Purification and characteristics of hydrophobic membrane protein(s) required for DCCD sensitivity of ATPase in mycobacterium phlei

Cohen, Nissim; Lee, Soonho; Brodie, Arnold F.

doi:10.1002/jss.400080109

Cited by 21 publications

(10 citation statements)

References 36 publications

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“…The mechanism of action and resistance against DCCD could provide an insight into this peculiar observation. Besides targeting the c-ring [62][63][64], DCCD also targets the β-subunit [65,66] of the F-ATP synthase. However, studies on E. coli [64] and Streptococcus faecalis [62] have shown that spontaneous DCCD resistance mutations can only be isolated in the c-subunit but not the β-subunit.…”

Section: Tbaj-876 Retained Bdq's Targeting Of Both the C-ring And ε-Smentioning

confidence: 99%

Re-Understanding the Mechanisms of Action of the Anti-Mycobacterial Drug Bedaquiline

2019

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Bedaquiline (BDQ) inhibits ATP generation in Mycobacterium tuberculosis by interfering with the F-ATP synthase activity. Two mechanisms of action of BDQ are broadly accepted. A direct mechanism involves BDQ binding to the enzyme's c-ring to block its rotation, thus inhibiting ATP synthesis in the enzyme's catalytic α 3 β 3 -headpiece. An indirect mechanism involves BDQ uncoupling electron transport in the electron transport chain from ATP synthesis at the F-ATP synthase. In a recently uncovered second direct mechanism, BDQ binds to the enzyme's ε-subunit to disrupt its ability to link c-ring rotation to ATP synthesis at the α 3 β 3 -headpiece. However, this mechanism is controversial as the drug's binding affinity for the isolated ε-subunit protein is moderate and spontaneous resistance mutants in the ε-subunit cannot be isolated. Recently, the new, structurally distinct BDQ analogue TBAJ-876 was utilized as a chemical probe to revisit BDQ's mechanisms of action. In this review, we first summarize discoveries on BDQ's mechanisms of action and then describe the new insights derived from the studies of TBAJ-876. The TBAJ-876 investigations confirm the c-ring as a target, while also supporting a functional role for targeting the ε-subunit. Surprisingly, the new findings suggest that the uncoupler mechanism does not play a key role in BDQ's anti-mycobacterial activity.Concentration (MIC) of 0.002-0.013 µg/mL [10,11]). This potent activity holds true in vivo where BDQ has demonstrated accelerated sterilizing activity [12], with four months of BDQ treatment being as efficacious as six months of first-line drug treatment of Mtb-infected mice [13]. Importantly, BDQ also displays bactericidal activity against non-replicating Mtb at therapeutically attainable concentrations [14,15]. In the clinical setting, the usage of BDQ has produced promising results. Studies have shown that the usage of the drug for drug-resistant TB treatment improved sputum conversion and reduced chemotherapy duration and relapse [16][17][18][19]. Hence, new BDQ-containing drug regimens are currently being explored in Phase III clinical trials, such as the STREAM and SimpliciTB trials [20]. One such trial, the Nix-TB trial, resulted in the recent US FDA approval of the BDQ-pretomanid-linezolid six months, all-oral regimen for the treatment of drug-resistant TB [21,22].

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Section: Tbaj-876 Retained Bdq's Targeting Of Both the C-ring And ε-Smentioning

confidence: 99%

Re-Understanding the Mechanisms of Action of the Anti-Mycobacterial Drug Bedaquiline

2019

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“…mol wt subunits and four to six of the proteolipid subunits per complex. Cohen et al(159) have purified an F o preparation from M phlei to which the FI-ATPase binds with restoration of DeeD sensitivity. The preparation is said to show three major subunits on SDS gels with apparent molecular weights of 24,000, 18,000, and 8,000.…”

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The Proton-Translocating Pumps of Oxidative Phosphorylation

Fillingame

1980

Annu. Rev. Biochem.

253

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“…The membrane-bound, energy-coupling ATPase complex has been purified from several species of bacteria, including the thermophilic bacterium PS3 (31), Escherichia coli (13,14), and Mycobacterium phlei (4,21). The enzyme complexes from these three sources probably consist of eight nonidentical subunits and are readily separated into two portions: Fl, containing five subunits Fo containing three subunits.…”

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confidence: 99%

Assembly of the adenosine triphosphatase complex in Escherichia coli: assembly of F0 is dependent on the formation of specific F1 subunits

Cox¹,

Downie²,

Langman³

et al. 1981

J Bacteriol

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A strain of Escherichia coli (AN1007) carrying the polar uncD436 allele which affects the operon coding for the F1-Fo adenosine triphosphatase (ATPase) complex was isolated and characterized. The uncD436 allele affected the two genes most distal to the operon promoter, i.e., uncD and uncC. Although the genes coding for the Fo portion of the ATPase complex were not affected in strains carrying this mutant allele, the lack of reconstitution of washed membranes by normal F1 ATPase suggested that a functional Fo might not be formed. This conclusion was supported by the observation that the 18,000-molecular-weight Fo subunit, coded for by the uncF gene, was absent from the membranes. Plasmid pAN36 (uncD+C+), when inserted into a strain carrying the uncD436 allele, resulted in the incorporation of the 18,000-molecular-weight Fo subunit into the membrane. A further series of experiments with Mu-induced polarity mutants, with and without plasmid pAN36, showed that the formation of both the a-and ,f-subunits of F1 ATPase was an essential prerequisite to the incorporation into the membrane of the 18,000-molecular-weight Fo subunit and to the formation of a functional Fo. Examination of the polypeptide composition of membranes from various unc mutants allowed a sequence for the normal assembly of the FI-Fo ATPase complex to be proposed.The membrane-bound, energy-coupling ATPase complex has been purified from several species of bacteria, including the thermophilic bacterium PS3 (31), Escherichia coli (13,14), and Mycobacterium phlei (4, 21). The enzyme complexes from these three sources probably consist of eight nonidentical subunits and are readily separated into two portions: Fl, containing five subunits Fo containing three subunits. There is some disagreement about the molecular weights of the Fo subunits of E. coli. However, those proposed by Foster and Fillingame (13) of 24,000, 19,000, and 8,400 agree well with the results of experiments with the cloned unc operon of E. coli in an in vitro transcription-translation system (7). Similar molecular weights have also been obtained for Fo components of M. phlei (4). t Present address: Fo portions from strain PS3 and E. coli have been incorporated into phospholipid vesicles and shown to function as a proton pore (24, 28). F1 ATPase is water soluble, has ATPase activity, and has been separated into its component subunits. These can be reassembled and combined with Fl-depleted membranes to give a fully functional F1-Fo ATPase complex (11,35). The similarity of the ATPase structures found in such diverse bacteria as strain PS3 and E. coli is emphasized by the experiments in which the ysubunits from the F1 ATPase from each species have been shown to be partly interchangeable (15). The Fo portion of the ATPase complex has not yet been dissociated into individual subunits and reassembled into a functional complex.Information concerning the assembly of the ATPase complex in strain PS3 has been obtained from experiments involving purified Fo incorporated into phospholipid vesicles an...

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Purification and characteristics of hydrophobic membrane protein(s) required for DCCD sensitivity of ATPase in mycobacterium phlei

Cited by 21 publications

References 36 publications

Re-Understanding the Mechanisms of Action of the Anti-Mycobacterial Drug Bedaquiline

Re-Understanding the Mechanisms of Action of the Anti-Mycobacterial Drug Bedaquiline

The Proton-Translocating Pumps of Oxidative Phosphorylation

Assembly of the adenosine triphosphatase complex in Escherichia coli: assembly of F0 is dependent on the formation of specific F1 subunits

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