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Literature summary extracted from

  • Shea, M.E.; Juarez, O.; Cho, J.; Barquera, B.
    Aspartic acid 397 in subunit B of the Na+-pumping NADH: quinone oxidoreductase from Vibrio cholerae forms part of a sodium binding site, is involved in cation selectivity and affects cation binding site cooperativity (2013), J. Biol. Chem., 288, 31241-31249.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
7.2.1.1 expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Vibrio cholerae serotype O1

Protein Variants

EC Number Protein Variants Comment Organism
7.2.1.1 D397A site-directed mutagenesis of subunit NqrB residue, part of the sodium binding site, the kinetics of the NqrB-D397A mutant show an unsaturable behavior for sodium in contrast to the Michaelis-Menten kinetics of the wild-type enzyme. The mutant is not stimulated by lithium Vibrio cholerae serotype O1
7.2.1.1 D397A subunit NqrB, mutant is completely insensitive to sodium and also not stimulated by lithium Vibrio cholerae serotype O1
7.2.1.1 D397C site-directed mutagenesis of subunit NqrB residue, part of the sodium binding site Vibrio cholerae serotype O1
7.2.1.1 D397C site-directed mutagenesis of subunit NqrB residue, part of the sodium binding site, the mutation that eliminates half of the negative charge, is stimulated only 2.6fold by sodium Vibrio cholerae serotype O1
7.2.1.1 D397C subunit NqrB, mutant is stimulated by Li+. Mutation results in negative cooperativity Vibrio cholerae serotype O1
7.2.1.1 D397E site-directed mutagenesis of subunit NqrB residue, part of the sodium binding site Vibrio cholerae serotype O1
7.2.1.1 D397E subunit NqrB, mutant is stimulated by Li+. Km value is not affected. Mutant contains approximately the same conformational flexibility as the wild type enzyme and is able to undergo a series of conformational changes induced by the redox reaction and by the addition of different cations Vibrio cholerae serotype O1
7.2.1.1 D397K site-directed mutagenesis of subunit NqrB residue, part of the sodium binding site, the introduction of a positive charge abates completely the stimulatory effect of sodium, the mutant is not stimulated by lithium Vibrio cholerae serotype O1
7.2.1.1 D397K subunit NqrB, mutant is completely insensitive to sodium and also not stimulated by lithium Vibrio cholerae serotype O1
7.2.1.1 D397N site-directed mutagenesis of subunit NqrB residue, part of the sodium binding site, the mutation that eliminates the negative charge, but that introduces a polar residue with a partial negative charge, is stimulated only 2fold by sodium Vibrio cholerae serotype O1
7.2.1.1 D397N subunit NqrB, mutant is stimulated by Li+. Km value is not affected. Sodium-binding site II is inactive Vibrio cholerae serotype O1
7.2.1.1 D397S site-directed mutagenesis of subunit NqrB residue, part of the sodium binding site, the mutation that eliminates the negative charge, but that introduces a polar residue with a partial negative charge, is stimulated only 2fold by sodium Vibrio cholerae serotype O1
7.2.1.1 D397S subunit NqrB, mutant is stimulated by Li+. Km value is not affected Vibrio cholerae serotype O1
7.2.1.1 additional information construction of a Vibrio cholerae deletion strain lacking the genomic nqr operon, DELTAnqr Vibrio cholerae serotype O1
7.2.1.1 additional information residue D397 in subunit NqrB forms part of one of the at least two sodium-binding sites. Both size and charge at this position are critical for the function of the enzyme. The residue is involved in cation selectivity, has a critical role in the communication between sodium-binding sites, by promoting cooperativity, and controls the electron transfer step involved in sodium uptake Vibrio cholerae serotype O1

Inhibitors

EC Number Inhibitors Comment Organism Structure
7.2.1.1 iodoacetamide inactivation Vibrio cholerae serotype O1

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
7.2.1.1 additional information
-
additional information in wild-type Na+-NQR, the sodium dependence of the steady state turnover follows a Michaelis-Menten behavior, while the kinetics of the NqrB-D397A mutant show an unsaturable behavior for sodium. Kinetic mechanism, modeling, overview Vibrio cholerae serotype O1
7.2.1.1 0.25
-
n Na+/in mutant D397C, presence of 2-mercaptoethanol, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 0.7
-
n Na+/in mutant D397C, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 1.8
-
n Na+/in mutant D397E, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 2.4
-
n Li+/in mutant D397N, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 2.5
-
n Na+/in wild-type, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 2.5
-
n Li+/in mutant D397E, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 2.7
-
n Li+/in mutant D397C, presence of 2-mercaptoethanol, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 2.8
-
n Na+/in mutant D397S, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 2.9
-
n Li+/in mutant D397S, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 3.1
-
n Na+/in mutant D397N, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 3.5
-
n Li+/in wild-type, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
7.2.1.1 membrane
-
Vibrio cholerae serotype O1 16020
-

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
7.2.1.1 Fe2+ 2 [2Fe-2S] clusters in the NqrF subunit Vibrio cholerae serotype O1
7.2.1.1 Li+ the wild-type enzyme is stimulated 3fold by lithium, while the mutants NqrBD397A and NqrB-D397K, that are completely insensitive to sodium, are also not stimulated by lithium. The activities of the mutants NqrBD397C, NqrB-D397E, NqrB-D397N and NqrB-D397S are stimulated by lithium, and interestingly the fraction of stimulationis greater for lithium compared to sodium Vibrio cholerae serotype O1
7.2.1.1 Na+ the wild-type enzyme's physiologic activity is stimulated 8fold in the presence of saturating amounts of sodium Vibrio cholerae serotype O1

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
7.2.1.1 NADH + H+ + ubiquinone + n Na+/in Vibrio cholerae serotype O1
-
NAD+ + ubiquinol + n Na+/out
-
?
7.2.1.1 NADH + H+ + ubiquinone + n Na+/in Vibrio cholerae serotype O1 ATCC 39541
-
NAD+ + ubiquinol + n Na+/out
-
?

Organism

EC Number Organism UniProt Comment Textmining
7.2.1.1 Vibrio cholerae serotype O1 A5F5X1 and A5F5X0 and A5F5Y7 and A5F5Y6 and A5F5Y3 and A5F5Y4 A5F5X1: subunit NqrA, A5F5X0: subunit NqrB, A5F5Y7: subunit NqrC, A5F5Y6: subunit NqrD; A5F5Y3: subunit NqrE, A5F5Y4: subunit NqrF. The enzyme consists of six subunits encoded by the NQR operon.
-
7.2.1.1 Vibrio cholerae serotype O1 ATCC 39541 A5F5X1 and A5F5X0 and A5F5Y7 and A5F5Y6 and A5F5Y3 and A5F5Y4 A5F5X1: subunit NqrA, A5F5X0: subunit NqrB, A5F5Y7: subunit NqrC, A5F5Y6: subunit NqrD; A5F5Y3: subunit NqrE, A5F5Y4: subunit NqrF. The enzyme consists of six subunits encoded by the NQR operon.
-

Purification (Commentary)

EC Number Purification (Comment) Organism
7.2.1.1 recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by affinity chromatography Vibrio cholerae serotype O1

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
7.2.1.1 NADH + H+ + ubiquinone + n Na+/in
-
Vibrio cholerae serotype O1 NAD+ + ubiquinol + n Na+/out
-
?
7.2.1.1 NADH + H+ + ubiquinone + n Na+/in the electrons move through the different redox centers in the enzyme in a linear pathway. During the first step of electron transfer, the non-covalently bound FAD accepts two electrons from NADH. Subsequently, the electrons are transferred stepwise by passing to the 2Fe-2S center, the two FMN molecules covalently attached to NqrC and NqrB (in this order), riboflavin, and finally to ubiquinone-8. The one-electron reduction of FMN in NqrC is the step involved in sodium uptake, and the reduction of riboflavin is involved in sodium translocation Vibrio cholerae serotype O1 NAD+ + ubiquinol + n Na+/out
-
?
7.2.1.1 NADH + H+ + ubiquinone + n Na+/in
-
Vibrio cholerae serotype O1 ATCC 39541 NAD+ + ubiquinol + n Na+/out
-
?
7.2.1.1 NADH + H+ + ubiquinone + n Na+/in the electrons move through the different redox centers in the enzyme in a linear pathway. During the first step of electron transfer, the non-covalently bound FAD accepts two electrons from NADH. Subsequently, the electrons are transferred stepwise by passing to the 2Fe-2S center, the two FMN molecules covalently attached to NqrC and NqrB (in this order), riboflavin, and finally to ubiquinone-8. The one-electron reduction of FMN in NqrC is the step involved in sodium uptake, and the reduction of riboflavin is involved in sodium translocation Vibrio cholerae serotype O1 ATCC 39541 NAD+ + ubiquinol + n Na+/out
-
?
7.2.1.1 NADH + H+ + ubiquinone-1 + n Li+/in
-
Vibrio cholerae serotype O1 NAD+ + ubiquinol-1 + n Li+/out
-
?
7.2.1.1 NADH + H+ + ubiquinone-1 + n Li+/in
-
Vibrio cholerae serotype O1 ATCC 39541 NAD+ + ubiquinol-1 + n Li+/out
-
?
7.2.1.1 NADH + H+ + ubiquinone-1 + n Na+/in
-
Vibrio cholerae serotype O1 NAD+ + ubiquinol-1 + n Na+/out
-
?
7.2.1.1 NADH + H+ + ubiquinone-1 + n Na+/in
-
Vibrio cholerae serotype O1 ATCC 39541 NAD+ + ubiquinol-1 + n Na+/out
-
?

Subunits

EC Number Subunits Comment Organism
7.2.1.1 monomer
-
Vibrio cholerae serotype O1

Synonyms

EC Number Synonyms Comment Organism
7.2.1.1 Na+-NQR
-
Vibrio cholerae serotype O1
7.2.1.1 Na+-pumping NADH: quinone oxidoreductase
-
Vibrio cholerae serotype O1
7.2.1.1 NqrB
-
Vibrio cholerae serotype O1

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
7.2.1.1 57.7
-
ubiquinone-1 mutant D397S, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 58.8
-
ubiquinone-1 mutant D397K, pH 8.0, temperature not specified in the publication, presence of 100 mM Na+ Vibrio cholerae serotype O1
7.2.1.1 59.7
-
ubiquinone-1 mutant D397N, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 59.8
-
ubiquinone-1 mutant D397K, pH 8.0, temperature not specified in the publication, presence of 100 mM Li+ Vibrio cholerae serotype O1
7.2.1.1 61.3
-
ubiquinone-1 mutant D397K, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 61.9
-
ubiquinone-1 mutant D397C, presence of 2-mercaptoethanol, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 65.7
-
ubiquinone-1 mutant D397C, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 66
-
ubiquinone-1 mutant D397E, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 66.3
-
ubiquinone-1 wild-type, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 70.6
-
ubiquinone-1 mutant D397A, pH 8.0, temperature not specified in the publication, presence of 100 mM Na+ Vibrio cholerae serotype O1
7.2.1.1 72.3
-
ubiquinone-1 mutant D397A, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 72.3
-
ubiquinone-1 mutant D397A, pH 8.0, temperature not specified in the publication, presence of 100 mM Li+ Vibrio cholerae serotype O1
7.2.1.1 80.7
-
ubiquinone-1 mutant D397S, pH 8.0, temperature not specified in the publication, presence of 100 mM Li+ Vibrio cholerae serotype O1
7.2.1.1 95.5
-
ubiquinone-1 mutant D397N, pH 8.0, temperature not specified in the publication, presence of 100 mM Li+ Vibrio cholerae serotype O1
7.2.1.1 96.4
-
ubiquinone-1 mutant D397E, pH 8.0, temperature not specified in the publication, presence of 100 mM Li+ Vibrio cholerae serotype O1
7.2.1.1 100
-
n Li+/in mutant D397N, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 101.3
-
ubiquinone-1 mutant D397S, pH 8.0, temperature not specified in the publication, presence of 100 mM Na+ Vibrio cholerae serotype O1
7.2.1.1 102.9
-
ubiquinone-1 mutant D397C, pH 8.0, temperature not specified in the publication, presence of 100 mM Li+ Vibrio cholerae serotype O1
7.2.1.1 110
-
n Li+/in mutant D397E, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 110.6
-
ubiquinone-1 mutant D397C, presence of 2-mercaptoethanol, pH 8.0, temperature not specified in the publication, presence of 100 mM Li+ Vibrio cholerae serotype O1
7.2.1.1 115
-
n Li+/in mutant D397C, presence of 2-mercaptoethanol, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 115
-
n Na+/in mutant D397S, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 122.9
-
ubiquinone-1 mutant D397N, pH 8.0, temperature not specified in the publication, presence of 100 mM Na+ Vibrio cholerae serotype O1
7.2.1.1 124
-
n Li+/in mutant D397S, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 127
-
n Na+/in mutant D397E, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 127
-
n Na+/in mutant D397N, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 129.6
-
ubiquinone-1 mutant D397E, pH 8.0, temperature not specified in the publication, presence of 100 mM Na+ Vibrio cholerae serotype O1
7.2.1.1 152.9
-
ubiquinone-1 mutant D397C, pH 8.0, temperature not specified in the publication, presence of 100 mM Na+ Vibrio cholerae serotype O1
7.2.1.1 162.2
-
ubiquinone-1 mutant D397C, presence of 2-mercaptoethanol, pH 8.0, temperature not specified in the publication, presence of 100 mM Na+ Vibrio cholerae serotype O1
7.2.1.1 170
-
n Na+/in mutant D397C, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 170
-
n Na+/in mutant D397C, presence of 2-mercaptoethanol, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 180
-
n Li+/in wild-type, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 205.5
-
ubiquinone-1 wild-type, pH 8.0, temperature not specified in the publication, presence of 100 mM Li+ Vibrio cholerae serotype O1
7.2.1.1 448.9
-
NADH wild-type, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 487.7
-
NADH mutant D397K, pH 8.0, temperature not specified in the publication, presence of 100 mM Na+ Vibrio cholerae serotype O1
7.2.1.1 495.4
-
NADH mutant D397C, presence of 2-mercaptoethanol, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 500
-
n Na+/in wild-type, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 501.4
-
NADH mutant D397C, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 511.7
-
NADH mutant D397S, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 521.9
-
NADH mutant D397K, pH 8.0, temperature not specified in the publication, presence of 100 mM Li+ Vibrio cholerae serotype O1
7.2.1.1 528.5
-
ubiquinone-1 wild-type, pH 8.0, temperature not specified in the publication, presence of 100 mM Na+ Vibrio cholerae serotype O1
7.2.1.1 544.7
-
NADH mutant D397E, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 560.8
-
NADH mutant D397N, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 580.2
-
NADH mutant D397A, pH 8.0, temperature not specified in the publication, presence of 100 mM Na+ Vibrio cholerae serotype O1
7.2.1.1 587.2
-
NADH wild-type, pH 8.0, temperature not specified in the publication, presence of 100 mM Li+ Vibrio cholerae serotype O1
7.2.1.1 600.1
-
NADH wild-type, pH 8.0, temperature not specified in the publication, presence of 100 mM Na+ Vibrio cholerae serotype O1
7.2.1.1 603.2
-
NADH mutant D397S, pH 8.0, temperature not specified in the publication, presence of 100 mM Li+ Vibrio cholerae serotype O1
7.2.1.1 622.2
-
NADH mutant D397A, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1
7.2.1.1 629.8
-
NADH mutant D397A, pH 8.0, temperature not specified in the publication, presence of 100 mM Li+ Vibrio cholerae serotype O1
7.2.1.1 656.6
-
NADH mutant D397C, pH 8.0, temperature not specified in the publication, presence of 100 mM Na+ Vibrio cholerae serotype O1
7.2.1.1 660.4
-
NADH mutant D397N, pH 8.0, temperature not specified in the publication, presence of 100 mM Li+ Vibrio cholerae serotype O1
7.2.1.1 662.9
-
NADH mutant D397S, pH 8.0, temperature not specified in the publication, presence of 100 mM Na+ Vibrio cholerae serotype O1
7.2.1.1 673.7
-
NADH mutant D397E, pH 8.0, temperature not specified in the publication, presence of 100 mM Na+ Vibrio cholerae serotype O1
7.2.1.1 681.6
-
NADH mutant D397N, pH 8.0, temperature not specified in the publication, presence of 100 mM Na+ Vibrio cholerae serotype O1
7.2.1.1 690.9
-
NADH mutant D397C, pH 8.0, temperature not specified in the publication, presence of 100 mM Li+ Vibrio cholerae serotype O1
7.2.1.1 694.6
-
NADH mutant D397C, presence of 2-mercaptoethanol, pH 8.0, temperature not specified in the publication, presence of 100 mM Na+ Vibrio cholerae serotype O1
7.2.1.1 695.4
-
NADH mutant D397E, pH 8.0, temperature not specified in the publication, presence of 100 mM Li+ Vibrio cholerae serotype O1
7.2.1.1 696.7
-
NADH mutant D397C, presence of 2-mercaptoethanol, pH 8.0, temperature not specified in the publication, presence of 100 mM Li+ Vibrio cholerae serotype O1
7.2.1.1 5199
-
NADH mutant D397K, pH 8.0, temperature not specified in the publication Vibrio cholerae serotype O1

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
7.2.1.1 8
-
assay at Vibrio cholerae serotype O1

Cofactor

EC Number Cofactor Comment Organism Structure
7.2.1.1 FAD non-covalently bound in the NqrF subunit Vibrio cholerae serotype O1
7.2.1.1 FMN two covalently bound in subunits NqrB und NqrC Vibrio cholerae serotype O1
7.2.1.1 NADH
-
Vibrio cholerae serotype O1
7.2.1.1 riboflavin one non-covalently bound in the subunit NqrB Vibrio cholerae serotype O1

General Information

EC Number General Information Comment Organism
7.2.1.1 additional information aspartate 397 in subunit B of the Na+-pumping NADH: quinone oxidoreductase forms part of a sodium binding site, is involved in cation selectivity and affects cation binding site cooperativity. The enzyme is partially functional with only one sodium-binding site, which indicates that the sites are connected to independent and probably parallel sodium pumping pathways Vibrio cholerae serotype O1
7.2.1.1 physiological function the enzyme is the entry site of electrons into the aerobic respiratory chain, catalyzing the electron transfer from NADH to ubiquinone, which is coupled to the pumping of sodium ions across the membrane. The sodium gradient produced by Na+-NQR is used by the cell for ATP synthesis, transport of nutrients, rotation of the flagellum, among other processes Vibrio cholerae serotype O1