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Literature summary for 7.2.1.1 extracted from
- 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.
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Vibrio cholerae serotype O1 |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| 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 |
| D397A | subunit NqrB, mutant is completely insensitive to sodium and also not stimulated by lithium | Vibrio cholerae serotype O1 |
| D397C | site-directed mutagenesis of subunit NqrB residue, part of the sodium binding site | Vibrio cholerae serotype O1 |
| 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 |
| D397C | subunit NqrB, mutant is stimulated by Li+. Mutation results in negative cooperativity | Vibrio cholerae serotype O1 |
| D397E | site-directed mutagenesis of subunit NqrB residue, part of the sodium binding site | Vibrio cholerae serotype O1 |
| 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 |
| 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 |
| D397K | subunit NqrB, mutant is completely insensitive to sodium and also not stimulated by lithium | Vibrio cholerae serotype O1 |
| 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 |
| D397N | subunit NqrB, mutant is stimulated by Li+. Km value is not affected. Sodium-binding site II is inactive | Vibrio cholerae serotype O1 |
| 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 |
| D397S | subunit NqrB, mutant is stimulated by Li+. Km value is not affected | Vibrio cholerae serotype O1 |
| additional information | construction of a Vibrio cholerae deletion strain lacking the genomic nqr operon, DELTAnqr | Vibrio cholerae serotype O1 |
| 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
| Inhibitors | Comment | Organism | Structure |
|---|---|---|---|
| iodoacetamide | inactivation | Vibrio cholerae serotype O1 |  |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 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 | |
| 0.25 | - |
n Na+/in | mutant D397C, presence of 2-mercaptoethanol, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 0.7 | - |
n Na+/in | mutant D397C, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 1.8 | - |
n Na+/in | mutant D397E, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 2.4 | - |
n Li+/in | mutant D397N, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 2.5 | - |
n Na+/in | wild-type, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 2.5 | - |
n Li+/in | mutant D397E, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 2.7 | - |
n Li+/in | mutant D397C, presence of 2-mercaptoethanol, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 2.8 | - |
n Na+/in | mutant D397S, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 2.9 | - |
n Li+/in | mutant D397S, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 3.1 | - |
n Na+/in | mutant D397N, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 3.5 | - |
n Li+/in | wild-type, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
Localization
| Localization | Comment | Organism | GeneOntology No. | Textmining |
|---|---|---|---|---|
| membrane | - |
Vibrio cholerae serotype O1 | 16020 | - |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Fe2+ | 2 [2Fe-2S] clusters in the NqrF subunit | Vibrio cholerae serotype O1 | |
| 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 | |
| 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)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| NADH + H+ + ubiquinone + n Na+/in | Vibrio cholerae serotype O1 | - |
NAD+ + ubiquinol + n Na+/out | - |
? | |
| NADH + H+ + ubiquinone + n Na+/in | Vibrio cholerae serotype O1 ATCC 39541 | - |
NAD+ + ubiquinol + n Na+/out | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| 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. | - |
| 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)
| Purification (Comment) | Organism |
|---|---|
| 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)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| NADH + H+ + ubiquinone + n Na+/in | - |
Vibrio cholerae serotype O1 | NAD+ + ubiquinol + n Na+/out | - |
? | |
| 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 | - |
? | |
| NADH + H+ + ubiquinone + n Na+/in | - |
Vibrio cholerae serotype O1 ATCC 39541 | NAD+ + ubiquinol + n Na+/out | - |
? | |
| 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 | - |
? | |
| NADH + H+ + ubiquinone-1 + n Li+/in | - |
Vibrio cholerae serotype O1 | NAD+ + ubiquinol-1 + n Li+/out | - |
? | |
| NADH + H+ + ubiquinone-1 + n Li+/in | - |
Vibrio cholerae serotype O1 ATCC 39541 | NAD+ + ubiquinol-1 + n Li+/out | - |
? | |
| NADH + H+ + ubiquinone-1 + n Na+/in | - |
Vibrio cholerae serotype O1 | NAD+ + ubiquinol-1 + n Na+/out | - |
? | |
| NADH + H+ + ubiquinone-1 + n Na+/in | - |
Vibrio cholerae serotype O1 ATCC 39541 | NAD+ + ubiquinol-1 + n Na+/out | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| monomer | - |
Vibrio cholerae serotype O1 |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| Na+-NQR | - |
Vibrio cholerae serotype O1 |
| Na+-pumping NADH: quinone oxidoreductase | - |
Vibrio cholerae serotype O1 |
| NqrB | - |
Vibrio cholerae serotype O1 |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 57.7 | - |
ubiquinone-1 | mutant D397S, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 58.8 | - |
ubiquinone-1 | mutant D397K, pH 8.0, temperature not specified in the publication, presence of 100 mM Na+ | Vibrio cholerae serotype O1 | |
| 59.7 | - |
ubiquinone-1 | mutant D397N, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 59.8 | - |
ubiquinone-1 | mutant D397K, pH 8.0, temperature not specified in the publication, presence of 100 mM Li+ | Vibrio cholerae serotype O1 | |
| 61.3 | - |
ubiquinone-1 | mutant D397K, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 61.9 | - |
ubiquinone-1 | mutant D397C, presence of 2-mercaptoethanol, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 65.7 | - |
ubiquinone-1 | mutant D397C, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 66 | - |
ubiquinone-1 | mutant D397E, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 66.3 | - |
ubiquinone-1 | wild-type, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 70.6 | - |
ubiquinone-1 | mutant D397A, pH 8.0, temperature not specified in the publication, presence of 100 mM Na+ | Vibrio cholerae serotype O1 | |
| 72.3 | - |
ubiquinone-1 | mutant D397A, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 72.3 | - |
ubiquinone-1 | mutant D397A, pH 8.0, temperature not specified in the publication, presence of 100 mM Li+ | Vibrio cholerae serotype O1 | |
| 80.7 | - |
ubiquinone-1 | mutant D397S, pH 8.0, temperature not specified in the publication, presence of 100 mM Li+ | Vibrio cholerae serotype O1 | |
| 95.5 | - |
ubiquinone-1 | mutant D397N, pH 8.0, temperature not specified in the publication, presence of 100 mM Li+ | Vibrio cholerae serotype O1 | |
| 96.4 | - |
ubiquinone-1 | mutant D397E, pH 8.0, temperature not specified in the publication, presence of 100 mM Li+ | Vibrio cholerae serotype O1 | |
| 100 | - |
n Li+/in | mutant D397N, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 101.3 | - |
ubiquinone-1 | mutant D397S, pH 8.0, temperature not specified in the publication, presence of 100 mM Na+ | Vibrio cholerae serotype O1 | |
| 102.9 | - |
ubiquinone-1 | mutant D397C, pH 8.0, temperature not specified in the publication, presence of 100 mM Li+ | Vibrio cholerae serotype O1 | |
| 110 | - |
n Li+/in | mutant D397E, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 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 | |
| 115 | - |
n Li+/in | mutant D397C, presence of 2-mercaptoethanol, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 115 | - |
n Na+/in | mutant D397S, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 122.9 | - |
ubiquinone-1 | mutant D397N, pH 8.0, temperature not specified in the publication, presence of 100 mM Na+ | Vibrio cholerae serotype O1 | |
| 124 | - |
n Li+/in | mutant D397S, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 127 | - |
n Na+/in | mutant D397E, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 127 | - |
n Na+/in | mutant D397N, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 129.6 | - |
ubiquinone-1 | mutant D397E, pH 8.0, temperature not specified in the publication, presence of 100 mM Na+ | Vibrio cholerae serotype O1 | |
| 152.9 | - |
ubiquinone-1 | mutant D397C, pH 8.0, temperature not specified in the publication, presence of 100 mM Na+ | Vibrio cholerae serotype O1 | |
| 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 | |
| 170 | - |
n Na+/in | mutant D397C, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 170 | - |
n Na+/in | mutant D397C, presence of 2-mercaptoethanol, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 180 | - |
n Li+/in | wild-type, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 205.5 | - |
ubiquinone-1 | wild-type, pH 8.0, temperature not specified in the publication, presence of 100 mM Li+ | Vibrio cholerae serotype O1 | |
| 448.9 | - |
NADH | wild-type, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 487.7 | - |
NADH | mutant D397K, pH 8.0, temperature not specified in the publication, presence of 100 mM Na+ | Vibrio cholerae serotype O1 | |
| 495.4 | - |
NADH | mutant D397C, presence of 2-mercaptoethanol, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 500 | - |
n Na+/in | wild-type, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 501.4 | - |
NADH | mutant D397C, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 511.7 | - |
NADH | mutant D397S, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 521.9 | - |
NADH | mutant D397K, pH 8.0, temperature not specified in the publication, presence of 100 mM Li+ | Vibrio cholerae serotype O1 | |
| 528.5 | - |
ubiquinone-1 | wild-type, pH 8.0, temperature not specified in the publication, presence of 100 mM Na+ | Vibrio cholerae serotype O1 | |
| 544.7 | - |
NADH | mutant D397E, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 560.8 | - |
NADH | mutant D397N, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 580.2 | - |
NADH | mutant D397A, pH 8.0, temperature not specified in the publication, presence of 100 mM Na+ | Vibrio cholerae serotype O1 | |
| 587.2 | - |
NADH | wild-type, pH 8.0, temperature not specified in the publication, presence of 100 mM Li+ | Vibrio cholerae serotype O1 | |
| 600.1 | - |
NADH | wild-type, pH 8.0, temperature not specified in the publication, presence of 100 mM Na+ | Vibrio cholerae serotype O1 | |
| 603.2 | - |
NADH | mutant D397S, pH 8.0, temperature not specified in the publication, presence of 100 mM Li+ | Vibrio cholerae serotype O1 | |
| 622.2 | - |
NADH | mutant D397A, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
| 629.8 | - |
NADH | mutant D397A, pH 8.0, temperature not specified in the publication, presence of 100 mM Li+ | Vibrio cholerae serotype O1 | |
| 656.6 | - |
NADH | mutant D397C, pH 8.0, temperature not specified in the publication, presence of 100 mM Na+ | Vibrio cholerae serotype O1 | |
| 660.4 | - |
NADH | mutant D397N, pH 8.0, temperature not specified in the publication, presence of 100 mM Li+ | Vibrio cholerae serotype O1 | |
| 662.9 | - |
NADH | mutant D397S, pH 8.0, temperature not specified in the publication, presence of 100 mM Na+ | Vibrio cholerae serotype O1 | |
| 673.7 | - |
NADH | mutant D397E, pH 8.0, temperature not specified in the publication, presence of 100 mM Na+ | Vibrio cholerae serotype O1 | |
| 681.6 | - |
NADH | mutant D397N, pH 8.0, temperature not specified in the publication, presence of 100 mM Na+ | Vibrio cholerae serotype O1 | |
| 690.9 | - |
NADH | mutant D397C, pH 8.0, temperature not specified in the publication, presence of 100 mM Li+ | Vibrio cholerae serotype O1 | |
| 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 | |
| 695.4 | - |
NADH | mutant D397E, pH 8.0, temperature not specified in the publication, presence of 100 mM Li+ | Vibrio cholerae serotype O1 | |
| 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 | |
| 5199 | - |
NADH | mutant D397K, pH 8.0, temperature not specified in the publication | Vibrio cholerae serotype O1 | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 8 | - |
assay at | Vibrio cholerae serotype O1 |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| FAD | non-covalently bound in the NqrF subunit | Vibrio cholerae serotype O1 | |
| FMN | two covalently bound in subunits NqrB und NqrC | Vibrio cholerae serotype O1 | |
| NADH | - |
Vibrio cholerae serotype O1 | |
| riboflavin | one non-covalently bound in the subunit NqrB | Vibrio cholerae serotype O1 | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| 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 |
| 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 |
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