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 (Comment) | Organism |
---|---|
expression in Rosetta 2(DE3)pLySs | Synechocystis sp. PCC 6803 |
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 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 | UniProt | Comment | Textmining |
---|---|---|---|
Synechocystis sp. PCC 6803 | Q55949 | - |
- |
Purification (Comment) | Organism |
---|---|
- |
Synechocystis sp. PCC 6803 |
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 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 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 |