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Literature summary extracted from

  • Cheng, Z.; Cui, W.; Xia, Y.; Peplowski, L.; Kobayashi, M.; Zhou, Z.
    Modulation of nitrile hydratase regioselectivity towards dinitriles by tailoring the substrate binding pocket residues (2018), ChemCatChem, 10, 449-458 .
No PubMed abstract available

Cloned(Commentary)

EC Number Cloned (Comment) Organism
4.2.1.84 expression in Escherichia coli Rhodococcus rhodochrous

Protein Variants

EC Number Protein Variants Comment Organism
4.2.1.84 W72Y with adiponitrile as substrate the wild-type enzyme forms only adipamide after a 4 h reaction. Y68T and W72Y mutations cause a significant shift in product formation and form primarily 5-cyanovaleramide. The use of the wild-type enzyme leads to malonamide formation with 97.3% malononitrile conversion. Variants Y68T and W72Y show a drastic change in regiospecificity by producing mainly the omega-cyanocarboxamide, cyanoacetamide, at a relatively low malononitrile conversion. The wild-type enzyme prefers terephthalonitrile to catalyze mainly into terephthalamide (84.3%) with a conversion up to 99.2 %. Variants Y68T and W72Y show a change in regiospecificity by producing mainly the 4-cyanobenzamide. The wild-type enzyme forms 100% phthalamide from phthalodinitrile. The mutant enzymes Y68T and W72Y result in a higher 2-cyanobenzamide formation than their parent enzyme Rhodococcus rhodochrous
4.2.1.84 Y68T with adiponitrile as substrate the wild-type enzyme forms only adipamide after a 4 h reaction. Y68T and W72Y mutations cause a significant shift in product formation and form primarily 5-cyanovaleramide. The use of the wild-type enzyme leads to malonamide formation with 97.3% malononitrile conversion. Variants Y68T and W72Y show a drastic change in regiospecificity by producing mainly the omega-cyanocarboxamide, cyanoacetamide, at a relatively low malononitrile conversion. The wild-type enzyme prefers terephthalonitrile to catalyze mainly into terephthalamide (84.3%) with a conversion up to 99.2%. Variants Y68T and W72Y show a change in regiospecificity by producing mainly the 4-cyanobenzamide. The wild-type enzyme forms 100% phthalamide from phthalodinitrile. The mutant enzymes Y68T and W72Y result in a higher 2-cyanobenzamide formation than their parent enzyme Rhodococcus rhodochrous
4.2.1.84 Y68T/W72Y the mutant enzyme displays a totally different regioselectivity towards dinitriles than its parent enzyme. The use of the wild-type enzyme leads to malonamide formation with 97.3% malononitrile conversion. Mutant enzyme Y68T/W72Y produces 97.1% omega-cyanocarboxamide. The wild-type enzyme forms only adipamide after a 4 h reaction. Mutant enzyme Y68T/W72Y produces 100% 5-cyanovaleramide. The wild-type enzyme prefers terephthalonitrile to catalyze mainly into terephthalamide (84.3%) with a conversion up to 99.2 %. Mutant enzyme Y68T/W72Y produces 98.2% 4-cyanobenzamide from terephthalonitrile. The wild-type enzyme forms 100% phthalamide from phthalodinitrile. Mutant enzyme Y68T/W72Y produces 100% 2-cyanobenzamide from phthalodinitrile Rhodococcus rhodochrous

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
4.2.1.84 2.3
-
 4-cyanobenzamide pH 7.4, 35°C, wild-type enzyme Rhodococcus rhodochrous
4.2.1.84 2.5
-
 2-cyanobenzamide pH 7.4, 35°C, wild-type enzyme Rhodococcus rhodochrous
4.2.1.84 13.2
-
 5-cyanovaleramide pH 7.4, 35°C, wild-type enzyme Rhodococcus rhodochrous
4.2.1.84 20.7
-
 cyanoacetamide pH 7.4, 35°C, wild-type enzyme Rhodococcus rhodochrous
4.2.1.84 85.3
-
 cyanoacetamide pH 7.4, 35°C, mutant enzyme W72Y Rhodococcus rhodochrous
4.2.1.84 88.2
-
 cyanoacetamide pH 7.4, 35°C, mutant enzyme Y68T Rhodococcus rhodochrous
4.2.1.84 113.2
-
 5-cyanovaleramide pH 7.4, 35°C, mutant enzyme W72Y Rhodococcus rhodochrous
4.2.1.84 127.6
-
 5-cyanovaleramide pH 7.4, 35°C, mutant enzyme Y68T Rhodococcus rhodochrous

Organism

EC Number Organism UniProt Comment Textmining
4.2.1.84 Rhodococcus rhodochrous P29378 AND P29379 P29378: alpha-subunit, P29379: beta-subunit
-
4.2.1.84 Rhodococcus rhodochrous J1 P29378 AND P29379 P29378: alpha-subunit, P29379: beta-subunit
-

Purification (Commentary)

EC Number Purification (Comment) Organism
4.2.1.84
-
 Rhodococcus rhodochrous

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4.2.1.84 2-cyanobenzamide + H2O
-
 Rhodococcus rhodochrous benzene-1,2-dicarboxamide
-
 ?
4.2.1.84 2-cyanobenzamide + H2O
-
 Rhodococcus rhodochrous J1 benzene-1,2-dicarboxamide
-
 ?
4.2.1.84 4-cyanobenzamide + H2O
-
 Rhodococcus rhodochrous benzene-1,4-dicarboxamide
-
 ?
4.2.1.84 4-cyanobenzamide + H2O
-
 Rhodococcus rhodochrous J1 benzene-1,4-dicarboxamide
-
 ?
4.2.1.84 5-cyanovaleramide
-
 Rhodococcus rhodochrous adiponitrile + H2O
-
 r
4.2.1.84 5-cyanovaleramide
-
 Rhodococcus rhodochrous J1 adiponitrile + H2O
-
 r
4.2.1.84 adiponitrile + 2 H2O wild-type enzyme forms only adipamide after a 4 h reaction. Y68T and W72Y mutations cause a significant shift in product formation and form primarily 5-cyanovaleramide. Mutant enzyme Y68T/W72Y produces 100% 5-cyanovaleramide Rhodococcus rhodochrous adipic acid amide
-
 ?
4.2.1.84 adiponitrile + 2 H2O wild-type enzyme forms only adipamide after a 4 h reaction. Y68T and W72Y mutations cause a significant shift in product formation and form primarily 5-cyanovaleramide. Mutant enzyme Y68T/W72Y produces 100% 5-cyanovaleramide Rhodococcus rhodochrous J1 adipic acid amide
-
 ?
4.2.1.84 adiponitrile + H2O wild-type enzyme forms only adipamide after a 4 h reaction. Y68T and W72Y mutations cause a significant shift in product formation and form primarily 5-cyanovaleramide. Mutant enzyme Y68T/W72Y produces 100% 5-cyanovaleramide Rhodococcus rhodochrous 5-cyanovaleramide
-
 r
4.2.1.84 adiponitrile + H2O wild-type enzyme forms only adipamide after a 4 h reaction. Y68T and W72Y mutations cause a significant shift in product formation and form primarily 5-cyanovaleramide. Mutant enzyme Y68T/W72Y produces 100% 5-cyanovaleramide Rhodococcus rhodochrous J1 5-cyanovaleramide
-
 r
4.2.1.84 cyanoacetamide
-
 Rhodococcus rhodochrous malononitrile + H2O
-
 r
4.2.1.84 malononitrile + 2 H2O the use of the wild-type enzyme leads to malonamide formation with 97.3% malononitrile conversion. Variants Y68T and W72Y show a drastic change in regiospecificity by producing mainly the omega-cyanocarboxamide, cyanoacetamide, at a relatively low malononitrile conversion. Mutant enzyme Y68T/W72Y produces 97.1% omega-cyanocarboxamide Rhodococcus rhodochrous malonamide
-
 ?
4.2.1.84 malononitrile + H2O the use of the wild-type enzyme leads to malonamide formation with 97.3% malononitrile conversion. Variants Y68T and W72Y show a drastic change in regiospecificity by producing mainly the omega-cyanocarboxamide, cyanoacetamide, at a relatively low malononitrile conversion. Mutant enzyme Y68T/W72Y produces 97.1% omega-cyanocarboxamide Rhodococcus rhodochrous cyanoacetamide
-
 r
4.2.1.84 phthalodinitrile + 2 H2O the wild-type enzyme forms 100% phthalamide from phthalodinitrile. The mutant enzymes Y68T and W72Y result in a higher 2-cyanobenzamide formation than their parent enzyme. Mutant enzyme Y68T/W72Y produces 100% 2-cyanobenzamide from phthalodinitrile Rhodococcus rhodochrous phthalamide
-
 ?
4.2.1.84 phthalodinitrile + H2O the wild-type enzyme forms 100% phthalamide from phthalodinitrile. The mutant enzymes Y68T and W72Y result in a higher 2-cyanobenzamide formation than their parent enzyme. Mutant enzyme Y68T/W72Y produces 100% 2-cyanobenzamide from phthalodinitrile Rhodococcus rhodochrous 2-cyanobenzamide
-
 ?
4.2.1.84 terephthalonitrile + 2 H2O the wild-type enzyme prefers terephthalonitrile to catalyze mainly into terephthalamide (84.3%) with a conversion up to 99.2 %. Variants Y68T and W72Y show a change in regiospecificity by producing mainly the 4-cyanobenzamide. Mutant enzyme Y68T/W72Y produces 98.2% 4-cyanobenzamide from terephthalonitrile Rhodococcus rhodochrous terephthalamide
-
 ?
4.2.1.84 terephthalonitrile + H2O the wild-type enzyme prefers terephthalonitrile to catalyze mainly into terephthalamide (84.3%) with a conversion up to 99.2 %. Variants Y68T and W72Y show a change in regiospecificity by producing mainly the 4-cyanobenzamide. Mutant enzyme Y68T/W72Y produces 98.2% 4-cyanobenzamide from terephthalonitrile Rhodococcus rhodochrous 4-cyanobenzamide
-
 ?

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
4.2.1.84 35
-
 assay at Rhodococcus rhodochrous

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
4.2.1.84 19.6
-
 5-cyanovaleramide pH 7.4, 35°C, mutant enzyme Y68T Rhodococcus rhodochrous
4.2.1.84 21.3
-
 5-cyanovaleramide pH 7.4, 35°C, mutant enzyme W72Y Rhodococcus rhodochrous
4.2.1.84 39.1
-
 cyanoacetamide pH 7.4, 35°C, mutant enzyme Y68T Rhodococcus rhodochrous
4.2.1.84 42.8
-
 cyanoacetamide pH 7.4, 35°C, mutant enzyme W72Y Rhodococcus rhodochrous
4.2.1.84 107.7
-
 4-cyanobenzamide pH 7.4, 35°C, wild-type enzyme Rhodococcus rhodochrous
4.2.1.84 145.6
-
 2-cyanobenzamide pH 7.4, 35°C, wild-type enzyme Rhodococcus rhodochrous
4.2.1.84 417.2
-
 cyanoacetamide pH 7.4, 35°C, wild-type enzyme Rhodococcus rhodochrous
4.2.1.84 435.7
-
 5-cyanovaleramide pH 7.4, 35°C, wild-type enzyme Rhodococcus rhodochrous

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
4.2.1.84 7.4
-
 assay at Rhodococcus rhodochrous

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
4.2.1.84 0.2
-
 5-cyanovaleramide pH 7.4, 35°C, mutant enzyme W72Y Rhodococcus rhodochrous
4.2.1.84 0.2
-
 5-cyanovaleramide pH 7.4, 35°C, mutant enzyme Y68T Rhodococcus rhodochrous
4.2.1.84 0.4
-
 cyanoacetamide pH 7.4, 35°C, mutant enzyme Y68T Rhodococcus rhodochrous
4.2.1.84 0.5
-
 cyanoacetamide pH 7.4, 35°C, mutant enzyme W72Y Rhodococcus rhodochrous
4.2.1.84 20.2
-
 cyanoacetamide pH 7.4, 35°C, wild-type enzyme Rhodococcus rhodochrous
4.2.1.84 33
-
 5-cyanovaleramide pH 7.4, 35°C, wild-type enzyme Rhodococcus rhodochrous
4.2.1.84 46.8
-
 4-cyanobenzamide pH 7.4, 35°C, wild-type enzyme Rhodococcus rhodochrous
4.2.1.84 58.2
-
 2-cyanobenzamide pH 7.4, 35°C, wild-type enzyme Rhodococcus rhodochrous