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Literature summary for 3.5.5.1 extracted from

  • Yeom, S.J.; Lee, J.K.; Oh, D.K.
    A positively charged amino acid at position 129 in nitrilase from Rhodococcus rhodochrous ATCC 33278 is an essential residue for the activity with meta-substituted benzonitriles (2010), FEBS Lett., 584, 106-110.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli strain ER2566 Rhodococcus rhodochrous

Protein Variants

Protein Variants Comment Organism
G103A the mutant exhibits decreased specific activity with 3-methylbenzonitrile and increased specific activity with benzonitrile compared to the wild type enzyme Rhodococcus rhodochrous
I104A the mutant exhibits decreased specific activity with 3-methylbenzonitrile and increased specific activity with benzonitrile compared to the wild type enzyme Rhodococcus rhodochrous
M114A the mutant exhibits decreased specific activity with 3-methylbenzonitrile and increased specific activity with benzonitrile compared to the wild type enzyme Rhodococcus rhodochrous
N164A the mutant exhibits decreased specific activity with 3-methylbenzonitrile and increased specific activity with benzonitrile compared to the wild type enzyme Rhodococcus rhodochrous
Q116A the mutant exhibits decreased specific activity with 3-methylbenzonitrile and increased specific activity with benzonitrile compared to the wild type enzyme Rhodococcus rhodochrous
R129A the mutant exhibits no activity with 3-methylbenzonitrile and benzonitrile Rhodococcus rhodochrous
R129H the mutant enzyme is active only for meta-substituted benzonitriles Rhodococcus rhodochrous
R129K the mutant enzyme is active only for meta-substituted benzonitriles Rhodococcus rhodochrous
R130A the mutant exhibits increased specific activity with 3-methylbenzonitrile and benzonitrile compared to the wild type enzyme Rhodococcus rhodochrous
S188A the mutant exhibits decreased specific activity with 3-methylbenzonitrile and increased specific activity with benzonitrile compared to the wild type enzyme Rhodococcus rhodochrous
T115A the mutant exhibits decreased specific activity with 3-methylbenzonitrile and increased specific activity with benzonitrile compared to the wild type enzyme Rhodococcus rhodochrous
V49A the mutant exhibits decreased specific activity with 3-methylbenzonitrile and increased specific activity with benzonitrile compared to the wild type enzyme Rhodococcus rhodochrous
Y142A the mutant exhibits decreased specific activity with 3-methylbenzonitrile and increased specific activity with benzonitrile compared to the wild type enzyme Rhodococcus rhodochrous

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.456
-
3-aminobenzonitrile mutant enzyme R129K, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
0.483
-
3-aminobenzonitrile mutant enzyme R129H, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
0.55
-
3-methylbenzonitrile mutant enzyme R129K, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
0.592
-
3-methylbenzonitrile mutant enzyme R129H, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
1.068
-
Benzonitrile wild type enzyme, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
1.468
-
Benzonitrile mutant enzyme R129K, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
1.699
-
4-aminobenzonitrile wild type enzyme, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
1.851
-
3-methylbenzonitrile wild type enzyme, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
1.863
-
3-aminobenzonitrile wild type enzyme, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
2.426
-
4-methylbenzonitrile wild type enzyme, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
3.13
-
Benzonitrile mutant enzyme R129H, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous

Organism

Organism UniProt Comment Textmining
Rhodococcus rhodochrous
-
-
-
Rhodococcus rhodochrous ATCC 33278
-
-
-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
0.6
-
mutant enzyme N164A, using 3-methylbenzonitrile as substrate, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
5.1
-
mutant enzyme V49A, using 3-methylbenzonitrile as substrate, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
5.2
-
mutant enzyme M114A, using 3-methylbenzonitrile as substrate, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
7
-
wild type enzyme, using benzonitrile as substrate, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
7.5
-
mutant enzyme T115A, using 3-methylbenzonitrile as substrate, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
8
-
mutant enzyme S188A, using 3-methylbenzonitrile as substrate, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
8.2
-
mutant enzyme T115A, using benzonitrile as substrate, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
8.8
-
mutant enzyme G103A, using benzonitrile as substrate, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
8.8
-
mutant enzyme I104A, using 3-methylbenzonitrile as substrate, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
9
-
mutant enzyme Q116A, using benzonitrile as substrate, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
9.6
-
mutant enzyme G103A, using 3-methylbenzonitrile as substrate, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
9.7
-
mutant enzyme N164A, using benzonitrile as substrate, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
9.9
-
mutant enzyme M114A, using benzonitrile as substrate, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
9.9
-
mutant enzyme Q116A, using 3-methylbenzonitrile as substrate, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
10
-
mutant enzyme S188A, using benzonitrile as substrate, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
10.4
-
mutant enzyme I104A, using benzonitrile as substrate, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
10.7
-
wild type enzyme, using 3-methylbenzonitrile as substrate, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
11.6
-
mutant enzymeV49A, using benzonitrile as substrate, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
14.5
-
mutant enzyme R130A, using 3-methylbenzonitrile as substrate, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
17.7
-
mutant enzyme R130A, using benzonitrile as substrate, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
22.4
-
mutant enzyme Y142A, using benzonitrile as substrate, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
26
-
mutant enzyme Y142A, using 3-methylbenzonitrile as substrate, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3-aminobenzonitrile + H2O
-
Rhodococcus rhodochrous 3-aminobenzoic acid + NH3
-
?
3-aminobenzonitrile + H2O
-
Rhodococcus rhodochrous ATCC 33278 3-aminobenzoic acid + NH3
-
?
3-methylbenzonitrile + H2O
-
Rhodococcus rhodochrous 3-methylbenzoic acid + NH3
-
?
3-methylbenzonitrile + H2O
-
Rhodococcus rhodochrous ATCC 33278 3-methylbenzoic acid + NH3
-
?
4-aminobenzonitrile + H2O
-
Rhodococcus rhodochrous 4-aminobenzoic acid + NH3
-
?
4-aminobenzonitrile + H2O
-
Rhodococcus rhodochrous ATCC 33278 4-aminobenzoic acid + NH3
-
?
4-methylbenzonitrile + H2O
-
Rhodococcus rhodochrous 4-methylbenzoic acid + NH3
-
?
benzonitrile + 2 H2O
-
Rhodococcus rhodochrous benzoic acid + NH3
-
?
benzonitrile + 2 H2O
-
Rhodococcus rhodochrous ATCC 33278 benzoic acid + NH3
-
?
additional information the wild type enzyme containing Arg129 is active only for meta- and para-substituted benzonitriles with a methyl or amino group, but shows no activity for ortho-substituted benzonitriles Rhodococcus rhodochrous ?
-
?
additional information the wild type enzyme containing Arg129 is active only for meta- and para-substituted benzonitriles with a methyl or amino group, but shows no activity for ortho-substituted benzonitriles Rhodococcus rhodochrous ATCC 33278 ?
-
?

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.000129
-
Benzonitrile wild type enzyme, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
0.00014
-
3-aminobenzonitrile mutant enzyme R129K, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
0.00015
-
3-aminobenzonitrile mutant enzyme R129H, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
0.000158
-
3-methylbenzonitrile mutant enzyme R129K, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
0.000162
-
Benzonitrile mutant enzyme R129K, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
0.00018
-
3-methylbenzonitrile mutant enzyme R129H, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
0.000198
-
3-methylbenzonitrile wild type enzyme, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
0.000283
-
4-aminobenzonitrile wild type enzyme, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
0.000288
-
3-aminobenzonitrile wild type enzyme, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
0.00047
-
Benzonitrile mutant enzyme R129H, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
0.000695
-
4-methylbenzonitrile wild type enzyme, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.001068
-
3-methylbenzonitrile wild type enzyme, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
0.0011
-
Benzonitrile mutant enzyme R129K, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
0.001223
-
Benzonitrile wild type enzyme, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
0.0015
-
Benzonitrile mutant enzyme R129H, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
0.001548
-
3-aminobenzonitrile wild type enzyme, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
0.001667
-
4-aminobenzonitrile wild type enzyme, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
0.002867
-
4-methylbenzonitrile wild type enzyme, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
0.0029
-
3-methylbenzonitrile mutant enzyme R129K, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
0.003
-
3-methylbenzonitrile mutant enzyme R129H, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
0.00308
-
3-aminobenzonitrile mutant enzyme R129K, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous
0.0032
-
3-aminobenzonitrile mutant enzyme R129H, in 50 mM potassium phosphate (pH 7.5), at 25°C Rhodococcus rhodochrous