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1.6.2.4: NADPH-hemoprotein reductase

This is an abbreviated version!
For detailed information about NADPH-hemoprotein reductase, go to the full flat file.

Word Map on EC 1.6.2.4

Reaction

NADPH
+
oxidized hemoprotein
=
NADP+
 +
reduced hemoprotein
 +
H+

Synonyms

107366505, aldehyde reductase (NADPH-dependent), ATR2, CPR, CPR I, CPR-X1, CPR-X2, CPR1, CPR2, CYPOR, cytochrome c reductase (reduced nicotinamide adenine dinucleotide phosphate, NADPH, NADPH-dependent), cytochrome P450 NADPH reductase, cytochrome P450 oxidoreductase, cytochrome P450 reductase, cytochrome P450-diflavin reductase, dihydroxynicotinamide adenine dinucleotide phosphate-cytochrome c reductase, FAD-cytochrome c reductase, ferrihemprotein P450 reductase, flavocytochrome P450 BM3, NAD(P)H cytochrome P450 reductase, NADP-cytochrome c reductase, NADP-cytochrome P-450 reductase, NADP-cytochrome reductase, NADPH cytochrome P450 reductase, NADPH dependent cytochrome P450 reductase, NADPH P450 oxidoreductase, NADPH P450 reductase, NADPH-CPR, NADPH-CYP reductase, NADPH-CYP450 reductase, NADPH-cytochrome c oxidoreductase, NADPH-cytochrome c reductase, NADPH-cytochrome p-450 reductase, NADPH-cytochrome P450 (CYP) oxidoreductase, NADPH-cytochrome P450 oxidoreductase, NADPH-cytochrome P450 reductase, NADPH-cytochrome P450 reductase 2, NADPH-dependent cytochrome c reductase, NADPH-dependent cytochrome P450 reductase, NADPH-dependent P450 reductase, NADPH-ferricytochrome c oxidoreductase, NADPH-ferrihemoprotein reductase, NADPH-P450 reductase, NADPH: cytochrome P450 oxidoreductase, NADPH: cytochrome P450 reductase, NADPH:CYP oxidoreductase, NADPH:cytochrome P450 oxidoreductase, NADPH:cytochrome P450 reductase, NADPH:ferrihemoprotein oxidoreductase, NCP1, neotetrazolin reductase, NPR, NP_000932, P-450 reductase, P450 BM3, P450 oxidoreductase, P450 Red, P450 reductase, P450R, P450REd, POR, RedA, reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase, reductase, cytochrome c (reduced nicotinamide adenine dinucleotide phosphate), SbCPR2, TcCPR-A, TcCPR-B, TcCPR-C, TPNH-cytochrome c reductase, TPNH2 cytochrome c reductase, WsCPR1, WsCPR2

ECTree

     1 Oxidoreductases
         1.6 Acting on NADH or NADPH
             1.6.2 With a heme protein as acceptor
                1.6.2.4 NADPH-hemoprotein reductase

Engineering

Engineering on EC 1.6.2.4 - NADPH-hemoprotein reductase

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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
L219F
L86F/L219F
-
the mutant upon reconstitution with the Anopheles minimus cytochrome P450 CYP6AA3 and a NADPH-regenerating system, increases CYP6AA3-mediated deltamethrin degradation compared to the wild type enzyme
L86F/L219F/P456A
-
increased stability with significant reduction in aggregation compared to the wild-type enzyme. The triple mutant is purified in high yield with stoichiometries of 0.97 FMN and 0.55 FAD (compared to 0.92 FMN and 1.05 FAD for wild-type enzyme). Deficiency in FAD content is overcome by addition of exogenous FAD to the enzyme. Both wild-type and the triple mutant follow a two-site ping-pong mechanism with similar kinetic constants
P456A
-
mutant enzyme contains 0.28 FMN and 0.5 FAD compared to 0.5 FMN and 1.05 FAD for wild-type enzyme
A115V
-
Vmax/Km for cytochrome c is 63% of wild-type value, Vmax/Km for NADPH is 41% of wild-type value
A287P
A457H
-
the POR mutant is associated with total loss of heme oxygenase-1 activity
A462T
-
vmax/Km for ferricytochrome c is 85% of the wild-type value. Vmax/Km for NADPH is 69% of the wild-type value
A485T
-
vmax/Km for ferricytochrome c is 36% of the wild-type value. Vmax/Km for NADPH is 51% of the wild-type value
A503V
A635G/R636S
the mutations lead to a modest increase in cytochrome c reduction, which is linked to weaker coenzyme binding and faster interflavin electron transfer
C569Y
D211L
-
vmax/Km for ferricytochrome c is 27% of the wild-type value. Vmax/Km for NADPH is 59% of the wild-type value
D634A
the variant elicits a modest increase in coenzyme binding affinity coupled with a 36fold reduction in cytochrome c turnover and a 17fold decrease in the pre-steady state rate of flavin reduction
D634N
the variant elicits a modest increase in coenzyme binding affinity coupled with a 10fold reduction in cytochrome c turnover and a 3fold decrease in the pre-steady state rate of flavin reduction
delE53
-
vmax/Km for ferricytochrome c is 57% of the wild-type value. Vmax/Km for NADPH is 93% of the wild-type value
delT236_I242
-
either deletion of a 7 amino acid long segment or its replacement by polyproline repeats (5 and 10 residues) results in a significant increase in 2',5'-ADP enthalpy of binding. This is accompanied by a decrease in the number of thermodynamic microstates available for the ligand-cytochrome P450 reductase complex. The estimated heat capacity change for this interaction changes from -220 cal/mol*K in the wild-type enzyme to -580 cal/mol*K in the deletion mutant. Presteady-state kinetics measurements reveal a 50fold decrease in the microscopic rate for interdomain (FAD/FMN) electron transfer in the deletion mutant. Multiple turnover cytochome c reduction assays indicate that these mutations impair the ability of the FMN-binding domain to shuttle electrons from the FAD-binding domain to the cytochrome partner
delT236_I242ins(Pro)10
-
either deletion of a 7 amino acid long segment or its replacement by polyproline repeats (5 and 10 residues) results in a significant increase in 2',5'-ADP enthalpy of binding. This is accompanied by a decrease in the number of thermodynamic microstates available for the ligand-cytochrome P450 reductase complex
delT236_I242ins(Pro)5
-
either deletion of a 7 amino acid long segment or its replacement by polyproline repeats (5 and 10 residues) results in a significant increase in 2',5'-ADP enthalpy of binding. This is accompanied by a decrease in the number of thermodynamic microstates available for the ligand-cytochrome P450 reductase complex
DELTAD675/DELTAV676
tandem deletion variant, about 16% of wild-type activity
DELTAS678
deletion of Ser678 has no effect on cytochrome c reductase activity or Km values for NADPH or cytochrome c
DELTAV676
repositioning of Trp677 by deletion of Val676 decreases cytochrome c reductase activity, to about 6% of wild-type levels
E300K
-
vmax/Km for ferricytochrome c is 93% of the wild-type value. Vmax/Km for NADPH is 104% of the wild-type value
F646del
-
Vmax/Km for cytochrome c is 36% of wild-type value, Vmax/Km for NADPH is 94% of wild-type value
G213E
-
vmax/Km for ferricytochrome c is 111% of the wild-type value. Vmax/Km for NADPH is 105% of the wild-type value
G240P
mutation in the hinge segment, ionic strength profile is shifted to lower salt concentrations
G413S
G504R
G539R
-
Vmax/Km for cytochrome c is 9% of wild-type value, Vmax/Km for NADPH is 0.2% of wild-type value
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+
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
H628P
-
mutation results in similar disruption of CYP17A1 and CYP21A2 activities
I245A
mutation in the hinge segment, ionic strength profile is shifted to lower salt concentrations
I245A/R246A
mutation in the hinge segment, ionic strength profile is shifted to lower salt concentrations
I245P
mutation in the hinge segment, ionic strength profile is shifted to lower salt concentrations
I245R/R246I
mutation in the hinge segment, ionic strength profile is shifted to lower salt concentrations
L565P
-
Vmax/Km for cytochrome c is 14% of wild-type value, Vmax/Km for NADPH is 1.4% of wild-type value
M263V
-
Vmax/Km for cytochrome c is 76% of wild-type value, Vmax/Km for NADPH is 57% of wild-type value
P228L
P284L
-
vmax/Km for ferricytochrome c is 104% of the wild-type value. Vmax/Km for NADPH is 153% of the wild-type value
P284T
-
vmax/Km for ferricytochrome c is 16% of the wild-type value. Vmax/Km for NADPH is 32% of the wild-type value
P452L
-
vmax/Km for ferricytochrome c is 16% of the wild-type value. Vmax/Km for NADPH is 12% of the wild-type value
P55L
-
vmax/Km for ferricytochrome c is 67% of the wild-type value. Vmax/Km for NADPH is 123% of the wild-type value
Q153R
-
Vmax/Km for cytochrome c is 9% of wild-type value, Vmax/Km for NADPH is 11% of wild-type value
R246A
mutation in the hinge segment, ionic strength profile is shifted to lower salt concentrations
R246P
mutation in the hinge segment, ionic strength profile is shifted to lower salt concentrations
R316W
R406H
-
vmax/Km for ferricytochrome c is 62% of the wild-type value. Vmax/Km for NADPH is 78% of the wild-type value
R457H
R600W
-
vmax/Km for ferricytochrome c is 18% of the wild-type value. Vmax/Km for NADPH is 7% of the wild-type value
R616X
-
the POR mutant is associated with total loss of heme oxygenase-1 activity
R636A
the mutation leads to a modest increase in cytochrome c reduction, which is linked to weaker coenzyme binding and faster interflavin electron transfer
R636S
the mutation leads to a modest increase in cytochrome c reduction, which is linked to weaker coenzyme binding and faster interflavin electron transfer
S243P
mutation in the hinge segment, ionic strength profile is shifted to lower salt concentrations
S244C
-
mutation results in similar disruption of CYP17A1 and CYP21A2 activities
T142A
-
Vmax/Km for cytochrome c is 49% of wild-type value, Vmax/Km for NADPH is 52% of wild-type value
V472M
-
vmax/Km for ferricytochrome c is 23% of the wild-type value. Vmax/Km for NADPH is 24% of the wild-type value
V492E
V608F
V631I
-
Vmax/Km for cytochrome c is 74% of wild-type value, Vmax/Km for NADPH is 23% of wild-type value
W676H
-
rate of FAD-reduction is modestly affected, enzyme is reduced only to the two-electron level in rapid mixing experiments
W677A
substitution of Trp677 decreases NADPH-dependent cytochrome c reductase activity 25- to 55fold. W677A substitution abolishes the decrease in cytochrome c reductase activity at low pH
W677G
substitution of Trp677 decreases NADPH-dependent cytochrome c reductase activity 25- to 55fold. W677G substitution exhibits only a slight decrease in activity at low pH
W677G/S678X
W677G/S678X substitution exhibits only a slight decrease in activity at low pH
Y181D
Y459H
Y607C
-
Vmax/Km for NADPH is 20% of the wild-type value
DELTA1-23
-
truncated enzyme
A264C
no notable decreases in expression of the respective haem domains or flavocytochromes. For A264C haem domain, fatty-acid-induced perturbation of the spinstate equilibrium is observed in some cases, but the extent of high-spin conversion is substantially less than observed for wild-type with arachidonate
A264M
higher dodecanoate affinity than wild-type BM3. For the A264M mutant, a more substantial fatty-acid-induced high-spin conversion of haem iron is observed than for A264C, albeit slightly less than for wild-type with the same fatty acids.
A264Q
no notable decreases in expression of the respective haem domains or flavocytochromes
C136A
site-directed mutagenesis
C228A
site-directed mutagenesis
C363T
site-directed mutagenesis
C445L
site-directed mutagenesis
C472T
C566A
site-directed mutagenesis, the mutant shows full catalytic activity and a 2.5fold increased Km for NADPH compared to the wild-type enzyme
D113A
-
the mutation increases kcat approximately 2fold, but does not affect Km at the lowest ionic strength (10 mM), the mutant displays no change in catalytic efficiency compared to the wild type enzyme
E115A/E116A
-
the mutations increase kcat approximately 2fold, but does not affect Km at the lowest ionic strength (10 mM), the mutant displays a slight decrease in catalytic efficiency at higher ionic strengths due to a larger increase in Km than observed for kcat
E238A/E239A
hinge region connecting the FMN domain to the rest of the protein
G237/+AA/E238
hinge region connecting the FMN domain to the rest of the protein
G237/+AAAA/238
hinge region connecting the FMN domain to the rest of the protein
G488L
substitution decreases FAD binding by approximately 80% but does not affect FMN incorporation, 42fold decrease in catalytic activity compared to wild type, substitution does not affect either Km for NADPH or Km for cytochrome c, addition of FAD to the mutant results in partial restoration of catalytic activity
K56Q
-
the full-length mutant enzyme is stable to spontaneopus proteolysis but possesses spectral and catzalytic properties of the wild-type flavoprotein
R454E
substitution decreases both FAD binding and FMN incorporation, suggesting interaction between the two flavin domains and/or the interconnecting region, FAD content ranged from undetectable to approximately 0.1 mol of FAD/mol of enzyme, 338fold decrease in catalytic activity compared to wild type, substitution does not affect either Km for NADPH or Km for cytochrome c, addition of FAD to the mutant resulted in partial restoration of catalytic activity
S457A/C630A/D675N
site-directed mutagenesis, catalytically inactive mutant possessing a structure almost identical to that of the wild-type
S678X
substitution does not affect FAD or FMN incorporation, substitution has no effect on the catalytic activity or kinetic properties
T236A/G237A/E238A/E239A
hinge region connecting the FMN domain to the rest of the protein
T491V
substitution decreases FAD binding by approximately 50% but does not affect FMN incorporation, 2fold decrease in catalytic activity compared to wild type, substitution does not affect either Km for NADPH or Km for cytochrome c, addition of FAD to the mutant results in full restoration of catalytic activity
W677X
substitution does not affect FAD or FMN incorporation, 34fold decrease in catalytic activity compared to wild type, substitution does not alter significantly Km for cytochrome c but decreases Km for NADPH
W677Y
substitution does not affect FAD or FMN incorporation, 2fold decrease in catalytic activity compared to wild type, substitution does not alter significantly Km for cytochrome c but decreases Km for NADPH
Y140D
-
substitution does not eliminate FMN binding but reduces cytochrome c reductase activity, Km value for cytochrome c or NADPH similar to wild type
Y140D/178D
-
substitution abolishes FMN binding and cytochrome c reductase activity
Y140F
-
substitution has no effect on FMN content or catalytic activity, Km value for cytochrome c or NADPH similar to wild type
Y140F/178F
-
substitution has no effect on FMN content or catalytic activity, slightly decreases Km for cytochrome c, NADPH Km value slightly higher than wild type
Y178D
-
substitution abolishes FMN binding and cytochrome c reductase activity, Km value for cytochrome c similar to wild type, NADPH Km value slightly higher than wild type
Y178F
-
substitution has no effect on FMN content or catalytic activity, slightly decreases Km for cytochrome c, NADPH Km value similar to wild type
Y456S
substitution decreases FAD binding but did not affect FMN incorporation, 250fold decrease in catalytic activity compared to wild type, substitution increases Km for cytochrome c, addition of FAD to the mutant results in full restoration of catalytic activity
additional information