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Literature summary for 1.6.2.2 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
Search for more literature for 1.6.2.2

Cloned(Commentary)

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

Crystallization (Commentary)

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

Protein Variants

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

KM Value [mM]

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

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
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

Organism UniProt Comment Textmining
Rattus norvegicus P20070
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant enzymes Rattus norvegicus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2 ferricyanide + NADPH
-
 Rattus norvegicus 2 ferrocyanide + NADP+ + H+
-
 ?
2 ferricytochrome b5 + NADH
-
 Rattus norvegicus 2 ferrocytochrome b5 + NAD+ + H+
-
 ?
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]

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

Cofactor

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