shows wild type activity but forms significantly decreased relative amounts of atrolactamide from 2-hydroxy-2-phenylpropionitrile	Pseudomonas fluorescens
3.5.5.5	Tyr54V	the mutant shows wild type activity but forms significantly decreased relative amounts of atrolactamide from 2-hydroxy-2-phenylpropionitrile	Pseudomonas fluorescens
3.5.5.5	Y54A	the mutant shows wild type activity but forms significantly decreased relative amounts of atrolactamide from 2-hydroxy-2-phenylpropionitrile	Pseudomonas fluorescens
3.5.5.5	Y54F	the mutant shows wild type activity but forms significantly decreased relative amounts of atrolactamide from 2-hydroxy-2-phenylpropionitrile	Pseudomonas fluorescens
3.5.5.5	Y54H	the mutant shows wild type activity but forms significantly decreased relative amounts of atrolactamide from 2-hydroxy-2-phenylpropionitrile	Pseudomonas fluorescens
3.5.5.5	Y54I	inactive	Pseudomonas fluorescens
3.5.5.5	Y54K	inactive	Pseudomonas fluorescens
3.5.5.5	Y54L	mutant with significantly reduced activity compared to the wild type enzyme	Pseudomonas fluorescens
3.5.5.5	Y54M	the mutant shows wild type activity but forms significantly decreased relative amounts of atrolactamide from 2-hydroxy-2-phenylpropionitrile	Pseudomonas fluorescens
3.5.5.5	Y54R	inactive	Pseudomonas fluorescens

KM Value [mM]

EC Number	KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
3.5.5.1	additional information	-	additional information	kinetic and chiral analysis, overview	Alcaligenes faecalis
3.5.5.1	additional information	-	additional information	kinetic and chiral analysis, overview	Rhodococcus sp.
3.5.5.1	additional information	-	additional information	kinetic and chiral analysis, overview	Synechocystis sp.
3.5.5.1	additional information	-	additional information	kinetic and chiral analysis, overview	Pseudomonas fluorescens
3.5.5.5	additional information	-	additional information	kinetic and chiral analysis, overview	Pseudomonas fluorescens

Natural Substrates/ Products (Substrates)

EC Number	Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
3.5.5.1	2-methyl-2-phenylpropionitrile + 2 H2O	Alcaligenes faecalis	-	2-methyl-2-phenylpropionic acid + NH3	-	?
3.5.5.1	2-methyl-2-phenylpropionitrile + 2 H2O	Rhodococcus sp.	-	2-methyl-2-phenylpropionic acid + NH3	-	?
3.5.5.1	2-methyl-2-phenylpropionitrile + 2 H2O	Synechocystis sp.	-	2-methyl-2-phenylpropionic acid + NH3	-	?
3.5.5.1	2-methyl-2-phenylpropionitrile + 2 H2O	Pseudomonas fluorescens	-	2-methyl-2-phenylpropionic acid + NH3	-	?
3.5.5.1	2-methyl-2-phenylpropionitrile + 2 H2O	Pseudomonas fluorescens EBC191	-	2-methyl-2-phenylpropionic acid + NH3	-	?
3.5.5.1	2-methyl-2-phenylpropionitrile + 2 H2O	Alcaligenes faecalis ATCC 8750	-	2-methyl-2-phenylpropionic acid + NH3	-	?
3.5.5.5	2-acetyloxy-2-methylphenylacetonitrile + 2 H2O	Pseudomonas fluorescens	-	2-acetyloxy-2-methylphenylacetic acid + NH3	-	?
3.5.5.5	2-acetyloxy-2-methylphenylacetonitrile + 2 H2O	Pseudomonas fluorescens EBC191	-	2-acetyloxy-2-methylphenylacetic acid + NH3	-	?

Organism

EC Number	Organism	UniProt	Comment	Textmining
3.5.5.1	Alcaligenes faecalis	-	-	-
3.5.5.1	Alcaligenes faecalis ATCC 8750	-	-	-
3.5.5.1	Pseudomonas fluorescens	Q5EG61	-	-
3.5.5.1	Pseudomonas fluorescens EBC191	Q5EG61	-	-
3.5.5.1	Rhodococcus sp.	-	-	-
3.5.5.1	Synechocystis sp.	-	-	-
3.5.5.5	Pseudomonas fluorescens	-	-	-
3.5.5.5	Pseudomonas fluorescens EBC191	-	-	-

Specific Activity [micromol/min/mg]

EC Number	Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
3.5.5.1	0.1	-	enzyme in cells, substrate benzonitrile in Na/K-phosphate buffer, pH 7.0, 30°C	Synechocystis sp.
3.5.5.1	0.4	-	enzyme in cells, substrate 2-methyl-2-phenylpropionitrile in Na/K-phosphate buffer, pH 7.0, 30°C	Alcaligenes faecalis
3.5.5.1	1.9	-	enzyme in cells, substrate 2-methyl-2-phenylpropionitrile in Na/K-phosphate buffer, pH 7.0, 30°C	Pseudomonas fluorescens
3.5.5.1	14.1	-	enzyme in cells, substrate benzonitrile in Na/K-phosphate buffer, pH 7.0, 30°C	Rhodococcus sp.

Substrates and Products (Substrate)

EC Number	Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
3.5.5.1	2-hydroxy-2-phenylpropionitrile + 2 H2O	acetophenone cyanohydrin, the substrate is transformed in cells to the corresponding acid (atrolactate) and amide (atrolactamide) at a ratio of about 3.4:1. The (R)-acid and the (S)-amide are formed preferentially from acetophenone cyanohydrin	Alcaligenes faecalis	2-hydroxy-2-phenylpropionic acid + NH3	i.e. atrolactic acid	?
3.5.5.1	2-hydroxy-2-phenylpropionitrile + 2 H2O	acetophenone cyanohydrin, the substrate is transformed in cells to the corresponding acid (atrolactate) and amide (atrolactamide) at a ratio of about 3.4:1. The (R)-acid and the (S)-amide are formed preferentially from acetophenone cyanohydrin	Rhodococcus sp.	2-hydroxy-2-phenylpropionic acid + NH3	i.e. atrolactic acid	?
3.5.5.1	2-hydroxy-2-phenylpropionitrile + 2 H2O	acetophenone cyanohydrin, the substrate is transformed in cells to the corresponding acid (atrolactate) and amide (atrolactamide) at a ratio of about 3.4:1. The (R)-acid and the (S)-amide are formed preferentially from acetophenone cyanohydrin	Synechocystis sp.	2-hydroxy-2-phenylpropionic acid + NH3	i.e. atrolactic acid	?
3.5.5.1	2-hydroxy-2-phenylpropionitrile + 2 H2O	acetophenone cyanohydrin, the substrate is transformed in cells to the corresponding acid (atrolactate) and amide (atrolactamide) at a ratio of about 3.4:1. The (R)-acid and the (S)-amide are formed preferentially from acetophenone cyanohydrin	Pseudomonas fluorescens	2-hydroxy-2-phenylpropionic acid + NH3	i.e. atrolactic acid	?
3.5.5.1	2-hydroxy-2-phenylpropionitrile + 2 H2O	acetophenone cyanohydrin, the substrate is transformed in cells to the corresponding acid (atrolactate) and amide (atrolactamide) at a ratio of about 3.4:1. The (R)-acid and the (S)-amide are formed preferentially from acetophenone cyanohydrin	Pseudomonas fluorescens EBC191	2-hydroxy-2-phenylpropionic acid + NH3	i.e. atrolactic acid	?
3.5.5.1	2-hydroxy-2-phenylpropionitrile + 2 H2O	acetophenone cyanohydrin, the substrate is transformed in cells to the corresponding acid (atrolactate) and amide (atrolactamide) at a ratio of about 3.4:1. The (R)-acid and the (S)-amide are formed preferentially from acetophenone cyanohydrin	Alcaligenes faecalis ATCC 8750	2-hydroxy-2-phenylpropionic acid + NH3	i.e. atrolactic acid	?
3.5.5.1	2-methyl-2-phenylpropionitrile + 2 H2O	-	Alcaligenes faecalis	2-methyl-2-phenylpropionic acid + NH3	-	?
3.5.5.1	2-methyl-2-phenylpropionitrile + 2 H2O	-	Rhodococcus sp.	2-methyl-2-phenylpropionic acid + NH3	-	?
3.5.5.1	2-methyl-2-phenylpropionitrile + 2 H2O	-	Synechocystis sp.	2-methyl-2-phenylpropionic acid + NH3	-	?
3.5.5.1	2-methyl-2-phenylpropionitrile + 2 H2O	-	Pseudomonas fluorescens	2-methyl-2-phenylpropionic acid + NH3	-	?
3.5.5.1	2-methyl-2-phenylpropionitrile + 2 H2O	substrate contains a quaternary carbon atom in the alpha-position toward the nitrile group	Alcaligenes faecalis	2-methyl-2-phenylpropionic acid + NH3	-	?
3.5.5.1	2-methyl-2-phenylpropionitrile + 2 H2O	substrate contains a quaternary carbon atom in the alpha-position toward the nitrile group	Rhodococcus sp.	2-methyl-2-phenylpropionic acid + NH3	-	?
3.5.5.1	2-methyl-2-phenylpropionitrile + 2 H2O	substrate contains a quaternary carbon atom in the alpha-position toward the nitrile group	Synechocystis sp.	2-methyl-2-phenylpropionic acid + NH3	-	?
3.5.5.1	2-methyl-2-phenylpropionitrile + 2 H2O	substrate contains a quaternary carbon atom in the alpha-position toward the nitrile group	Pseudomonas fluorescens	2-methyl-2-phenylpropionic acid + NH3	-	?
3.5.5.1	2-methyl-2-phenylpropionitrile + 2 H2O	-	Pseudomonas fluorescens EBC191	2-methyl-2-phenylpropionic acid + NH3	-	?
3.5.5.1	2-methyl-2-phenylpropionitrile + 2 H2O	substrate contains a quaternary carbon atom in the alpha-position toward the nitrile group	Pseudomonas fluorescens EBC191	2-methyl-2-phenylpropionic acid + NH3	-	?
3.5.5.1	2-methyl-2-phenylpropionitrile + 2 H2O	-	Alcaligenes faecalis ATCC 8750	2-methyl-2-phenylpropionic acid + NH3	-	?
3.5.5.1	2-methyl-2-phenylpropionitrile + 2 H2O	substrate contains a quaternary carbon atom in the alpha-position toward the nitrile group	Alcaligenes faecalis ATCC 8750	2-methyl-2-phenylpropionic acid + NH3	-	?
3.5.5.1	benzonitrile + 2 H2O	-	Alcaligenes faecalis	benzoate + NH3	-	?
3.5.5.1	benzonitrile + 2 H2O	-	Rhodococcus sp.	benzoate + NH3	-	?
3.5.5.1	benzonitrile + 2 H2O	-	Synechocystis sp.	benzoate + NH3	-	?
3.5.5.1	benzonitrile + 2 H2O	-	Alcaligenes faecalis ATCC 8750	benzoate + NH3	-	?
3.5.5.1	additional information	the enzyme catalyzes the reactions of nitrilase, EC 3.5.5.1, and arylacetonitrilase, EC 3.5.5.5, substrate specificity and enantioselectivity, overview. Steric hindrance with amino acid residue Tyr54 impairs the binding or conversion of sterically demanding substrates, homology modelling	Alcaligenes faecalis	?	-	?
3.5.5.1	additional information	the enzyme catalyzes the reactions of nitrilase, EC 3.5.5.1, and arylacetonitrilase, EC 3.5.5.5, substrate specificity and enantioselectivity, overview. Steric hindrance with amino acid residue Tyr54 impairs the binding or conversion of sterically demanding substrates, homology modelling	Rhodococcus sp.	?	-	?
3.5.5.1	additional information	the enzyme catalyzes the reactions of nitrilase, EC 3.5.5.1, and arylacetonitrilase, EC 3.5.5.5, substrate specificity and enantioselectivity, overview. Steric hindrance with amino acid residue Tyr54 impairs the binding or conversion of sterically demanding substrates, homology modelling	Synechocystis sp.	?	-	?
3.5.5.1	additional information	the enzyme catalyzes the reactions of nitrilase, EC 3.5.5.1, and arylacetonitrilase, EC 3.5.5.5, substrate specificity and enantioselectivity, overview. Steric hindrance with amino acid residue Tyr54 impairs the binding or conversion of sterically demanding substrates, homology modelling	Pseudomonas fluorescens	?	-	?
3.5.5.1	additional information	the enzyme catalyzes the reactions of nitrilase, EC 3.5.5.1, and arylacetonitrilase, EC 3.5.5.5, substrate specificity and enantioselectivity, overview. Steric hindrance with amino acid residue Tyr54 impairs the binding or conversion of sterically demanding substrates, homology modelling	Pseudomonas fluorescens EBC191	?	-	?
3.5.5.1	additional information	the enzyme catalyzes the reactions of nitrilase, EC 3.5.5.1, and arylacetonitrilase, EC 3.5.5.5, substrate specificity and enantioselectivity, overview. Steric hindrance with amino acid residue Tyr54 impairs the binding or conversion of sterically demanding substrates, homology modelling	Alcaligenes faecalis ATCC 8750	?	-	?
3.5.5.5	2-acetyloxy-2-methylphenylacetonitrile + 2 H2O	-	Pseudomonas fluorescens	2-acetyloxy-2-methylphenylacetic acid + NH3	-	?
3.5.5.5	2-acetyloxy-2-methylphenylacetonitrile + 2 H2O	-	Pseudomonas fluorescens EBC191	2-acetyloxy-2-methylphenylacetic acid + NH3	-	?
3.5.5.5	2-acetyloxy-2-methylphenylacetonitrile + H2O	-	Pseudomonas fluorescens	2-acetyloxy-2-methylphenylacetic acid + NH3	-	?
3.5.5.5	2-hydroxy-2-phenylpropionitrile + H2O	i.e. acetophenone cyanohydrin	Pseudomonas fluorescens	2-hydroxy-2-phenylpropionic acid + 2-hydroxy-2-phenylpropionamide + NH3	product ratio of about 3.4:1 for 2-hydroxy-2-phenylpropionic acid and 2-hydroxy-2-phenylpropionamide	?
3.5.5.5	2-methyl-2-phenylpropionitrile + H2O	-	Pseudomonas fluorescens	2-methyl-2-phenylpropionic acid + NH3	-	?
3.5.5.5	2-phenylbutyronitrile + 2 H2O	-	Pseudomonas fluorescens	2-phenylbutyric acid + NH3	-	?
3.5.5.5	2-phenylbutyronitrile + 2 H2O	-	Pseudomonas fluorescens EBC191	2-phenylbutyric acid + NH3	-	?
3.5.5.5	2-phenylpropionitrile + 2 H2O	-	Pseudomonas fluorescens	2-phenylpropionic acid + NH3	-	?
3.5.5.5	2-phenylpropionitrile + 2 H2O	-	Pseudomonas fluorescens EBC191	2-phenylpropionic acid + NH3	-	?
3.5.5.5	2-phenylvaleronitrile + 2 H2O	-	Pseudomonas fluorescens	2-phenylvaleric acid + NH3	-	?
3.5.5.5	mandelonitrile + 2 H2O	-	Pseudomonas fluorescens	mandelic acid + NH3	-	?
3.5.5.5	mandelonitrile + 2 H2O	-	Pseudomonas fluorescens EBC191	mandelic acid + NH3	-	?
3.5.5.5	additional information	the nitrilase from strain EBC191 hydrolyzes a wide range of arylacetonitriles, such as 2-phenylpropionitrile (2-methylphenylacetonitrile), 2-phenylbutyronitrile, or 2-phenylvaleronitrile, and also alpha-hydroxynitriles, such as mandelonitrile, with rather high specific activities. The enzyme catalyzes the reactions of nitrilase, EC 3.5.5.1, and arylacetonitrilase, EC 3.5.5.5, substrate specificity and enantioselectivity, overview. Steric hindrance with amino acid residue Tyr54 impairs the binding or conversion of sterically demanding substrates, homology modelling	Pseudomonas fluorescens	?	-	?
3.5.5.5	additional information	the nitrilase from strain EBC191 hydrolyzes a wide range of arylacetonitriles, such as 2-phenylpropionitrile (2-methylphenylacetonitrile), 2-phenylbutyronitrile, or 2-phenylvaleronitrile, and also alpha-hydroxynitriles, such as mandelonitrile, with rather high specific activities. The enzyme catalyzes the reactions of nitrilase, EC 3.5.5.1, and arylacetonitrilase, EC 3.5.5.5, substrate specificity and enantioselectivity, overview. Steric hindrance with amino acid residue Tyr54 impairs the binding or conversion of sterically demanding substrates, homology modelling	Pseudomonas fluorescens EBC191	?	-	?

Synonyms

EC Number	Synonyms	Comment	Organism
3.5.5.5	Arylacetonitrilase	-	Pseudomonas fluorescens

Temperature Optimum [°C]

EC Number	Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
3.5.5.1	30	-	assay at	Alcaligenes faecalis
3.5.5.1	30	-	assay at	Rhodococcus sp.
3.5.5.1	30	-	assay at	Synechocystis sp.
3.5.5.1	30	-	assay at	Pseudomonas fluorescens
3.5.5.5	30	-	assay at	Pseudomonas fluorescens

pH Optimum

EC Number	pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
3.5.5.1	7	-	with substrate 2-methyl-2-phenylpropionitrile in Na/K-phosphate buffer	Alcaligenes faecalis
3.5.5.1	7	-	with substrate 2-methyl-2-phenylpropionitrile in Na/K-phosphate buffer	Pseudomonas fluorescens
3.5.5.1	7	-	with substrate benzonitrile in Na/K-phosphate buffer	Rhodococcus sp.
3.5.5.1	7	-	with substrate benzonitrile in Na/K-phosphate buffer	Synechocystis sp.
3.5.5.5	4.5	-	assay at	Pseudomonas fluorescens
3.5.5.5	7	-	-	Pseudomonas fluorescens

General Information

EC Number	General Information	Comment	Organism
3.5.5.1	additional information	generation of a model of the nitrilase by homology modeling, overview	Alcaligenes faecalis
3.5.5.1	additional information	generation of a model of the nitrilase by homology modeling, overview	Rhodococcus sp.
3.5.5.1	additional information	generation of a model of the nitrilase by homology modeling, overview	Synechocystis sp.
3.5.5.1	additional information	generation of a model of the nitrilase by homology modeling, overview	Pseudomonas fluorescens