Cloning and sequencing of four structural genes for the Na⁺‐translocating NADH‐ubiquinone oxidoreductase of Vibrio alginolyticus

Abstract

Oligonucleotide probes based on the N-terminal amino acid sequences of the NqrA and NqrC subunits were used to clone genes for the Na'-dependent NADH-ubiquinone oxidoreductase complex from Vibrio alginolyticus. Four consecutive ORFs were identified encoding subunit proteins of 48.6,46.8, 27.7 and 22.6 kDa, respectively (NqrA-D). A further ORF, showing 71% homology to the BolA protein of Escherichio coli, was located upstream. From sequence comparisons, we conclude that the Na'-dependent NADH-ubiquinone oxidoreductase complex of I! alginolyticus is clearly distinct from the corresponding H'-dependent enzymes of both prokaryotes and eukaryotes.

NADH:Ubiquinone Oxidoreductase of Vibrio alginolyticus: Purification, Properties, and Reconstitution of the Na+ Pump

“…The polypeptides with apparent molecular masses of 50.7, 45.9, and 31.7 kDa correspond to the three (hydrophilic) subunits R, , and γ noted in a previous study of the enzyme (Hayashi & Unemoto, 1987). According to the DNA sequence (Beattie et al, 1994;Hayashi et al, 1995) the enzyme presumably contains three additional hydrophobic subunits which will be termed a, b, and c according to decreasing molecular mass in analogy to the terminology of the F 1 F o ATPase subunits (see also Table 2). The polypetpide with the apparent molecular mass of 33.3 kDa ( Figure 1) was tentatively assigned as the a subunit.…”

“…In Table 2 the polypeptides predicted from the recently determined DNA sequence (Beattie et al, 1994;Hayashi et al, 1995) were compared with those present in the purified NQR-1. The three hydrophilic polypeptides of the complex (R, , γ) correlate with NqrA, NqrF, and NqrC according to the N-terminal sequences (R, γ), and the presence of NADH and FAD binding motifs ( ), respectively (see Results).…”

“…We have shown recently that the enzyme is probably more complex than anticipated, consisting of more than three subunits, and that FAD is the only flavin-containing prosthetic group (Pfenninger-Li & Dimroth, 1995). The genes encoding the 52 and 32 kDa subunits of the V. alginolyticus NQR-1 were shown by two different groups to be physically connected to a cluster of together six open reading frames that was tentatively designated the nqr operon (Beattie et al, 1994;Hayashi et al, 1995). Further biochemical characterization of the protein is required to assess the participation of all of the gene products in the enzyme complex and to gain more insight into the catalytic mechanism.…”

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

“…A possible mechanism of ion-coupling was proposed on this basis [18]. However, the operon encoding these subunits has now been sequenced by two groups and six open reading frames (ORFs) have been identified [21][22][23]. It is, therefore, timely to reassess the possible structure and mechanism of this unusual member of the NQR family of enzymes.…”

Existence of Na+‐translocating NADH‐quinone reductase in Haemophilus influenzae

“…Recently, we have cloned and sequenced a part of nqr operon from V. alginolyticus [7,8]. At the same time, Beattie et al [9] reported the presence of four consecutive open reading frames in the nqr operon. In combination with these results, the nqr operon was found to be constructed from six consecutive structural genes, where nqrl, nqr3 and nqr6 corresponded to ct-, y-, and 13-subunits, respectively, of the NQR complex.…”