Use this URL to cite or link to this record in EThOS:
Title: Molecular characterization of a sodium-dependent NADH-ubiquinone oxidoreductase from Vibrio alginolyticus
Author: Tan, Karen Ai Ling
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1997
Availability of Full Text:
Access from EThOS:
Full text unavailable from EThOS. Please try the link below.
Access from Institution:
The sodium-dependent NADH-ubiquinone oxidoreductase (Na+-NQR) was discovered first in the marine bacterium, Vibrio alginolyticus. 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+-NQR was composed of 3 subunits: α, β and γ, with apparent Mr 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+-independent one-electron transfer reaction to produce the semiquinone. The subsequent reduction of the semiquinone is dependent on Na+ and is catalyzed by the α subunit. Degenerate oligonucleotides designed from the N-terminal sequences obtained from partially purified α and γ subunits were used to isolate clones from an EcoRI library of wild-type V. alginolyticus DNA. Six genes which comprise the nqr operon, nqrA-nqrF, 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+-translocating NADH-ubiquinone oxidoreductase. Na+-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 N-terminal region. Pulse-chase radiolabelling of various clones expressing nqrB-nqrF verified that the putative products of the nqr operon identified from sequencing, were indeed transcribed and translated in vivo. The nqrF gene was cloned into pET16-b and expressed in Escherichia coli, BL21(DE3)pLysS as a 46 kDa polypeptide.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available