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Literature summary extracted from

  • Marohnic, C.C.; Bewley, M.C.; Barber, M.J.
    Engineering and characterization of a NADPH-utilizing cytochrome b5 reductase (2003), Biochemistry, 42, 11170-11182.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.6.2.2 mutant enzymes expressed in Escherichia coli BL21(DE3)-RIL as His-tag fusion proteins Rattus norvegicus

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.6.2.2 sitting drop method, complete data set collected for the D239T mutant enzyme Rattus norvegicus

Protein Variants

EC Number Protein Variants Comment Organism
1.6.2.2 D239E decreased activity with NADH and NADPH Rattus norvegicus
1.6.2.2 D239S significantly increased activity with NADPH Rattus norvegicus
1.6.2.2 D239S/F251R specific for NADPH Rattus norvegicus
1.6.2.2 D239S/F251Y bispecific for NADH and NADPH Rattus norvegicus
1.6.2.2 D239T specific for NADPH, 11fold preference for NADPH over NADH Rattus norvegicus
1.6.2.2 D239T/F251R specific for NADPH Rattus norvegicus
1.6.2.2 F251R minor effects on activity Rattus norvegicus
1.6.2.2 F251Y minor effects on activity Rattus norvegicus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.6.2.2 0.003
-
 NADH F251Y mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 0.005
-
 NADH D239S/F251Y mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 0.006
-
 NADH D239E mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 0.006
-
 NADH wild type enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 0.009
-
 NADPH D239T mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 0.012
-
 NADH F251R mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 0.017
-
 NADH D239S mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 0.022
-
 NADPH D239S/F251R mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 0.025
-
 NADPH D239S/F251Y mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 0.044
-
 NADPH D239T/F251R mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 0.094
-
 NADPH D239E mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 0.119
-
 NADH D239T mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 0.138
-
 NADPH F251R mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 0.224
-
 NADH D239S/F251R mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 0.268
-
 NADPH D239S mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 0.512
-
 NADH D239T/F251R mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 0.617
-
 NADPH F251Y mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 0.924
-
 NADPH wild type enzyme, pH 7.0, 25°C Rattus norvegicus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.6.2.2 2 ferricytochrome b5 + NADH Rattus norvegicus involved in the synthesis of fatty acids and cholesterol, and in the oxidation of xenobiotics 2 ferrocytochrome b5 + NAD+ + H+
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.6.2.2 Rattus norvegicus P20070
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.6.2.2 recombinant enzymes Rattus norvegicus

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.6.2.2 2 ferricyanide + NADPH
-
 Rattus norvegicus 2 ferrocyanide + NADP+ + H+
-
 ?
1.6.2.2 2 ferricytochrome b5 + NADH
-
 Rattus norvegicus 2 ferrocytochrome b5 + NAD+ + H+
-
 ?
1.6.2.2 2 ferricytochrome b5 + NADH involved in the synthesis of fatty acids and cholesterol, and in the oxidation of xenobiotics Rattus norvegicus 2 ferrocytochrome b5 + NAD+ + H+
-
 ?

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.6.2.2 5.2
-
 NADPH D239E mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 33
-
 NADPH wild type enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 50
-
 NADPH F251R mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 50
-
 NADPH F251Y mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 200
-
 NADPH D239S/F251R mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 217
-
 NADPH D239S mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 250
-
 NADH D239S/F251R mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 267
-
 NADPH D239T mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 333
-
 NADH D239T mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 367
-
 NADH F251Y mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 417
-
 NADPH D239S/F251Y mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 433
-
 NADH D239S mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 467
-
 NADH D239S/F251Y mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 500
-
 NADH F251R mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 517
-
 NADH D239E mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 550
-
 NADPH D239T/F251R mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 717
-
 NADH D239T/F251R mutant enzyme, pH 7.0, 25°C Rattus norvegicus
1.6.2.2 800
-
 NADH wild type enzyme, pH 7.0, 25°C Rattus norvegicus

Cofactor

EC Number Cofactor Comment Organism Structure
1.6.2.2 FAD
-
 Rattus norvegicus
1.6.2.2 NADH native enzyme shows 3700fold preference for NADH over NADPH Rattus norvegicus
1.6.2.2 NADPH preferred cofactor for some engineered enzymes Rattus norvegicus