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Literature summary for 3.5.5.7 extracted from

  • Zhang, L.; Yin, B.; Wang, C.; Jiang, S.; Wang, H.; Yuan, Y.A.; Wei, D.
    Structural insights into enzymatic activity and substrate specificity determination by a single amino acid in nitrilase from Synechocystis sp. PCC6803 (2014), J. Struct. Biol., 188, 93-101.
    View publication on PubMed
Search for more literature for 3.5.5.7

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli BL21(DE3) cells Synechocystis sp.

Crystallization (Commentary)

Crystallization (Comment) Organism
vapor diffusion method, using 15% (w/v) PEG 5000MME, 1 mM dithiothreitol, 3% tascimate and 100 mM HEPES, pH 6.8 Synechocystis sp.

Protein Variants

Protein Variants Comment Organism
C169A inactive Synechocystis sp.
E142A inactive Synechocystis sp.
E53A inactive Synechocystis sp.
K135A inactive Synechocystis sp.
N118A inactive Synechocystis sp.
W146A inactive Synechocystis sp.
W146C inactive Synechocystis sp.
W146D inactive Synechocystis sp.
W146E inactive Synechocystis sp.
W146F the mutant shows reduced activity compared to the wild type enzyme Synechocystis sp.
W146G inactive Synechocystis sp.
W146H the mutant shows reduced activity compared to the wild type enzyme Synechocystis sp.
W146I inactive Synechocystis sp.
W146K inactive Synechocystis sp.
W146L inactive Synechocystis sp.
W146M inactive Synechocystis sp.
W146N inactive Synechocystis sp.
W146P inactive Synechocystis sp.
W146Q inactive Synechocystis sp.
W146R inactive Synechocystis sp.
W146S inactive Synechocystis sp.
W146T inactive Synechocystis sp.
W146V inactive Synechocystis sp.
W146Y the mutant shows reduced activity compared to the wild type enzyme Synechocystis sp.
Y59A inactive Synechocystis sp.

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.98
-
 Benzonitrile mutant enzyme W146Y, at pH 7.0 and 30°C Synechocystis sp.
1.13
-
 2-Cyanopyridine mutant enzyme W146Y, at pH 7.0 and 30°C Synechocystis sp.
1.21
-
 Benzonitrile wild type enzyme, at pH 7.0 and 30°C Synechocystis sp.
1.26
-
 Benzonitrile mutant enzyme W146F, at pH 7.0 and 30°C Synechocystis sp.
1.26
-
 2-Cyanopyridine mutant enzyme W146F, at pH 7.0 and 30°C Synechocystis sp.
1.3
-
 2-Cyanopyridine mutant enzyme W146H, at pH 7.0 and 30°C Synechocystis sp.
1.33
-
 Benzonitrile mutant enzyme W146H, at pH 7.0 and 30°C Synechocystis sp.
1.47
-
 2-Cyanopyridine wild type enzyme, at pH 7.0 and 30°C Synechocystis sp.
3.16
-
 3-Cyanopyridine mutant enzyme W146Y, at pH 7.0 and 30°C Synechocystis sp.
3.2
-
 3-Cyanopyridine wild type enzyme, at pH 7.0 and 30°C Synechocystis sp.
3.94
-
 3-Cyanopyridine mutant enzyme W146H, at pH 7.0 and 30°C Synechocystis sp.
4.41
-
 3-Cyanopyridine mutant enzyme W146F, at pH 7.0 and 30°C Synechocystis sp.
7.95
-
 2-butenenitrile wild type enzyme, at pH 7.0 and 30°C Synechocystis sp.
9.01
-
 Fumaronitrile wild type enzyme, at pH 7.0 and 30°C Synechocystis sp.
9.16
-
 2-butenenitrile mutant enzyme W146F, at pH 7.0 and 30°C Synechocystis sp.
10.77
-
 Fumaronitrile mutant enzyme W146Y, at pH 7.0 and 30°C Synechocystis sp.
11.06
-
 Fumaronitrile mutant enzyme W146H, at pH 7.0 and 30°C Synechocystis sp.
11.45
-
 Fumaronitrile mutant enzyme W146F, at pH 7.0 and 30°C Synechocystis sp.

Organism

Organism UniProt Comment Textmining
Synechocystis sp. Q55949
-
-

Purification (Commentary)

Purification (Comment) Organism
Ni2+ affinity column chromatography and Superdex 75 gel filtration Synechocystis sp.

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
0.05
-
 mutant enzyme W146F, with 2-butenenitrile as substrate, at pH 7.0 and 30°C Synechocystis sp.
0.29
-
 wild type enzyme, with 2-butenenitrile as substrate, at pH 7.0 and 30°C Synechocystis sp.
0.68
-
 wild type enzyme, with 3-cyanopyridine as substrate, at pH 7.0 and 30°C Synechocystis sp.
0.97
-
 wild type enzyme, with benzonitrile as substrate, at pH 7.0 and 30°C Synechocystis sp.
1
-
 mutant enzyme W146F, with benzonitrile as substrate, at pH 7.0 and 30°C Synechocystis sp.
1.18
-
 mutant enzyme W146Y, with benzonitrile as substrate, at pH 7.0 and 30°C Synechocystis sp.
1.47
-
 mutant enzyme W146H, with benzonitrile as substrate, at pH 7.0 and 30°C Synechocystis sp.
1.57
-
 mutant enzyme W146Y, with fumaronitrile as substrate, at pH 7.0 and 30°C Synechocystis sp.
1.6
-
 mutant enzyme W146F, with 3-cyanopyridine as substrate, at pH 7.0 and 30°C Synechocystis sp.
1.69
-
 mutant enzyme W146Y, with 3-cyanopyridine as substrate, at pH 7.0 and 30°C Synechocystis sp.
1.82
-
 mutant enzyme W146H, with 3-cyanopyridine as substrate, at pH 7.0 and 30°C Synechocystis sp.
1.85
-
 mutant enzyme W146F, with fumaronitrile as substrate, at pH 7.0 and 30°C Synechocystis sp.
1.95
-
 mutant enzyme W146H, with fumaronitrile as substrate, at pH 7.0 and 30°C Synechocystis sp.
2.7
-
 wild type enzyme, with 2-cyanopyridine as substrate, at pH 7.0 and 30°C Synechocystis sp.
2.72
-
 mutant enzyme W146H, with 2-cyanopyridine as substrate, at pH 7.0 and 30°C Synechocystis sp.
2.93
-
 mutant enzyme W146F, with 2-cyanopyridine as substrate, at pH 7.0 and 30°C Synechocystis sp.
3.08
-
 mutant enzyme W146Y, with 2-cyanopyridine as substrate, at pH 7.0 and 30°C Synechocystis sp.
3.2
-
 wild type enzyme, with fumaronitrile as substrate, at pH 7.0 and 30°C Synechocystis sp.

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2-butenenitrile + H2O worst substrate Synechocystis sp. 2-butenic acid + NH3
-
 ?
2-cyanopyridine + 2 H2O second best substrate Synechocystis sp. pyridine 2-carboxylic acid + NH3
-
 ?
3-cyanopyridine + 2 H2O
-
 Synechocystis sp. pyridine 3-carboxylic acid + NH3
-
 ?
benzonitrile + 2 H2O
-
 Synechocystis sp. benzoate + NH3
-
 ?
fumaronitrile + H2O best substrate Synechocystis sp. fumarate + NH3
-
 ?

Synonyms

Synonyms Comment Organism
NIT
-
 Synechocystis sp.

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.03
-
 2-butenenitrile mutant enzyme W146F, at pH 7.0 and 30°C Synechocystis sp.
0.19
-
 2-butenenitrile wild type enzyme, at pH 7.0 and 30°C Synechocystis sp.
0.45
-
 3-Cyanopyridine wild type enzyme, at pH 7.0 and 30°C Synechocystis sp.
0.65
-
 Benzonitrile wild type enzyme, at pH 7.0 and 30°C Synechocystis sp.
0.67
-
 Benzonitrile mutant enzyme W146F, at pH 7.0 and 30°C Synechocystis sp.
0.79
-
 Benzonitrile mutant enzyme W146Y, at pH 7.0 and 30°C Synechocystis sp.
0.98
-
 Benzonitrile mutant enzyme W146H, at pH 7.0 and 30°C Synechocystis sp.
1.07
-
 3-Cyanopyridine mutant enzyme W146F, at pH 7.0 and 30°C Synechocystis sp.
1.13
-
 3-Cyanopyridine mutant enzyme W146Y, at pH 7.0 and 30°C Synechocystis sp.
1.21
-
 3-Cyanopyridine mutant enzyme W146H, at pH 7.0 and 30°C Synechocystis sp.
1.8
-
 2-Cyanopyridine wild type enzyme, at pH 7.0 and 30°C Synechocystis sp.
1.81
-
 2-Cyanopyridine mutant enzyme W146H, at pH 7.0 and 30°C Synechocystis sp.
1.95
-
 2-Cyanopyridine mutant enzyme W146F, at pH 7.0 and 30°C Synechocystis sp.
2.05
-
 2-Cyanopyridine mutant enzyme W146Y, at pH 7.0 and 30°C Synechocystis sp.
7.42
-
 Fumaronitrile mutant enzyme W146Y, at pH 7.0 and 30°C Synechocystis sp.
8.75
-
 Fumaronitrile mutant enzyme W146F, at pH 7.0 and 30°C Synechocystis sp.
9.22
-
 Fumaronitrile mutant enzyme W146H, at pH 7.0 and 30°C Synechocystis sp.
15.13
-
 Fumaronitrile wild type enzyme, at pH 7.0 and 30°C Synechocystis sp.

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.00328
-
 2-butenenitrile mutant enzyme W146F, at pH 7.0 and 30°C Synechocystis sp.
0.0239
-
 2-butenenitrile wild type enzyme, at pH 7.0 and 30°C Synechocystis sp.
0.141
-
 3-Cyanopyridine wild type enzyme, at pH 7.0 and 30°C Synechocystis sp.
0.243
-
 3-Cyanopyridine mutant enzyme W146F, at pH 7.0 and 30°C Synechocystis sp.
0.307
-
 3-Cyanopyridine mutant enzyme W146H, at pH 7.0 and 30°C Synechocystis sp.
0.357
-
 3-Cyanopyridine mutant enzyme W146Y, at pH 7.0 and 30°C Synechocystis sp.
0.531
-
 Benzonitrile mutant enzyme W146F, at pH 7.0 and 30°C Synechocystis sp.
0.537
-
 Benzonitrile wild type enzyme, at pH 7.0 and 30°C Synechocystis sp.
0.689
-
 Fumaronitrile mutant enzyme W146Y, at pH 7.0 and 30°C Synechocystis sp.
0.737
-
 Benzonitrile mutant enzyme W146H, at pH 7.0 and 30°C Synechocystis sp.
0.764
-
 Fumaronitrile mutant enzyme W146F, at pH 7.0 and 30°C Synechocystis sp.
0.806
-
 Benzonitrile mutant enzyme W146Y, at pH 7.0 and 30°C Synechocystis sp.
0.834
-
 Fumaronitrile mutant enzyme W146H, at pH 7.0 and 30°C Synechocystis sp.
1.224
-
 2-Cyanopyridine wild type enzyme, at pH 7.0 and 30°C Synechocystis sp.
1.392
-
 2-Cyanopyridine mutant enzyme W146H, at pH 7.0 and 30°C Synechocystis sp.
1.548
-
 2-Cyanopyridine mutant enzyme W146F, at pH 7.0 and 30°C Synechocystis sp.
1.679
-
 Fumaronitrile wild type enzyme, at pH 7.0 and 30°C Synechocystis sp.
1.814
-
 2-Cyanopyridine mutant enzyme W146Y, at pH 7.0 and 30°C Synechocystis sp.