Cloning, sequencing and expression of the nhaA and nhaR genes from Salmonella entiritidis

Pinner, Elhanan; Carmel, O.; Bercovier, Hervé; Sela, Shlomo; Padan, Etana; Schuldiner, Shimon

doi:10.1007/bf00248676

Cited by 37 publications

(33 citation statements)

References 17 publications

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“…Comparisons were also made with the subunits of a bacterial NDH-1 [25][26][27][28][29] and the bacterial form of NDH-2 from E. coli [8] using the program BESTFIT. In addition, the three hydrophobic proteins encoded by nqrb, nqrd and nqre were compared with bacterial Na+/H ÷ antiporters [24,[30][31][32]. Significant homologies with proteins in the SWISSPROT database were found only in the case of the NqrF subunit and this arose primarily from the presence of common binding motifs for NADH, FAD and the iron sulphur centre (see below).…”

Section: Sequence Homologies With Other Proteins and Cofactorbinding mentioning

confidence: 99%

Predicted structure and possible ionmotive mechanism of the sodium‐linked NADH‐ubiquinone oxidoreductase of Vibrio alginolyticus

1995

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Two groups have now published sequences of the six genes contained in the operon coding for the sodium-linked NADH-ubiquinone oxidoreductase of Vibrio aiginolyticus. Sequence analyses indicate that this enzyme is unrelated to other known respiratory NADH dehydrogenases. A search for cofactor motifs suggests that the enzyme contains only one FAD, a ferredoxin-type iron sulphur centre, and the NADH-binding site. These are all located on NqrF, a subunit that can be recognized as a new member of a large diverse family of NAD(P)H-oxidizing flavoenzymes. A possible model of ion-coupling is presented, based upon this new information.Key words." NADH-ubiquinone oxidoreductase; NADH dehydrogenase; Sodium ion translocation BackgroundSeveral types of enzyme (NQR) are known to catalyse the respiratory reaction of oxidation of NADH by membranebound ubiquinone. Only one, termed complex I, is found in mammalian mitochondria, where it oxidizes internally generated NADH. Bovine complex I is remarkably complicated, with seven mitochondrially encoded and at least 34 further nuclear encoded subunits [1]. Gene clusters coding for proton translocating NADH-ubiquinone oxidoreductases have also been identified in the prokaryotes Paracoccus denitrificans, Rhodobacter capsulatus and Escherichia coli [24]. These comprise homologues of only 14 of the mammalian subunits [5] and, presumably, represent a more minimal core catalytic structure. The purified enzyme from E. eoli can be subfractionated into three domains, viz. a fragment (FP) of three subunits which contains the NADH site, FMN and iron sulphur centres; a more amphipathic fragment (IP) of four subunits also containing iron sulphur centres; and a very hydrophobic fragment (HP) of seven proteins which are homologues of the mitochondrially encoded subunits of eukaryotes. In the mitochondrial enzyme, one HP subunit (ND 1) contains a ubiquinone-binding site while another (ND2) is reactive with rotenone and DCCD [1]. All homologues of complex I are presumed to be coupled to proton translocation and have been generically termed NDH-1 types [5,6]. *Corresponding author. Fax: (44) (1208) 821 575. E-mail: mbprr@seqnet.dl.ac.uk Two other types of NADH dehydrogenase can be found in mitochondria from fungal and plant sources [7]. One of these oxidizes internally generated NADH but, in contrast to complex I, it is not coupled to proton translocation. It is likely to be homologous to a non-protonmotive NQR in bacteria, termed NDH-2 [6]. The enzyme may be a single polypeptide, whose sequence has been established [8][9][10], and with FAD as the only redox cofactor [6]. A third NADH dehydrogenase in the inner membrane of plant and fungal mitochondria, which we propose might be termed NDH-3, has an externally facing NADH site and can directly oxidize cytosolic NADH [11]. This enzyme is also not coupled to proton translocation [12]. A bacterial homologue has not been identified and its detailed structure and composition remain unknown.A further type of NQR was first found in the marine bacter...

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Section: Sequence Homologies With Other Proteins and Cofactorbinding mentioning

confidence: 99%

Predicted structure and possible ionmotive mechanism of the sodium‐linked NADH‐ubiquinone oxidoreductase of Vibrio alginolyticus

1995

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“…NhaB was identified as the residual antiporter activity, specific to Na ÷ and Li ÷, after nhaA had been deleted from the chromosome [200]. The activity of NhaB is independent of pH [200,202], but the actual Na+/H ÷ stoichiometry of the exchange reaction is unknown at present. Since the variations in the apparent Na+/H + stoichiometry in membrane vesicles and cells, expressing both NhaA and NhaB [203,204], parallel the pH dependence of NhaA [196,197,199], an electroneutral exchange by NhaB would be most consistent with the observed stoichiometries.…”

Section: V-f Na + / H ÷ Antiporter (Nhaa) Of E Colimentioning

confidence: 99%

Secondary solute transport in bacteria

Poolman

Konings

1993

Biochimica et Biophysica Acta (BBA) - Bioenergetics

202

160

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“…ChaA is a Ca*+/I-I' antiporter which has a low affinity for Na+ but at high copy number can rather effectively transport Na'. NhaB displays the highest atfmity for Na+ ions reported by any antiporter thus far, 40 PM [4]. Its stoichiometry is still unknown and its activity seems to be important for the cell under conditions at which NhaA is the least active, i.e.…”

Section: Introductionmentioning

confidence: 99%

Mechanism of Na⁺/H⁺ exchange by Escherichia coli NhaA in reconstituted proteoliposomes

Dibrov

Taglicht

1993

FEBS Letters

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Purified NhaA, a Na%I' antiporter from Escherichia coli, reconstituted into proteoliposomes was used to study partial reactions catalyzed by this protein. Homologous Na'Ma' exchange as well as Na+/Li+ exchange via NhaA were detected by monitoring the effects of external Li+ and Na+ ions on the ApH-driven sodium uptake into NH, Cl-loaded vesicles. Furthermore, a sodium counterflow reaction was demonstrated in proteoliposomes preloaded with non-radioactive Na+ and placed into the experimental buffer containing low amounts of *%a' under experimental conditions when both components of protonmotive force generated by the antiporter. Av and ApH, were dissipated by corresponding ionophores. The apparent lu, for sodium counterflow is 1.1 mM, and V_ is 80 ~mol/min/mg of protein. External Na' accelerates the downhill efLIux of *%a+ suggesting that the translocation of the Na'-loaded form of the carrier is faster than the rest of the catalytic cycle.

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Cloning, sequencing and expression of the nhaA and nhaR genes from Salmonella entiritidis

Cited by 37 publications

References 17 publications

Predicted structure and possible ionmotive mechanism of the sodium‐linked NADH‐ubiquinone oxidoreductase of Vibrio alginolyticus

Predicted structure and possible ionmotive mechanism of the sodium‐linked NADH‐ubiquinone oxidoreductase of Vibrio alginolyticus

Secondary solute transport in bacteria

Mechanism of Na⁺/H⁺ exchange by Escherichia coli NhaA in reconstituted proteoliposomes

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Cloning, sequencing and expression of the nhaA and nhaR genes from Salmonella entiritidis

Cited by 37 publications

References 17 publications

Predicted structure and possible ionmotive mechanism of the sodium‐linked NADH‐ubiquinone oxidoreductase of Vibrio alginolyticus

Predicted structure and possible ionmotive mechanism of the sodium‐linked NADH‐ubiquinone oxidoreductase of Vibrio alginolyticus

Secondary solute transport in bacteria

Mechanism of Na+/H+ exchange by Escherichia coli NhaA in reconstituted proteoliposomes

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Mechanism of Na⁺/H⁺ exchange by Escherichia coli NhaA in reconstituted proteoliposomes