Literature summary for 4.2.1.84 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

Search for more literature for 4.2.1.84

Cloned(Commentary)

Cloned (Comment)	Organism
expression in Escherichia coli	Rhodococcus rhodochrous

Protein Variants

Protein Variants	Comment	Organism
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
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
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]

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

Organism

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

Purification (Commentary)

Purification (Comment)	Organism
-	Rhodococcus rhodochrous

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
2-cyanobenzamide + H2O	-	Rhodococcus rhodochrous	benzene-1,2-dicarboxamide	-	?
2-cyanobenzamide + H2O	-	Rhodococcus rhodochrous J1	benzene-1,2-dicarboxamide	-	?
4-cyanobenzamide + H2O	-	Rhodococcus rhodochrous	benzene-1,4-dicarboxamide	-	?
4-cyanobenzamide + H2O	-	Rhodococcus rhodochrous J1	benzene-1,4-dicarboxamide	-	?
5-cyanovaleramide	-	Rhodococcus rhodochrous	adiponitrile + H2O	-	r
5-cyanovaleramide	-	Rhodococcus rhodochrous J1	adiponitrile + H2O	-	r
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	-	?
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	-	?
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
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
cyanoacetamide	-	Rhodococcus rhodochrous	malononitrile + H2O	-	r
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	-	?
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
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	-	?
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	-	?
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	-	?
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]

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

Turnover Number [1/s]

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

pH Optimum

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

kcat/KM [mM/s]

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

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Release 2023.1 (January 2023)