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Literature summary for 3.5.5.1 extracted from

  • Yu, S.; Yao, P.; Li, J.; Feng, J.; Wu, Q.; Zhu, D.
    Improving the catalytic efficiency and stereoselectivity of a nitrilase from Synechocystis sp. PCC6803 by semi-rational engineering en route to chiral gamma-amino acids (2019), Catal. Sci. Technol., 9, 1504-1510 .
No PubMed abstract available

Application

Application Comment Organism
synthesis the use of mutant enzyme P194A/I201A/F202V is feasible for application in the production of (S)-3-(4-chlorophenyl)-4-cyanobutanoic acid en route to (R)-baclofen Synechocystis sp. PCC 6803

Cloned(Commentary)

Cloned (Comment) Organism
expression in Rosetta 2(DE3)pLySs Synechocystis sp. PCC 6803

Protein Variants

Protein Variants Comment Organism
E142A mutant enzyme shows almost no activity Synechocystis sp. PCC 6803
F193A mutant enzyme shows almost no activity Synechocystis sp. PCC 6803
F202V the mutant enzyme shows 6.25fold improvement in activity towards 3-(4-chlorophenyl) glutaronitrile relative to that of wild-type enzyme Synechocystis sp. PCC 6803
F64A lower activity as compared to wild-type enzyme Synechocystis sp. PCC 6803
H141A fold increase in activity as compared to wild-type enzyme. No tradeoff occurred in stereoselectivity Synechocystis sp. PCC 6803
I201A fold increase in activity as compared to wild-type enzyme. No tradeoff occurred in stereoselectivity Synechocystis sp. PCC 6803
M197A fold increase in activity as compared to wild-type enzyme. No tradeoff occurred in stereoselectivity Synechocystis sp. PCC 6803
P194A fold increase in activity as compared to wild-type enzyme. No tradeoff occurred in stereoselectivity Synechocystis sp. PCC 6803
P194A/I201A/F202V the mutant enzyme with a larger substrate-binding pocket displays significantly enhanced catalytic activity and enantioselectivity (S, 99% ee) toward 3-(4-chlorophenyl) glutaronitrile and other 3-substituted glutaronitriles Synechocystis sp. PCC 6803
P202A fold increase in activity as compared to wild-type enzyme. No tradeoff occurred in stereoselectivity Synechocystis sp. PCC 6803
Q205A lower activity as compared to wild-type enzyme Synechocystis sp. PCC 6803
T139A mutant enzyme shows almost no activity Synechocystis sp. PCC 6803
V198A lower activity as compared to wild-type enzyme. The enantiomeric excess value of mutant enzyme V198A is 75% (S) Synechocystis sp. PCC 6803
W170A mutant enzyme shows almost no activity Synechocystis sp. PCC 6803
Y140A lower activity as compared to wild-type enzyme Synechocystis sp. PCC 6803
Y59A mutant enzyme shows almost no activity Synechocystis sp. PCC 6803

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.13
-
3-(4-chlorophenyl)pentanedinitrile mutant F202V, sodium phosphate buffer, pH 7.0, 30°C Synechocystis sp. PCC 6803
0.25
-
3-(4-chlorophenyl)pentanedinitrile wild-type enzyme, sodium phosphate buffer, pH 7.0, 30°C Synechocystis sp. PCC 6803
0.31
-
3-(4-chlorophenyl)pentanedinitrile mutant P194A/F202V, sodium phosphate buffer, pH 7.0, 30°C Synechocystis sp. PCC 6803
0.38
-
3-(4-chlorophenyl)pentanedinitrile mutant P194A, sodium phosphate buffer, pH 7.0, 30°C Synechocystis sp. PCC 6803
0.44
-
3-(4-chlorophenyl)pentanedinitrile mutant P194A/I201A/F202V, sodium phosphate buffer, pH 7.0, 30°C Synechocystis sp. PCC 6803

Organism

Organism UniProt Comment Textmining
Synechocystis sp. PCC 6803 Q55949
-
-

Purification (Commentary)

Purification (Comment) Organism
-
Synechocystis sp. PCC 6803

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3-(2-chlorophenyl)pentanedinitrile + H2O the specific activity of mutant enzyme P194A/I201A/F202V is 5.1fold higher than activity of wild-type enzyme The S-enantiomer ist formed with 74% enantiomeric excess by wild-type enzyme and with 97% enantiomeric excess by the mutant enzyme P194A/I201A/F202V Synechocystis sp. PCC 6803 (3S)-3-(2-chlorophenyl)-4-cyanobutanoic acid
-
?
3-(3-chlorophenyl)pentanedinitrile + H2O the specific activity of mutant enzyme P194A/I201A/F202V is 3.1fold higher than activity of wild-type enzyme The S-enantiomer ist formed with 93% enantiomeric excess by wild-type enzyme and with 98% enantiomeric excess by the mutant enzyme P194A/I201A/F202V Synechocystis sp. PCC 6803 (3S)-3-(3-chlorophenyl)-4-cyanobutanoic acid
-
?
3-(4-bromophenyl)pentanedinitrile + H2O the specific activity of mutant enzyme P194A/I201A/F202V is 18.6fold higher than activity of wild-type enzyme The S-enantiomer ist formed with 97% enantiomeric excess by wild-type enzyme and with more than 99% enantiomeric excess by the mutant enzyme P194A/I201A/F202V Synechocystis sp. PCC 6803 (3S)-3-(4-bromophenyl)-4-cyanobutanoic acid
-
?
3-(4-chlorophenyl)pentanedinitrile + H2O the specific activity of mutant enzyme P194A/I201A/F202V is 11.0fold higher than activity of wild-type enzyme The S-enantiomer ist formed with 97% enantiomeric excess by wild-type enzyme and with 99% enantiomeric excess by the mutant enzyme P194A/I201A/F202V Synechocystis sp. PCC 6803 (3S)-3-(4-chlorophenyl)-4-cyanobutanoic acid
-
?
3-(4-fluorophenyl)pentanedinitrile + H2O the specific activity of mutant enzyme P194A/I201A/F202V is 8.8fold higher than activity of wild-type enzyme The S-enantiomer ist formed with 99% enantiomeric excess by wild-type enzyme and with more than 99% enantiomeric excess by the mutant enzyme P194A/I201A/F202V Synechocystis sp. PCC 6803 (3S)-4-cyano-3-(4-fluorophenyl)butanoic acid
-
?
3-(4-hydroxyphenyl)pentanedinitrile + H2O the specific activity of mutant enzyme P194A/I201A/F202V is 78fold higher than activity of wild-type enzyme Synechocystis sp. PCC 6803 (3S)-4-cyano-3-(4-hydroxyphenyl)butanoic acid
-
?
3-(4-methoxyphenyl)pentanedinitrile + H2O the specific activity of mutant enzyme P194A/I201A/F202V is 12.8fold higher than activity of wild-type enzyme The S-enantiomer ist formed with 98% enantiomeric excess by wild-type enzyme and with more than 99% enantiomeric excess by the mutant enzyme P194A/I201A/F202V Synechocystis sp. PCC 6803 (3S)-4-cyano-3-(4-methoxyphenyl)butanoic acid
-
?
3-(4-methylphenyl)pentanedinitrile + H2O the specific activity of mutant enzyme P194A/I201A/F202V is 6.7fold higher than activity of wild-type enzyme The S-enantiomer ist formed with 98% enantiomeric excess by wild-type enzyme and with 99% enantiomeric excess by the mutant enzyme P194A/I201A/F202V Synechocystis sp. PCC 6803 (3S)-4-cyano-3-(4-methylphenyl)butanoic acid
-
?
3-(4-tert-butylphenyl)pentanedinitrile + H2O the specific activity of mutant enzyme P194A/I201A/F202V is2.3fold higher than activity of wild-type enzyme Synechocystis sp. PCC 6803 (3S)-3-(4-tert-butylphenyl)-4-cyanobutanoic acid
-
?
3-phenylpentanedinitrile + H2O the specific activity of mutant enzyme P194A/I201A/F202V is 5.8fold higher than activity of wild-type enzyme The S-enantiomer ist formed with 96% enantiomeric excess by wild-type enzyme and with 96% enantiomeric excess by the mutant enzyme P194A/I201A/F202V Synechocystis sp. PCC 6803 (3S)-4-cyano-3-phenylbutanoic acid
-
?
3-[4-(dimethylamino)phenyl]pentanedinitrile + H2O the specific activity of mutant enzyme P194A/I201A/F202V is 28.0fold higher than activity of wild-type enzyme Synechocystis sp. PCC 6803 (3S)-4-cyano-3-[4-(dimethylamino)phenyl]butanoic acid
-
?
3-[4-(methylsulfanyl)phenyl]pentanedinitrile + H2O the specific activity of mutant enzyme P194A/I201A/F202V is 18.0fold higher than activity of wild-type enzyme The S-enantiomer ist formed with 97% enantiomeric excess by wild-type enzyme and with more than 99% enantiomeric excess by the mutant enzyme P194A/I201A/F202V Synechocystis sp. PCC 6803 (3S)-4-cyano-3-[4-(methylsulfanyl)phenyl]butanoic acid
-
?
3-[4-(propan-2-yl)phenyl]pentanedinitrile + H2O the specific activity of mutant enzyme P194A/I201A/F202V is 18.0fold higher than activity of wild-type enzyme The S-enantiomer ist formed with 95% enantiomeric excess by wild-type enzyme and with 98% enantiomeric excess by the mutant enzyme P194A/I201A/F202V Synechocystis sp. PCC 6803 (3S)-4-cyano-3-[4-(propan-2-yl)phenyl]butanoic acid
-
?
3-[4-(trifluoromethyl)phenyl]pentanedinitrile + H2O the specific activity of mutant enzyme P194A/I201A/F202V is 4.8fold higher than activity of wild-type enzyme The S-enantiomer ist formed with 97% enantiomeric excess by wild-type enzyme and with 96% enantiomeric excess by the mutant enzyme P194A/I201A/F202V Synechocystis sp. PCC 6803 (3S)-4-cyano-3-[4-(trifluoromethyl)phenyl]butanoic acid
-
?

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.37
-
3-(4-chlorophenyl)pentanedinitrile mutant P194A, sodium phosphate buffer, pH 7.0, 30°C Synechocystis sp. PCC 6803
0.43
-
3-(4-chlorophenyl)pentanedinitrile wild-type enzyme, sodium phosphate buffer, pH 7.0, 30°C Synechocystis sp. PCC 6803
0.7
-
3-(4-chlorophenyl)pentanedinitrile mutant F202V, sodium phosphate buffer, pH 7.0, 30°C Synechocystis sp. PCC 6803
3.48
-
3-(4-chlorophenyl)pentanedinitrile mutant P194A/F202V, sodium phosphate buffer, pH 7.0, 30°C Synechocystis sp. PCC 6803

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.97
-
3-(4-chlorophenyl)pentanedinitrile mutant P194A, sodium phosphate buffer, pH 7.0, 30°C Synechocystis sp. PCC 6803
1.69
-
3-(4-chlorophenyl)pentanedinitrile wild-type enzyme, sodium phosphate buffer, pH 7.0, 30°C Synechocystis sp. PCC 6803
5.96
-
3-(4-chlorophenyl)pentanedinitrile mutant F202V, sodium phosphate buffer, pH 7.0, 30°C Synechocystis sp. PCC 6803
11.02
-
3-(4-chlorophenyl)pentanedinitrile mutant P194A/F202V, sodium phosphate buffer, pH 7.0, 30°C Synechocystis sp. PCC 6803
11.17
-
3-(4-chlorophenyl)pentanedinitrile mutant P194A/I201A/F202V, sodium phosphate buffer, pH 7.0, 30°C Synechocystis sp. PCC 6803