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Literature summary extracted from

  • Meints, C.E.; Parke, S.M.; Wolthers, K.R.
    Proximal FAD histidine residue influences interflavin electron transfer in cytochrome P450 reductase and methionine synthase reductase (2014), Arch. Biochem. Biophys., 547, 18-26 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.16.1.8 recombinant expression of wild-type and mutant enzymes in Escherichia coli strain Rosetta2(DE3)pLysS Homo sapiens

Protein Variants

EC Number Protein Variants Comment Organism
1.6.2.4 H322Ala the mutation does not affect the rate of NADPH hydride transfer or the FAD redox potentials, but does impede interflavin electron transfer. The mutant elicits a 4fold decrease in cytochrome c reduction and a 1.5fold decrease in ferricyanide reduction. The H322A substitution also leads to a modest increase in NADP(H) binding affinity, evidenced by a 2-3fold reduction in the Km for NADPH and Ki for NADP+ Homo sapiens
1.6.2.4 H322Q the mutant shows a 50% decrease in cytochrome c and ferricyanide reduction and a marginal increase in NADP(H) binding affinity compared to the wild type enzyme Homo sapiens
1.16.1.8 A312H site-directed mutangenesis, mutation of the catalytic residue leads to the kinetic coupling of hydride and interflavin electron transfer, and eliminates the formation of the FAD hydroquinone intermediate, substitution of Ala312 for His in MSR weakens NADP(H) binding as the Km for NADPH and Ki for NADP+ increases 6 and 1.7fold, respectively. NADPH reduction of A312H resembles that of native cytochrome P450 reductase, in that it occurs in two discrete kinetic phases, without the transient formation of the E-FADH2-FMN intermediate Homo sapiens
1.16.1.8 A312Q site-directed mutangenesis, the catalytic site mutant shows a 2.5fold increased Km and a slightly decreased Ki for the coenzyme FAD Homo sapiens

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.6.2.4 NADP+
-
 Homo sapiens
1.16.1.8 NADP+
-
 Homo sapiens

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.6.2.4 0.00031
-
 NADPH mutant enzyme H322A, at pH 7.5 and 25°C Homo sapiens
1.6.2.4 0.00058
-
 NADPH mutant enzyme H322Q, at pH 7.5 and 25°C Homo sapiens
1.6.2.4 0.00071
-
 NADPH wild type enzyme, at pH 7.5 and 25°C Homo sapiens
1.16.1.8 additional information
-
 additional information stopped-flow and steady-state kinetic analysis Homo sapiens
1.16.1.8 0.0024
-
 NADPH pH 7.5, 25°C, recombinant wild-type enzyme Homo sapiens
1.16.1.8 0.0062
-
 NADPH pH 7.5, 25°C, recombinant mutant A312Q Homo sapiens
1.16.1.8 0.015
-
 NADPH pH 7.5, 25°C, recombinant mutant A312H Homo sapiens

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.16.1.8 2 [methionine synthase]-cob(II)alamin + NADPH + H+ + 2 S-adenosyl-L-methionine Homo sapiens
-
 2 [methionine synthase]-methylcob(I)alamin + 2 S-adenosylhomocysteine + NADP+
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.6.2.4 Homo sapiens
-
-
-
1.16.1.8 Homo sapiens Q9UBK8
-
-

Reaction

EC Number Reaction Comment Organism Reaction ID
1.16.1.8 2 [methionine synthase]-methylcob(III)alamin + 2 S-adenosyl-L-homocysteine + NADP+ = 2 [methionine synthase]-cob(II)alamin + NADPH + H+ + 2 S-adenosyl-L-methionine hydride transfer and interflavin electron transfer are two catalytic steps represented by two distinct kinetic phases leading to transient formation of the FAD hydroquinone Homo sapiens
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.6.2.4 NADPH + H+ + ferricyanide
-
 Homo sapiens NADP+ + ferrocyanide
-
 ?
1.6.2.4 NADPH + H+ + ferricytochrome c
-
 Homo sapiens NADP+ + ferrocytochrome c
-
 ?
1.16.1.8 2 [methionine synthase]-cob(II)alamin + NADPH + H+ + 2 S-adenosyl-L-methionine
-
 Homo sapiens 2 [methionine synthase]-methylcob(I)alamin + 2 S-adenosylhomocysteine + NADP+
-
 ?

Synonyms

EC Number Synonyms Comment Organism
1.6.2.4 CPR
-
 Homo sapiens
1.6.2.4 cytochrome P450 reductase
-
 Homo sapiens
1.16.1.8 Methionine synthase reductase
-
 Homo sapiens
1.16.1.8 MSR
-
 Homo sapiens

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.16.1.8 25
-
 assay at Homo sapiens

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.6.2.4 5.4
-
 NADPH mutant enzyme H322A, at pH 7.5 and 25°C Homo sapiens
1.6.2.4 11.3
-
 NADPH mutant enzyme H322Q, at pH 7.5 and 25°C Homo sapiens
1.6.2.4 18.1
-
 ferricyanide mutant enzyme H322A, at pH 7.5 and 25°C Homo sapiens
1.6.2.4 20
-
 NADPH wild type enzyme, at pH 7.5 and 25°C Homo sapiens
1.6.2.4 26.1
-
 ferricyanide mutant enzyme H322Q, at pH 7.5 and 25°C Homo sapiens
1.6.2.4 43.5
-
 ferricyanide wild type enzyme, at pH 7.5 and 25°C Homo sapiens
1.16.1.8 3.6
-
 NADPH pH 7.5, 25°C, recombinant mutant A312H Homo sapiens
1.16.1.8 7.2
-
 NADPH pH 7.5, 25°C, recombinant wild-type enzyme Homo sapiens
1.16.1.8 7.8
-
 NADPH pH 7.5, 25°C, recombinant mutant A312Q Homo sapiens

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.16.1.8 7.5
-
 assay at Homo sapiens

Cofactor

EC Number Cofactor Comment Organism Structure
1.6.2.4 FAD
-
 Homo sapiens
1.6.2.4 FMN
-
 Homo sapiens
1.16.1.8 FAD the proximal FAD histidine residue accelerates proton-coupled electron transfer from FADH2 to the higher potential FMN Homo sapiens
1.16.1.8 FMN the proximal FAD histidine residue accelerates proton-coupled electron transfer from FADH2 to the higher potential FMN Homo sapiens
1.16.1.8 additional information mechanism of NADPH reduction of a diflavin enzyme, overview Homo sapiens
1.16.1.8 NADPH
-
 Homo sapiens

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
1.6.2.4 0.0003
-
 NADP+ mutant enzyme H322A, at pH 7.5 and 25°C Homo sapiens
1.6.2.4 0.00065
-
 NADP+ mutant enzyme H322Q, at pH 7.5 and 25°C Homo sapiens
1.6.2.4 0.00095
-
 NADP+ wild type enzyme, at pH 7.5 and 25°C Homo sapiens
1.16.1.8 0.0291
-
 NADP+ pH 7.5, 25°C, recombinant mutant A312Q Homo sapiens
1.16.1.8 0.0369
-
 NADP+ pH 7.5, 25°C, recombinant wild-type enzyme Homo sapiens
1.16.1.8 0.0729
-
 NADP+ pH 7.5, 25°C, recombinant mutant A312H Homo sapiens

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.6.2.4 17400
-
 NADPH mutant enzyme H322A, at pH 7.5 and 25°C Homo sapiens
1.6.2.4 19400
-
 NADPH mutant enzyme H322Q, at pH 7.5 and 25°C Homo sapiens
1.6.2.4 28200
-
 NADPH wild type enzyme, at pH 7.5 and 25°C Homo sapiens