NB5R3_PIG
ID NB5R3_PIG Reviewed; 272 AA.
AC P83686;
DT 15-DEC-2003, integrated into UniProtKB/Swiss-Prot.
DT 15-DEC-2003, sequence version 1.
DT 03-AUG-2022, entry version 120.
DE RecName: Full=NADH-cytochrome b5 reductase 3 {ECO:0000305};
DE Short=B5R;
DE Short=Cytochrome b5 reductase;
DE EC=1.6.2.2 {ECO:0000269|PubMed:3967486};
DE AltName: Full=Diaphorase-1;
DE Flags: Fragment;
GN Name=CYB5R3; Synonyms=DIA1;
OS Sus scrofa (Pig).
OC Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia;
OC Eutheria; Laurasiatheria; Artiodactyla; Suina; Suidae; Sus.
OX NCBI_TaxID=9823 {ECO:0000312|PDB:1NDH};
RN [1] {ECO:0000305}
RP NUCLEOTIDE SEQUENCE, PROTEIN SEQUENCE OF 1-5, MUTAGENESIS OF HIS-49 AND
RP PHE-272, AND CIRCULAR DICHROISM ANALYSIS.
RC TISSUE=Liver {ECO:0000269|PubMed:10082957};
RX PubMed=10082957; DOI=10.1016/s0167-4838(99)00008-4;
RA Kimura S., Emi Y., Ikushiro S., Iyanagi T.;
RT "Systematic mutations of highly conserved His49 and carboxyl-terminal of
RT recombinant porcine liver NADH-cytochrome b5 reductase solubilized
RT domain.";
RL Biochim. Biophys. Acta 1430:290-301(1999).
RN [2] {ECO:0000305}
RP PROTEIN SEQUENCE OF 1-20.
RC TISSUE=Liver {ECO:0000269|PubMed:7417338};
RX PubMed=7417338; DOI=10.1016/s0006-291x(80)80088-x;
RA Crabb J.W., Tarr G.E., Yasunobu K.T., Iyanagi T., Coon M.J.;
RT "Terminal sequences of lysosome solubilized pig liver cytochrome b5
RT reductase.";
RL Biochem. Biophys. Res. Commun. 95:1650-1655(1980).
RN [3] {ECO:0000305}
RP CHARACTERIZATION, AND CATALYTIC ACTIVITY.
RX PubMed=3967486; DOI=10.1016/0305-0491(85)90440-7;
RA Ghesquier D., Robert J.C., Soumarmon A., Abastado M., Grelac F.,
RA Lewin M.J.M.;
RT "Gastric microsomal NADH-cytochrome b5 reductase: characterization and
RT solubilization.";
RL Comp. Biochem. Physiol. 80B:165-169(1985).
RN [4] {ECO:0000305}
RP FAD-BINDING.
RX PubMed=7890048; DOI=10.1016/0014-5793(95)00161-2;
RA Nishida H., Inaka K., Miki K.;
RT "Specific arrangement of three amino acid residues for flavin-binding
RT barrel structures in NADH-cytochrome b5 reductase and the other flavin-
RT dependent reductases.";
RL FEBS Lett. 361:97-100(1995).
RN [5] {ECO:0000305}
RP POTENTIOMETRIC TITRATION, AND EPR SPECTROSCOPY.
RX PubMed=19038; DOI=10.1021/bi00631a021;
RA Iyanagi T.;
RT "Redox properties of microsomal reduced nicotinamide adenine dinucleotide-
RT cytochrome b5 reductase and cytochrome b5.";
RL Biochemistry 16:2725-2730(1977).
RN [6] {ECO:0000305}
RP ENZYME KINETICS.
RX PubMed=3372498; DOI=10.1016/s0021-9258(18)68525-4;
RA Kobayashi K., Iyanagi T., Ohara H., Hayashi K.;
RT "One-electron reduction of hepatic NADH-cytochrome b5 reductase as studied
RT by pulse radiolysis.";
RL J. Biol. Chem. 263:7493-7499(1988).
RN [7] {ECO:0000305}
RP MUTAGENESIS OF ARG-63; TYR-65; LYS-97 AND SER-99, AND CIRCULAR DICHROISM
RP ANALYSIS.
RX PubMed=11574067; DOI=10.1093/oxfordjournals.jbchem.a003010;
RA Kimura S., Nishida H., Iyanagi T.;
RT "Effects of flavin-binding motif amino acid mutations in the NADH-
RT cytochrome b5 reductase catalytic domain on protein stability and
RT catalysis.";
RL J. Biochem. 130:481-490(2001).
RN [8] {ECO:0000305}
RP MUTAGENESIS OF THR-66, ENZYME KINETICS, AND CIRCULAR DICHROISM ANALYSIS.
RX PubMed=12459552; DOI=10.1074/jbc.m209838200;
RA Kimura S., Kawamura M., Iyanagi T.;
RT "Role of Thr(66) in porcine NADH-cytochrome b5 reductase in catalysis and
RT control of the rate-limiting step in electron transfer.";
RL J. Biol. Chem. 278:3580-3589(2003).
RN [9]
RP IDENTIFICATION IN A COMPLEX WITH CYTOCHROME B5 AND MTARC2.
RX PubMed=16973608; DOI=10.1074/jbc.m607697200;
RA Havemeyer A., Bittner F., Wollers S., Mendel R., Kunze T., Clement B.;
RT "Identification of the missing component in the mitochondrial benzamidoxime
RT prodrug converting system as a novel molybdenum enzyme.";
RL J. Biol. Chem. 281:34796-34802(2006).
RN [10] {ECO:0000312|PDB:1NDH}
RP X-RAY CRYSTALLOGRAPHY (2.4 ANGSTROMS) OF 2-273.
RX PubMed=7893687; DOI=10.1021/bi00009a004;
RA Nishida H., Inaka K., Yamanaka M., Kaida S., Kobayashi K., Miki K.;
RT "Crystal structure of NADH-cytochrome b5 reductase from pig liver at 2.4 A
RT resolution.";
RL Biochemistry 34:2763-2767(1995).
RN [11] {ECO:0000305}
RP X-RAY CRYSTALLOGRAPHY (2.1 ANGSTROMS), AND 3D-STRUCTURE MODELING OF COMPLEX
RP WITH CYTOCHROME B5.
RX PubMed=8880927;
RX DOI=10.1002/(sici)1097-0134(199609)26:1<32::aid-prot3>3.0.co;2-i;
RA Nishida H., Miki K.;
RT "Electrostatic properties deduced from refined structures of NADH-
RT cytochrome b5 reductase and the other flavin-dependent reductases: pyridine
RT nucleotide-binding and interaction with an electron-transfer partner.";
RL Proteins 26:32-41(1996).
CC -!- FUNCTION: Transfers two electrons from NADH to two molecules of
CC cytochrome b5 through an enzyme-bound FAD. Electrons from the reduced
CC cytochrome b5 are then transferred to various electron acceptors,
CC thereby participating in methemoglobin reduction in red blood cells and
CC in the desaturation and elongation of fatty acids, cholesterol
CC biosynthesis and cytochrome P-450-mediated drug metabolism other
CC tissues. {ECO:0000250|UniProtKB:P00387}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=2 Fe(III)-[cytochrome b5] + NADH = 2 Fe(II)-[cytochrome b5] +
CC H(+) + NAD(+); Xref=Rhea:RHEA:46680, Rhea:RHEA-COMP:10438, Rhea:RHEA-
CC COMP:10439, ChEBI:CHEBI:15378, ChEBI:CHEBI:29033, ChEBI:CHEBI:29034,
CC ChEBI:CHEBI:57540, ChEBI:CHEBI:57945; EC=1.6.2.2;
CC Evidence={ECO:0000269|PubMed:3967486};
CC PhysiologicalDirection=left-to-right; Xref=Rhea:RHEA:46681;
CC Evidence={ECO:0000305|PubMed:3967486};
CC -!- COFACTOR:
CC Name=FAD; Xref=ChEBI:CHEBI:57692;
CC Evidence={ECO:0000269|PubMed:7890048};
CC -!- SUBUNIT: Component of a complex composed of cytochrome b5, NADH-
CC cytochrome b5 reductase (CYB5R3) and MTARC2 (PubMed:16973608).
CC Interacts with MTLN; the interaction is required to maintain cellular
CC lipid composition and leads to stimulation of mitochondrial respiratory
CC complex I activity (By similarity). {ECO:0000250|UniProtKB:Q9DCN2,
CC ECO:0000269|PubMed:16973608}.
CC -!- SUBCELLULAR LOCATION: Endoplasmic reticulum membrane
CC {ECO:0000250|UniProtKB:P00387}; Lipid-anchor
CC {ECO:0000250|UniProtKB:P00387}; Cytoplasmic side
CC {ECO:0000250|UniProtKB:P00387}. Mitochondrion outer membrane
CC {ECO:0000250|UniProtKB:P00387}; Lipid-anchor
CC {ECO:0000250|UniProtKB:P00387}; Cytoplasmic side
CC {ECO:0000250|UniProtKB:P00387}. Cytoplasm
CC {ECO:0000250|UniProtKB:P00387}. Note=The enzyme exists in two forms, a
CC membrane-bound form on the cytoplasmic side of the endoplasmic
CC reticulum and also on the mitochondrial outer membrane and in soluble
CC form in erythrocytes. NADH-cytochrome b5 reductase 3 membrane-bound
CC form: Endoplasmic reticulum membrane; Lipid-anchor; Cytoplasmic side.
CC Mitochondrion outer membrane; Lipid-anchor; Cytoplasmic side. NADH-
CC cytochrome b5 reductase 3 soluble form: Cytoplasm.
CC {ECO:0000250|UniProtKB:P00387}.
CC -!- SIMILARITY: Belongs to the flavoprotein pyridine nucleotide cytochrome
CC reductase family. {ECO:0000305}.
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DR PDB; 1NDH; X-ray; 2.10 A; A=1-272.
DR PDB; 3W2E; X-ray; 2.10 A; A=2-272.
DR PDB; 3W2F; X-ray; 1.76 A; A=2-272.
DR PDB; 3W2G; X-ray; 1.68 A; A=2-272.
DR PDB; 3W2H; X-ray; 1.75 A; A=2-272.
DR PDB; 3W2I; X-ray; 1.81 A; A=2-272.
DR PDB; 3W5H; X-ray; 0.78 A; A=1-272.
DR PDB; 5GV7; X-ray; 0.80 A; A=1-272.
DR PDB; 5GV8; X-ray; 0.78 A; A=1-272.
DR PDBsum; 1NDH; -.
DR PDBsum; 3W2E; -.
DR PDBsum; 3W2F; -.
DR PDBsum; 3W2G; -.
DR PDBsum; 3W2H; -.
DR PDBsum; 3W2I; -.
DR PDBsum; 3W5H; -.
DR PDBsum; 5GV7; -.
DR PDBsum; 5GV8; -.
DR AlphaFoldDB; P83686; -.
DR SMR; P83686; -.
DR STRING; 9823.ENSSSCP00000000040; -.
DR PaxDb; P83686; -.
DR PeptideAtlas; P83686; -.
DR PRIDE; P83686; -.
DR eggNOG; KOG0534; Eukaryota.
DR InParanoid; P83686; -.
DR BRENDA; 1.6.2.2; 6170.
DR SABIO-RK; P83686; -.
DR EvolutionaryTrace; P83686; -.
DR Proteomes; UP000008227; Unplaced.
DR Proteomes; UP000314985; Unplaced.
DR GO; GO:0005789; C:endoplasmic reticulum membrane; IEA:UniProtKB-SubCell.
DR GO; GO:0005741; C:mitochondrial outer membrane; IEA:UniProtKB-SubCell.
DR GO; GO:0004128; F:cytochrome-b5 reductase activity, acting on NAD(P)H; IDA:UniProtKB.
DR GO; GO:0071949; F:FAD binding; IBA:GO_Central.
DR GO; GO:0050660; F:flavin adenine dinucleotide binding; IDA:UniProtKB.
DR GO; GO:0006695; P:cholesterol biosynthetic process; IEA:UniProtKB-KW.
DR Gene3D; 3.40.50.80; -; 1.
DR InterPro; IPR001834; CBR-like.
DR InterPro; IPR008333; Cbr1-like_FAD-bd_dom.
DR InterPro; IPR017927; FAD-bd_FR_type.
DR InterPro; IPR001709; Flavoprot_Pyr_Nucl_cyt_Rdtase.
DR InterPro; IPR039261; FNR_nucleotide-bd.
DR InterPro; IPR001433; OxRdtase_FAD/NAD-bd.
DR InterPro; IPR017938; Riboflavin_synthase-like_b-brl.
DR PANTHER; PTHR19370; PTHR19370; 1.
DR Pfam; PF00970; FAD_binding_6; 1.
DR Pfam; PF00175; NAD_binding_1; 1.
DR PRINTS; PR00371; FPNCR.
DR SUPFAM; SSF52343; SSF52343; 1.
DR SUPFAM; SSF63380; SSF63380; 1.
DR PROSITE; PS51384; FAD_FR; 1.
PE 1: Evidence at protein level;
KW 3D-structure; Acetylation; Cholesterol biosynthesis;
KW Cholesterol metabolism; Cytoplasm; Direct protein sequencing;
KW Endoplasmic reticulum; FAD; Flavoprotein; Lipid biosynthesis;
KW Lipid metabolism; Lipoprotein; Membrane; Mitochondrion;
KW Mitochondrion outer membrane; NAD; Oxidoreductase; Phosphoprotein;
KW Reference proteome; Steroid biosynthesis; Steroid metabolism;
KW Sterol biosynthesis; Sterol metabolism.
FT CHAIN <1..272
FT /note="NADH-cytochrome b5 reductase 3"
FT /id="PRO_0000167625"
FT DOMAIN 11..123
FT /note="FAD-binding FR-type"
FT /evidence="ECO:0000255|PROSITE-ProRule:PRU00716"
FT BINDING 103..118
FT /ligand="FAD"
FT /ligand_id="ChEBI:CHEBI:57692"
FT /evidence="ECO:0000250"
FT BINDING 142..177
FT /ligand="FAD"
FT /ligand_id="ChEBI:CHEBI:57692"
FT /evidence="ECO:0000250"
FT MOD_RES 13
FT /note="N6-acetyllysine"
FT /evidence="ECO:0000250|UniProtKB:P00387"
FT MOD_RES 14
FT /note="Phosphotyrosine"
FT /evidence="ECO:0000250|UniProtKB:P00387"
FT MOD_RES 21
FT /note="N6-acetyllysine"
FT /evidence="ECO:0000250|UniProtKB:Q9DCN2"
FT MOD_RES 91
FT /note="N6-acetyllysine"
FT /evidence="ECO:0000250|UniProtKB:Q9DCN2"
FT MUTAGEN 49
FT /note="H->A: Similar properties to wild-type."
FT /evidence="ECO:0000269|PubMed:10082957"
FT MUTAGEN 49
FT /note="H->E: Decreased kcat values for PB5, ferricyanide
FT and NADH. Increased Km values for all three substrates."
FT /evidence="ECO:0000269|PubMed:10082957"
FT MUTAGEN 49
FT /note="H->K: Decreased kcat and Km values for PB5 and
FT ferricyanide. Increased values for NADH."
FT /evidence="ECO:0000269|PubMed:10082957"
FT MUTAGEN 49
FT /note="H->Y: Similar properties to wild-type."
FT /evidence="ECO:0000269|PubMed:10082957"
FT MUTAGEN 63
FT /note="R->A,Q: Increased Km values for NADH and increased
FT dissociation constant for NAD(+)."
FT /evidence="ECO:0000269|PubMed:11574067"
FT MUTAGEN 63
FT /note="R->K: Little effect on Km and KD values."
FT /evidence="ECO:0000269|PubMed:11574067"
FT MUTAGEN 65
FT /note="Y->A,F: Protein destabilized, release of FAD
FT accelerated, and kcat values decreased."
FT /evidence="ECO:0000269|PubMed:11574067"
FT MUTAGEN 66
FT /note="T->A: Similar properties to wild-type."
FT /evidence="ECO:0000269|PubMed:12459552"
FT MUTAGEN 66
FT /note="T->S: Similar properties to wild-type."
FT /evidence="ECO:0000269|PubMed:12459552"
FT MUTAGEN 66
FT /note="T->V: 10% of wild-type kcat values."
FT /evidence="ECO:0000269|PubMed:12459552"
FT MUTAGEN 97
FT /note="K->A: Little effect on absorption or CD spectra."
FT /evidence="ECO:0000269|PubMed:11574067"
FT MUTAGEN 97
FT /note="K->R: Little effect on absorption or CD spectra."
FT /evidence="ECO:0000269|PubMed:11574067"
FT MUTAGEN 99
FT /note="S->A,V: Increased Km values for NADH and increased
FT dissociation constant for NAD(+)."
FT /evidence="ECO:0000269|PubMed:11574067"
FT MUTAGEN 99
FT /note="S->T: Little effect on Km and KD values."
FT /evidence="ECO:0000269|PubMed:11574067"
FT MUTAGEN 272
FT /note="F->FG: 10% of wild-type kcat values."
FT /evidence="ECO:0000269|PubMed:10082957"
FT MUTAGEN 272
FT /note="Missing: 54% of wild-type kcat values."
FT /evidence="ECO:0000269|PubMed:10082957"
FT CONFLICT 39
FT /note="E -> Q (in Ref. 1)"
FT /evidence="ECO:0000305"
FT NON_TER 1
FT STRAND 14..25
FT /evidence="ECO:0007829|PDB:3W5H"
FT STRAND 28..34
FT /evidence="ECO:0007829|PDB:3W5H"
FT STRAND 49..56
FT /evidence="ECO:0007829|PDB:3W5H"
FT STRAND 59..65
FT /evidence="ECO:0007829|PDB:3W5H"
FT STRAND 67..69
FT /evidence="ECO:0007829|PDB:3W2E"
FT STRAND 75..82
FT /evidence="ECO:0007829|PDB:3W5H"
FT TURN 86..88
FT /evidence="ECO:0007829|PDB:3W2G"
FT STRAND 89..91
FT /evidence="ECO:0007829|PDB:1NDH"
FT HELIX 97..104
FT /evidence="ECO:0007829|PDB:3W5H"
FT STRAND 110..117
FT /evidence="ECO:0007829|PDB:3W5H"
FT STRAND 119..124
FT /evidence="ECO:0007829|PDB:3W5H"
FT STRAND 127..130
FT /evidence="ECO:0007829|PDB:3W5H"
FT STRAND 132..135
FT /evidence="ECO:0007829|PDB:1NDH"
FT STRAND 139..142
FT /evidence="ECO:0007829|PDB:3W5H"
FT STRAND 144..151
FT /evidence="ECO:0007829|PDB:3W5H"
FT HELIX 152..154
FT /evidence="ECO:0007829|PDB:3W5H"
FT HELIX 155..166
FT /evidence="ECO:0007829|PDB:3W5H"
FT STRAND 174..183
FT /evidence="ECO:0007829|PDB:3W5H"
FT HELIX 184..186
FT /evidence="ECO:0007829|PDB:3W5H"
FT HELIX 190..200
FT /evidence="ECO:0007829|PDB:3W5H"
FT TURN 201..203
FT /evidence="ECO:0007829|PDB:3W5H"
FT STRAND 204..212
FT /evidence="ECO:0007829|PDB:3W5H"
FT STRAND 218..223
FT /evidence="ECO:0007829|PDB:3W5H"
FT HELIX 226..232
FT /evidence="ECO:0007829|PDB:3W5H"
FT HELIX 236..238
FT /evidence="ECO:0007829|PDB:3W5H"
FT STRAND 241..246
FT /evidence="ECO:0007829|PDB:3W5H"
FT HELIX 248..253
FT /evidence="ECO:0007829|PDB:3W5H"
FT HELIX 256..262
FT /evidence="ECO:0007829|PDB:3W5H"
FT HELIX 266..268
FT /evidence="ECO:0007829|PDB:3W5H"
FT STRAND 269..271
FT /evidence="ECO:0007829|PDB:5GV7"
SQ SEQUENCE 272 AA; 30831 MW; 16B08682FC365090 CRC64;
STPAITLENP DIKYPLRLID KEVVNHDTRR FRFALPSPEH ILGLPVGQHI YLSARIDGNL
VIRPYTPVSS DDDKGFVDLV IKVYFKDTHP KFPAGGKMSQ YLESMKIGDT IEFRGPNGLL
VYQGKGKFAI RPDKKSSPVI KTVKSVGMIA GGTGITPMLQ VIRAIMKDPD DHTVCHLLFA
NQTEKDILLR PELEELRNEH SARFKLWYTV DRAPEAWDYS QGFVNEEMIR DHLPPPEEEP
LVLMCGPPPM IQYACLPNLE RVGHPKERCF AF
