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1.
J Biol Chem ; 288(20): 14276-14286, 2013 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-23558683

RESUMO

Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) contains two flavin residues as redox-active prosthetic groups attached by a phosphoester bond to threonine residues in subunits NqrB and NqrC. We demonstrate here that flavinylation of truncated Vibrio harveyi NqrC at Thr-229 in Escherichia coli cells requires the presence of a co-expressed Vibrio apbE gene. The apbE genes cluster with genes for Na(+)-NQR and other FMN-binding flavoproteins in bacterial genomes and encode proteins with previously unknown function. Experiments with isolated NqrC and ApbE proteins confirmed that ApbE is the only protein factor required for NqrC flavinylation and also indicated that the reaction is Mg(2+)-dependent and proceeds with FAD but not FMN. Inactivation of the apbE gene in Klebsiella pneumoniae, wherein the nqr operon and apbE are well separated in the chromosome, resulted in a complete loss of the quinone reductase activity of Na(+)-NQR, consistent with its dependence on covalently bound flavin. Our data thus identify ApbE as a novel modifying enzyme, flavin transferase.


Assuntos
Mononucleotídeo de Flavina/metabolismo , Flavinas/metabolismo , Klebsiella pneumoniae/enzimologia , Nucleotidiltransferases/química , Pirimidinas/biossíntese , Vibrio/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Catálise , Transporte de Elétrons , Escherichia coli/genética , Escherichia coli/metabolismo , Vetores Genéticos , Klebsiella pneumoniae/genética , Magnésio/metabolismo , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Nucleotidiltransferases/metabolismo , Óperon , Ligação Proteica , Processamento de Proteína Pós-Traducional , Homologia de Sequência de Aminoácidos
2.
Biochemistry ; 48(27): 6291-8, 2009 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-19496621

RESUMO

Redox properties of all EPR-detectable prosthetic groups of Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) from Vibrio harveyi were studied at pH 7.5 using cryo-EPR spectroelectrochemistry. Titration shows five redox transitions. One with E(m) = -275 mV belongs to the reduction of the [2Fe-2S] cluster, and the four others reflect redox transitions of flavin cofactors. Two transitions (E(m)(1) = -190 mV and E(m)(2) = -275 mV) originate from the formation of FMN anion radical, covalently bound to the NqrC subunit, and its subsequent reduction. The remaining two transitions arise from the two other flavin cofactors. A high potential (E(m) = -10 mV) transition corresponds to the reduction of riboflavin neutral radical, which is stable at rather high redox potentials. An E(m) = -130 mV transition reflects the formation of FMN anion radical from a flavin covalently bound to the NqrB subunit, which stays as a radical down to very low potentials. Taking into account the EPR-silent, two-electron transition of noncovalently bound FAD located in the NqrF subunit, there are four flavins in Na(+)-NQR all together. Defined by dipole-dipole magnetic interaction measurements, the interspin distance between the [2Fe-2S](+) cluster and the NqrB subunit-bound FMN anion radical is found to be 22.5 +/- 1.5 A, which means that for the functional electron transfer between these two centers another cofactor, most likely FMN bound to the NqrC subunit, should be located.


Assuntos
Quinona Redutases/química , Elétrons , Mononucleotídeo de Flavina/química , Concentração de Íons de Hidrogênio , Oxirredução , Quinonas/química , Riboflavina/química , Sódio/química , Vibrio/enzimologia
3.
FEMS Microbiol Lett ; 279(1): 116-23, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18300384

RESUMO

The catalytic properties of sodium-translocating NADH:quinone oxidoreductases (Na+-NQRs) from the marine bacterium Vibrio harveyi, the enterobacterium Klebsiella pneumoniae, and the soil microorganism Azotobacter vinelandii have been comparatively analyzed. It is shown that these enzymes drastically differ in their affinity to sodium ions. The enzymes also possess different sensitivity to inhibitors. Na+-NQR from A. vinelandii is not sensitive to low 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO) concentrations, while Na+-NQR from K. pneumoniae is fully resistant to either Ag+ or N-ethylmaleimide. All the Na+-NQR-type enzymes are sensitive to diphenyliodonium, which is shown to modify the noncovalently bound FAD of the enzyme.


Assuntos
Azotobacter vinelandii/enzimologia , Proteínas de Bactérias/metabolismo , Klebsiella pneumoniae/enzimologia , Quinona Redutases/metabolismo , Vibrio/enzimologia , Proteínas de Bactérias/antagonistas & inibidores , Compostos de Bifenilo/farmacologia , Inibidores Enzimáticos/farmacologia , Etilmaleimida/farmacologia , Hidroxiquinolinas/farmacologia , Oniocompostos/farmacologia , Quinona Redutases/antagonistas & inibidores , Prata/farmacologia
4.
Arch Microbiol ; 188(4): 341-8, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17551713

RESUMO

The expression of genes encoding sodium-translocating NADH:quinone oxidoreductase (Na(+)-NQR) was studied in the marine bacterium Vibrio harveyi and in the enterobacterium Klebsiella pneumoniae. It has been shown that such parameters as NaCl concentration, pH value, and presence of an uncoupler in the growth media do not influence significantly the level of nqr expression. However, nqr expression depends on the growth substrates used by these bacteria. Na(+)-NQR is highly repressed in V. harveyi during anaerobic growth, and nqr expression is modulated by electron acceptors and values of their redox potentials. The latter effect was shown to be independent of the ArcAB regulatory system.


Assuntos
Proteínas de Bactérias/biossíntese , Regulação Bacteriana da Expressão Gênica , Klebsiella pneumoniae/metabolismo , Quinona Redutases/biossíntese , Vibrio/metabolismo , Anaerobiose , Fusão Gênica Artificial , Proteínas de Bactérias/genética , Carbono/metabolismo , Genes Reporter , Concentração de Íons de Hidrogênio , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/genética , Dados de Sequência Molecular , Nitratos/metabolismo , Oxigênio/metabolismo , Quinona Redutases/genética , Cloreto de Sódio/metabolismo , Desacopladores/farmacologia , Vibrio/enzimologia , Vibrio/genética , beta-Galactosidase/análise , beta-Galactosidase/genética
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