Molecular characterization of a sodium-dependent NADH-ubiquinone oxidoreductase from Vibrio alginolyticus

• Main Author: Tan, Karen Ai Ling
• Published: University of Edinburgh 1997
• Subjects: 579
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.662715

The sodium-dependent NADH-ubiquinone oxidoreductase (Na<SUP>+</SUP>-NQR) was discovered first in the marine bacterium, <I>Vibrio alginolyticus. </I>It acts as a primary electrogenic pump for sodium translocation during aerobic respiration, generating a sodium motive force which drives ATP synthesis, solute transport and flagellar motion. Early biochemical studies indicated that Na<SUP>+</SUP>-NQR was composed of 3 subunits: α, β and γ, with apparent M<SUB>r</SUB> values of 52, 46 and 32 kDa. A proposed model suggested that the β subunit, a NADH dehydrogenase, accepts electrons from NADH and reduces menadione or quinone by a Na<SUP>+</SUP>-independent one-electron transfer reaction to produce the semiquinone. The subsequent reduction of the semiquinone is dependent on Na<SUP>+</SUP> and is catalyzed by the α subunit. Degenerate oligonucleotides designed from the <I>N-</I>terminal sequences obtained from partially purified α and γ subunits were used to isolate clones from an <I>Eco</I>RI library of wild-type <I>V. alginolyticus </I>DNA. Six genes which comprise the <I>nqr </I>operon, <I>nqr</I>A-<I>nqr</I>F, were sequenced and identified. Sequence analysis and database comparisons led to the conclusion that this enzyme complex is both structurally and evolutionarily distinct from the H<SUP>+</SUP>-translocating NADH-ubiquinone oxidoreductase. Na<SUP>+</SUP>-NQR comprises 3 hydrophilic subunits, NqrA, NqrC and NqrF and 3 highly hydrophobic membrane-spanning subunits, NqrB, NqrD and NqrE. The 3 hydrophilic subunits, NqrA, NqrC and NqrF correspond to the previously identified α, γ and β subunits respectively. Based on sequence comparisons, a [2Fe-2S] cluster region, a FAD binding site and an NADH binding domain were identified in NqrF, the proposed NADH dehydrogenase subunit. From hydropathy plots, NqrF also appeared to possess a hydrophobic <I>N</I>-terminal region. Pulse-chase radiolabelling of various clones expressing <I>nqr</I>B-<I>nqr</I>F verified that the putative products of the<I> nqr </I>operon identified from sequencing, were indeed transcribed and translated <I>in vivo. </I>The <I>nqr</I>F gene was cloned into pET16-b and expressed in <I>Escherichia coli, </I>BL21(DE3)p<I>LysS </I>as a 46 kDa polypeptide.