Construction, expression and characterization of a plasmid‐encoded Na⁺‐specific ATPase hybrid consisting of Propionigenium modestum F₀‐ATPase and Escherichia coli F₁‐ATPase

Abstract

The Escherichia coli strain DK8, a deletion mutant lacking the complete unc operon, was transformed with a plasmid containing the genes encoding the a, b, c, 6 and part of the a subunit of the Na+-dependent ATPase of Propionigenium modestum and the genes encoding the a, y , p and E subunits of the H'-dependent E. coli ATPase. The transformants showed Na'-dependent growth on succinate as non-fermentable carbon source. The functionally expressed hybrid ATPase was activated 13-fold at pH 7.5 by the addition of Na' and inhibited by 1,3-dicyclohexylcarbodiimide, azide and tributyltin chloride. At pH 7.5 and pH 9.0, the hybrid enzyme was protected from inhibition by 1,3-dicyclohexylcarbodiimide in the presence of 50 mM NaCl and 5 mM NaCl, respectively. The hybrid ATPase was reconstituted into proteoliposomes and catalyzed the transport of Na' upon ATP addition. ATP-dependent fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine proved that the ATPase hybrid was able to pump protons in the absence of Na'. Furthermore, ATP synthesis could be measured under conditions where a valinomycin-mediated K+ diffusion potential ( A lu) and a Na' concentration gradient (ApNa') were imposed.The strictly anaerobic, rod-shaped bacterium Propionigenium modestum is able to grow using the decarboxylation of succinate to propionate and CO, as an energy source. The small free energy of the decarboxylation reaction (AGO' =

Voltage-generated torque drives the motor of the ATP synthase

“…This kinetic effect is expected because the efficiency of Na + binding to the stator and release from the rotor depends upon these Na + concentrations. Our results agree with previous reports on ΔμNa + ‐driven ATP synthesis by reconstituted proteoliposomes containing the ATP synthase of P.modestum (Dmitriev et al ., 1993; Kaim and Dimroth, 1994). We show here for the first time that ATP synthesis can be energized by ΔΨ in the absence of ΔpNa + .…”

“…The ATP synthase was purified and determined by its ATP hydrolysis activity as described (Kaim and Dimroth, 1993, 1994). The 22 Na + occlusion experiments by the ATP synthase were performed as described (Kaim et al ., 1998).…”

“…ATP synthesis was determined by the luciferin–luciferase assay as described (Kaim and Dimroth, 1994). The ATP synthase of P.modestum was reconstituted into proteoliposomes by the freeze–thaw sonication procedure (Kluge and Dimroth, 1992).…”

ATP synthesis by the F1Fo ATP synthase of Escherichia coli is obligatorily dependent on the electric potential

“…Liposomes were formed by sonicating a suspension of 44 mg phosphatidylcholine (Sigma; type II‐S) in 1 ml 5 mM sodium phosphate buffer, pH 7.2, for 2×1 min in a water bath sonicator. E. coli strain DH5α (Bethesda Research Laboratories) was grown in M13 minimal medium with 10 mM glucose, supplemented with thiamine‐HCl (0.1 mg/l) and the ATP synthase was purified as described [13]. To obtain a protein/phospholipid ratio of 1:80, 0.55 mg purified F 1 F o (1 nmol) in sodium phosphate buffer, pH 7.2, containing 0.1 mM diisopropylfluorophosphate and 0.05% Triton X‐100 was added to the liposomes and the suspension was frozen in liquid nitrogen for 15 min.…”

Functional and idling rotatory motion within F 1 -ATPase

“…The enzyme is present in most prokaryotic and eukaryotic organisms, which diverged in evolution probably more than 3 billion years ago. Astoundingly, the recombination of subunits from different sources has yielded functional chimeric constructs (7)(8)(9)(10)(11)(12). Solubilized F 1 is composed of five different subunits, (␣␤) 3 ␥␦, and is active as an ATPase (2,3).…”