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Information on EC 1.6.2.2 - cytochrome-b5 reductase and Organism(s) Sus scrofa and UniProt Accession P83686
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1.6.2.2 cytochrome-b5 reductaseIUBMB Comments
A flavoprotein (FAD).
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Word Map
- 1.6.2.2
- erythrocyte
- methemoglobinemia
-
nadph-cytochrome
- hereditary
- reductases
-
cyanosis
- fad
- ferricyanide
- flavoproteins
-
desaturation
- dt-diaphorase
- aniline
-
amidoxime
-
methb
-
hemolysate
-
bioreductive
- n-demethylase
- aminopyrine
-
marc
-
n-hydroxylated
-
mixed-function
-
nadh-binding
- methaemoglobin
- medicine
-
oxyhemoglobin
- diagnostics
- analysis
- industry
- drug development
The taxonomic range for the selected organisms is: Sus scrofa
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria, Archaea
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria, Archaea
Synonyms
nadh-cytochrome b5 reductase, cytochrome b5 reductase, methemoglobin reductase, cyb5r3, ncb5or, nadh cytochrome b5 reductase, nadh:cytochrome b5 reductase, cyb5r2, cytochrome-b5 reductase, cyb5r, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
B5R
cytochrome b5 reductase
-
-
-
-
dihydronicotinamide adenine dinucleotide-cytochrome b5 reductase
-
-
-
-
NADH-cytochrome b5 reductase
NADH-cytochrome-b5 reductase
-
-
-
-
NADH-ferricytochrome b5 oxidoreductase
-
-
-
-
P34/P32
-
-
-
-
P35
-
-
-
-
reduced nicotinamide adeninedinucleotide-cytochrome b5 reductase
-
-
-
-
reductase, cytochrome b5
-
-
-
-
B5R
-
-
-
-
NADH-cytochrome b5 reductase
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
NADH + 2 ferricytochrome b5 = NAD+ + H+ + 2 ferrocytochrome b5
hypothetical mechanism
-
NADH + 2 ferricytochrome b5 = NAD+ + H+ + 2 ferrocytochrome b5
electron-transport chain from NADH to a terminal oxidase desaturase, proposed electron transfer mechanism
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
redox reaction
-
-
-
-
oxidation
-
-
-
-
reduction
-
-
-
-
PATHWAY SOURCE
PATHWAYS
SYSTEMATIC NAME
IUBMB Comments
NADH:ferricytochrome-b5 oxidoreductase
A flavoprotein (FAD).
CAS REGISTRY NUMBER
COMMENTARY
9032-25-1
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
NADH + oxidized nitroblue tetrazolium
NAD+ + H+ + reduced nitroblue tetrazolium
-
-
-
-
?
2 ferricytochrome b5 + NADH
2 ferrocytochrome b5 + NAD+ + H+
-
-
-
?
2 ferricytochrome b5 + NADH
2 ferrocytochrome b5 + NAD+ + H+
involved in the synthesis of fatty acids and cholesterol, and in the oxidation of xenobiotics
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
2 ferricytochrome b5 + NADH
2 ferrocytochrome b5 + NAD+ + H+
involved in the synthesis of fatty acids and cholesterol, and in the oxidation of xenobiotics
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
FAD
redox state of FAD during the b5R catalytic cycle and crystal structures comparion of the fully reduced form and the oxidized form, overview
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.00104
ferricyanide
-
mutant enzyme P247L, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
0.00723
ferricyanide
-
wild type enzyme, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
0.01096
ferricyanide
-
mutant enzyme P247A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
0.01563
ferricyanide
-
mutant enzyme P248A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
0.01568
ferricyanide
-
mutant enzyme P248L, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
0.02646
ferricyanide
-
mutant enzyme P249A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
0.03309
ferricyanide
-
mutant enzyme P249L, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
0.01353
ferricytochrome b5
-
wild type enzyme, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
0.0207
ferricytochrome b5
-
mutant enzyme P247A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
0.03434
ferricytochrome b5
-
mutant enzyme P248A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
0.04481
ferricytochrome b5
-
mutant enzyme P249A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
0.01097
NADH
-
wild type enzyme, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
0.0135
NADH
-
mutant enzyme P248L, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
0.0155
NADH
-
mutant enzyme P249L, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
0.01713
NADH
-
mutant enzyme P247A, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
0.02407
NADH
-
mutant enzyme P248A, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
0.02467
NADH
-
mutant enzyme P247L, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
0.02928
NADH
-
mutant enzyme P249A, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
30.8
ferricyanide
-
mutant enzyme P247L, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
337
ferricyanide
-
wild type enzyme, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
357
ferricyanide
-
mutant enzyme P247A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
399
ferricyanide
-
mutant enzyme P248A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
504
ferricyanide
-
mutant enzyme P248L, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
838
ferricyanide
-
mutant enzyme P249A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
1241
ferricyanide
-
mutant enzyme P249L, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
147
ferricytochrome b5
-
wild type enzyme, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
167
ferricytochrome b5
-
mutant enzyme P247A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
301
ferricytochrome b5
-
mutant enzyme P248A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
540
ferricytochrome b5
-
mutant enzyme P249A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
21.7
NADH
-
mutant enzyme P247L, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
354
NADH
-
mutant enzyme P247A, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
540
NADH
-
mutant enzyme P248L, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
559
NADH
-
mutant enzyme P248A, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
681
NADH
-
mutant enzyme P249A, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
742
NADH
-
wild type enzyme, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
1059
NADH
-
mutant enzyme P249L, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
25530
ferricyanide
-
mutant enzyme P248A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
29620
ferricyanide
-
mutant enzyme P247L, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
31670
ferricyanide
-
mutant enzyme P249A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
32140
ferricyanide
-
mutant enzyme P248L, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
32570
ferricyanide
-
mutant enzyme P247A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
37500
ferricyanide
-
mutant enzyme P249L, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
46610
ferricyanide
-
wild type enzyme, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
80700
ferricytochrome b5
-
mutant enzyme P247A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
87700
ferricytochrome b5
-
mutant enzyme P248A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
108600
ferricytochrome b5
-
wild type enzyme, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
120500
ferricytochrome b5
-
mutant enzyme P249A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
880
NADH
-
mutant enzyme P247L, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
20670
NADH
-
mutant enzyme P247A, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
23220
NADH
-
mutant enzyme P248A, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
23260
NADH
-
mutant enzyme P249A, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
40000
NADH
-
mutant enzyme P248L, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
67640
NADH
-
wild type enzyme, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
68320
NADH
-
mutant enzyme P249L, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
the enzyme catalyzes the electron transfer from NADH to cytochrome b5 and participates in fatty acid synthesis, cholesterol synthesis, and xenobiotic oxidation as a member of the electron transport chain on the endoplasmic reticulum. In erythrocytes, the enzyme also participates in the reduction of methemoglobin
additional information
additional information
the NADH-cytochrome b5 reductase is a flavoprotein consisting of NADH and FAD binding domains, that catalyzes electron transfer from the two-electron carrier NADH to the one-electron carrier cytochrome b5
additional information
-
the NADH-cytochrome b5 reductase is a flavoprotein consisting of NADH and FAD binding domains, that catalyzes electron transfer from the two-electron carrier NADH to the one-electron carrier cytochrome b5
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). NB5R3_PIG
272
0
30831
Swiss-Prot
other Location (Reliability: 1)
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
fully reduced form and the oxidized form of the purified liver enzyme, X-ray diffraction structure determination and analysis at 1.68 A resolution
hanging drop vapor diffusion method, using 9-12% (w/v) PEG 4,000, 100 mM potassium phosphate (pH 7.7) and 5 mM dithiothreitol
vapor diffusion method, 5 mg/ml protein, 12.5% polyethylenglycol 4000, 50 mM potassium phosphate pH 6.0-8.0, 0.1 mM EDTA, preliminary X-ray data
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
T66A
Km for NADH is not affected, Km for cytochrome b5 is significantly enhanced
T66V
turnover is reduced to 10% of the native enzyme, Km for NADH is not affected, Km for cytochrome b5 is significantly enhanced
P247A
P247L
P248A
P248L
P249A
P249L
P247A
-
the mutant shows increased Km and decreased kcat values for NADH and cytochrome b5, as well as increased Km and kcat values for ferricyanide compared to the wild type enzyme
P247L
-
the mutation significantly decreases kcat with slight increase (about 2fold) in Km for the physiological electron donor NADH. However, Km and kcat values for the electron acceptors (both cytochrome b5 and ferricyanide) are decreased significantly
P248A
-
the mutant shows increased Km and decreased kcat values for NADH and cytochrome b5, as well as increased Km and kcat values for ferricyanide compared to the wild type enzyme
P248L
-
the mutant shows increased Km and decreased kcat values for NADH and ferricyanide compared to the wild type enzyme
P249A
-
the mutation affects the Km (NADH) values to increase slightly by a factor of 3 compared to the wild type enzyme
P249L
-
the mutant shows increased Km and kcat values for NADH, ferricyanide and cytochrome b5 compared to the wild type enzyme
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate precipitation, DE-52 cellulose column chromatography, hydroxylapatite column chromatography, and NAD+-agarose column chromatography
-
ammonium sulfate precipitation, DEAE-Sepharose CL-6B column chromatography, 5'-ADP-agarose (or 5'-AMP-agarose) gel column chromatography, and Ni-NTA-agarose column chromatography
-
recombinant wild-type, K97A, K97R, S99A, S99T, S99V, R63A, R63Q, R63K, Y65A and Y65F mutant enzymes
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression of wild-type, H49A, H49E, H49K, H49Y, and DELTAF272 mutant enzymes in Escherichia coli
-
expression of wild-type, K97A, K97R, S99A, S99T, S99V, R63A, R63Q, R63K, Y65A and Y65F mutant enzymes in Escherichia coli
-
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Tamura, M.; Yoshida, S.; Tamura, T.; Saitoh, T.; Takeshita, M.
Effect of divalent cations on NADH-dependent and NADPH-dependent cytochrome b5 reduction by hepatic microsomes
Arch. Biochem. Biophys.
280
313-319
1990
Sus scrofa
Miki, K.; Kaida, S.; Kasai, N.; Iyanagi, T.; Kobayashi, K.; Hayashi, K.
Crystallization and preliminary x-ray crystallographic study of NADH-cytochrome b5 reductase from pig liver microsomes
J. Biol. Chem.
262
11801-11802
1987
Sus scrofa
Iyanagi, T.; Watanabe, S.; Anan, K.F.
One-electron oxidation-reduction properties of hepatic NADH-cytochrome b5 reductase
Biochemistry
23
1418-1425
1984
Sus scrofa
Strittmatter, P.
Microsomal cytochrome b5 and cytochrome b5 reductase
The Enzymes, 2nd Ed. (Boyer, P. D. , Lardy, H. , Myrbck, K. , eds. )
8
113-145
1963
Bos taurus, Oryctolagus cuniculus, Rattus norvegicus, Sus scrofa
-
Oshino, N.; Sato, R.
Stimulation by phenols of the reoxidation microsomal bound cytochrome b5 and its implication to fatty acid desaturation
J. Biochem.
69
169-180
1971
Rattus norvegicus, Sus scrofa
Nishida, H.; Inaka, K.; Yamanaka, M.; Kaida, S.; Kobayashi, K.; Miki, K.
Crystal structure of NADH-cytochrome b5 reductase from pig liver at 2.4 A resolution
Biochemistry
34
2763-2767
1995
Sus scrofa
Nishida, H.; Miki, K.
Electrostatic properties deduced from refined structures of NADH-cytochrome b5 reductase and the other flavin-dependent reductases: pyridine nucleotide-binding and interaction with an electron-transfer partner
Proteins Struct. Funct. Genet.
26
32-41
1996
Sus scrofa
Kimura, S.; Emi, Y.; Ikushiro, S.; Iyanagi, T.
Systematic mutations of highly conserved His49 and carboxyl-terminal of recombinant porcine liver NADH-cytochrome b5 reductase solubilized domain
Biochim. Biophys. Acta
1430
290-301
1999
Sus scrofa
Kimura, S.; Nishida, H.; Iyanagi, T.
Effects of flavin-binding motif amino acid mutations in the NADH-cytochrome b5 reductase catalytic domain on protein stability and catalysis
J. Biochem.
130
481-490
2001
Sus scrofa
Kimura, S.; Kawamura, M.; Iyanagi, T.
Role of Thr(66) in porcine NADH-cytochrome b5 reductase in catalysis and control of the rate-limiting step in electron transfer
J. Biol. Chem.
278
3580-3589
2003
Sus scrofa (P83686), Sus scrofa
Nishimura, Y.; Shibuya, M.; Muraki, A.; Takeuchi, F.; Park, S.; Tsubaki, M.
Structural and mechanistic roles of three consecutive Pro residues of porcine NADH-cytochrome b5 reductase for the binding of beta-NADH
J. Biosci. Bioeng.
108
286-292
2009
Sus scrofa
Yamada, M.; Tamada, T.; Takeda, K.; Matsumoto, F.; Ohno, H.; Kosugi, M.; Takaba, K.; Shoyama, Y.; Kimura, S.; Kuroki, R.; Miki, K.
Elucidations of the catalytic cycle of NADH-cytochrome b5 reductase by X-ray crystallography: new insights into regulation of efficient electron transfer
J. Mol. Biol.
425
4295-4306
2013
Sus scrofa (P83686), Sus scrofa
Samhan-Arias, A.K.; Gutierrez-Merino, C.
Purified NADH-cytochrome b5 reductase is a novel superoxide anion source inhibited by apocynin sensitivity to nitric oxide and peroxynitrite
Free Radic. Biol. Med.
73
174-189
2014
Sus scrofa
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