Cloning and sequencing of the gene encoding the beta subunit of the sodium ion translocating ATP synthase of Propionigenium modestum

Abstract The gene coding for the β-subunit of the sodium ion translocating ATP synthase from Propionigenium modestum was cloned and sequenced. The predicted amino acid sequence resembles that of the β-subunits from proton-translocating ATP synthases. The same conserved regions are found in both types of ATP synthases. This is a good indication that the β-subunits of the proton and sodium ion translocating ATP synthases have evolved from a common ancestor.     

NMR studies of subunit c of the ATP synthase from Propionigenium modestum in dodecylsulphate micelles.

The structure of the Na+, Li+ or H+-binding c subunit of the ATP synthase from Propionigenium modestum was studied by NMR. Subunit c in dodecylsulphate micelles consists of four alpha-helical segments, I-IV, that are connected by short linker peptides with non-regular secondary structures. We propose that helices I (V4-I26) and IV (I69-V85) are membrane-spanning structures, and that helices II and III and the intervening hydrophilic loop are located in the cytoplasm. The Na+-binding residues Q32, E65 and S66 are located in the I-->II and III-->IV helix connections, probably near the membrane surface on the cytoplasmic side.     

Sequence of subunit c of the sodium ion translocating adenosine triphosphate synthase of Propionigenium modestum

The 30 N-terminal amino acid residues of the purified ATPase c subunit of Propionigenium modestum have been determined. An oligonucleotide mixture was derived from this sequence and used as probe for cloning the corresponding gene in Escherichia coli. The nucleotide sequence of the gene has been determined and compared with those of ATPase c subunits from other bacteria and chloroplasts. Peculiar sequence similarities are found only at the C-terminus between the c subunits of the ATPases from P. modestum and from Vibrio alginolyticus, another putative Na(+)-translocating ATPase.     

Reconstitution of Fo of the sodium ion translocating ATP synthase of Propionigenium modestum from its heterologously expressed and purified subunits.

The atpB and atpF genes of Propionigenium modestum were cloned as His-tag fusion constructs and expressed in Escherichia coli. Both recombinant subunits a and b were purified via Ni(2+) chelate affinity chromatography. A functionally active Fo complex was reassembled in vitro from subunits a, b and c, and incorporated into liposomes. The F(o) liposomes catalysed (22)Na(+) uptake in response to an inside negative potassium diffusion potential, and the uptake was prevented by modification of the c subunits with N,N'-dicyclohexylcarbodiimide (DCCD). In the absence of a membrane potential the Fo complexes catalysed (22)Na(+)(out)/Na(+)(in)-exchange. After F(1) addition the F(1)F(o) complex was formed and the holoenzyme catalysed ATP synthesis, ATP dependent Na(+) pumping, and ATP hydrolysis, which was inhibited by DCCD. Functional F(o) hybrids were reconstituted with recombinant subunits a and b from P. modestum and c(11) from Ilyobacter tartaricus. These Fo hybrids had Na(+) translocation activities that were not distinguishable from that of P. modestum F(o).     

Osmomechanics of the Propionigenium modestum Fo Motor

In Propionigenium modestum, ATP is manufactured from ADP and phosphate by the enzyme ATP synthase using the free energy of an electrochemical gradient of Na⁺ ions. The P. modestum ATP synthase is a clear member of the family of F-type ATP synthases and the only major distinction is an extension of the coupling ion specificity to H⁺, Li⁺, or Na⁺, depending on the conditions. The use of Na⁺ as a coupling ion offers unique experimental options to decipher the ion-translocation mechanism and the osmotic and mechanical behavior of the enzyme. The single a subunit and the oligomer of c subunits are part of the stator and rotor, respectively, and operate together in the ion-translocation mechanism. During ATP synthesis, Na⁺ diffuses from the periplasm through the a subunit channel onto the Na⁺ binding site on a c subunit. From there it dissociates into the cytoplasm after the site has rotated out of the interface with subunit a. In the absence of a membrane potential, the rotor performs Brownian motions into either direction and Na⁺ ions are exchanged between the two compartments separated by the membrane. Upon applying voltage, however, the direction of Na⁺ flux and of rotation is biased by the potential. The motor generates torque to drive the rotation of the subunit, thereby releasing tightly bound ATP from catalytic sites in F₁. Hence, the membrane potential plays a pivotal role in the torque-generating mechanism. This is corroborated by the fact that for ATP synthesis, at physiological rates, the membrane potential is indispensable. We propose a catalytic mechanism for torque generation by the F_o motor that is in accord with all experimental data and is in quantitative agreement with the requirement for ATP synthesis.     

The sodium ion translocating adenosinetriphosphatase of Propionigenium modestum pumps protons at low sodium ion concentrations

The purified ATPase (F1F0) of Propionigenium modestum has its pH optimum at pH 7.0 or at pH 6.0 in the presence or absence of 5 mM NaCl, respectively. The activation by 5 mM NaCl was 12-fold at pH 7.0, 3.5-fold at pH 6.0, and 1.5-fold at pH 5.0. In addition to its function as a primary Na+ pump, the ATPase was capable of pumping protons. This activity was demonstrated with reconstituted proteoliposomes by the ATP-dependent quenching of the fluorescence of 9-amino-6-chloro-2-methoxyacridine. No delta pH was formed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone or by blocking the ATPase with dicyclohexylcarbodiimide. In the presence of valinomycin and K+, the delta pH increased, in accord with the operation of an electrogenic proton pump. The proton pump was only operative at low Na+ concentrations (less than 1 mM), and its activity increased as the Na+ concentration decreased. Parallel to the decrease of H+ pumping, the velocity of the Na+ transport increased about 6-fold from 0.1 to 4 mM NaCl, indicating a switch from H+ to Na+ pumping, as the Na+ concentration increases. Due to proton leaks in the proteoliposomal membranes, fluorescence quenching was released after blocking the ATPase with dicyclohexylcarbodiimide, by trapping residual ATP with glucose and hexokinase, or by the Na+-induced conversion of the proton pump onto a Na+ pump. Amiloride, an inhibitor of various Na+-coupled transport systems, was without effect on the kinetics of Na+ transport by the P. modestum ATPase.     

A hybrid adenosinetriphosphatase composed of F1 of Escherichia coli and F0 of Propionigenium modestum is a functional sodium ion pump

Analyses on immunoblots indicated strong binding of the alpha- and beta-subunits of the ATPase of Propionigenium modestum to antibodies raised against the corresponding subunits of the F1F0 ATPase of Escherichia coli. Cross-reactivities of antibodies against the other ATPase subunits were not observed. The use of Na+ or H+ as alternate coupling ions, observed previously for the P. modestum ATPase [Laubinger, W., & Dimroth, P. (1989) Biochemistry 28, 7194-7198], is not found for the F1F0 ATPase of E. coli, which is specific for protons. However, a hybrid consisting of the F1 moiety of the E. coli ATPase and F0 of that from P. modestum performed Na+ or H+ transport in a reconstituted system. As with the homologous ATPase of P. modestum, H+ pumping of the hybrid was abolished at Na+ concentrations of greater than 1 mM. The F0 sector and not F1, therefore, determines the cation specificity of these F1F0 ATPases.     

Propionigenium modestum: a separate line of descent within the eubacteria

The complete nucleotide sequence of 16S rRNA from Propionigenium modestum was determined and compared with 380 16S rRNA sequences from representatives of all eu- and archaebacterial phyla known so far. The phylogenetic analysis of this data set indicated P. modestum to represent a new separated line of descent within the radiation of eubacterial phyla moderately related to cyanobacteria and Gram-positive bacteria with low DNA GC content.     

Characterization of the Na+-stimulated ATPase of Propionigenium modestum as an enzyme of the F1F0 type

The ATP-hydrolyzing activity of Propionigenium modestum was extracted from the membranes with Triton X-100 or by incubation with EDTA at low ionic strength. The ATPase in the Triton extract was highly sensitive to dicyclohexylcarbodiimide but not to vanadate. These properties are characteristic for enzymes of the F1 F0 type. The ATPase was specifically activated by Na+ ions yielding a 15-fold increase in catalytic activity at 5 mM Na+ concentration. The additional presence of 1% Triton X-100 caused a further 1.5-fold activation. In the absence of Na+ Triton stimulated the ATPase about 13-fold. The Triton-stimulated ATPase was further activated about 1.5-2-fold by Na+ addition. The ATPase extracted by the low-ionic-strength treatment was purified to homogeneity by fractionation with poly(ethylene glycol) and gel chromatography. The enzyme had the characteristic F1-ATPase subunit structure with Mr values of 58,000 (alpha), 56,000 (beta), 37,600 (gamma), 22,700 (delta), and 14,000 (epsilon). The F1-ATPase was not stimulated by Na+ ions. The membrane-bound ATPase was reconstituted from the purified F1 part and F1-depleted membranes, thus further indicating an F1 F0 structure for the ATPase of P. modestum. Upon reconstitution the ATPase recovered its stimulation by Na+ ions, suggesting that the binding site for Na+ is localized on the membrane-bound F0 part of the enzyme complex.     

NMR investigations of subunit c of the ATP synthase from Propionigenium modestum in chloroform/methanol/water (4 : 4 : 1).

The subunit c from the ATP synthase of Propionigenium modestum was studied by NMR in chloroform/methanol/water (4 : 4 : 1). In this solvent, subunit c consists of two helical segments, comprised of residues L5 to I26 and G29 to N82, respectively. On comparing the secondary structure of subunit c from P. modestum in the organic solvent mixture with that in dodecylsulfate micelles several deviations became apparent: in the organic solvent, the interruption of the alpha helical structure within the conserved GXGXGXGX motif was shortened from five to two residues, the prominent interruption of the alpha helical structure in the cystoplasmic loop region was not apparent, and neither was there a break in the alpha helix after the sodium ion-binding Glu65 residue. The folding of subunit c of P. modestum in the organic solvent also deviated from that of Escherichia coli in the same environment, the most important difference being that subunit c of P. modestum did not adopt a stable hairpin structure like subunit c of E. coli.     

The Na(+)-translocating F(1)F(0) ATP synthase of Propionigenium modestum: mechanochemical insights into the F(0) motor that drives ATP synthesis

The ATP synthase of Propionigenium modestum encloses a rotary motor involved in the production of ATP from ADP and inorganic phosphate utilizing the free energy of an electrochemical Na(+) ion gradient. This enzyme clearly belongs to the family of F(1)F(0) ATP synthases and uses exclusively Na(+) ions as the physiological coupling ion. The motor domain, F(0), comprises subunit a and the b subunit dimer which are part of the stator and the subunit c oligomer acting as part of the rotor. During ATP synthesis, Na(+) translocation through F(0) proceeds from the periplasm via the stator channel (subunit a) onto a Na(+) binding site of the rotor (subunit c). Upon rotation of the subunit c oligomer versus subunit a, the occupied rotor site leaves the interface with the stator and the Na(+) ion can freely dissociate into the cytoplasm. Recent experiments demonstrate that the membrane potential is crucial for ATP synthesis under physiological conditions. These findings support the view that voltage generates torque in F(0), which drives the rotation of the gamma subunit thus liberating tightly bound ATP from the catalytic sites in F(1). We suggest a mechanochemical model for the transduction of transmembrane Na(+)-motive force into rotary torque by the F(0) motor that can account quantitatively for the experimental data.     

The ATPases of Propionigenium modestum and Bacillus alcalophilus. Strategies for ATP synthesis under low energy conditions.

In Propionigenium modestum, ATP synthesis is coupled via delta mu Na+ to the decarboxylation of (S)-methylmalonyl-CoA. The low energy yield of this reaction implies that approx. 4 decarboxylation cycles are necessary to synthesize 1 molecule of ATP. Theoretical considerations in accord with experimental results suggest ATP synthesis in P. modestum at delta mu Na+ = -110 mV. Other anaerobic bacteria synthesize ATP at a delta mu H+ of similar size and alkaliphilic bacteria at pH 10.3 have a delta mu H+ of only -103 mV. In these cases, the H+(Na+) to ATP stoichiometry must be at least 4.     

Expression of subunits a and c of the sodium-dependent ATPase of Propionigenium modestum in Escherichia coli

The aim of the present study was to construct functional hybrid ATPases consisting of all Escherichia coli ATPase subunits excepts the F₀ subunits a or c which were replaced by the respective subunits of the Propionigenium modestum ATPase. This would give valuable information on the subunit(s) conferring the coupling ion specificity. Plasmids were constructed that carried the gene for subunit c (uncE) or subunit a (uncB) behind a tac promoter. These plasmids were transformed into E. coli strains which differed with respect to the unc operon and the expression of the P. modestum genes was verified biochemically. Enhanced expression of the P. modestum genes led to strong growth inhibition of all E. coli strains tested. However, the expressed P. modestum proteins could not functionally complement E. coli strains that lacked the homologous subunit.Abbreviations PCR Polymerase chain reaction - ACMA 9-amino-6-chloro-2-methoxyacridine - SDS sodium dodecyl sulfate - DCCD N,N-dicyclohexylcarbodiimide - PMSF pnenylmethyl sulfornyl fluoride - DFP dirsopropylfluorphosphat - TCA trichloroacetic acid     

Efficiency of ATP synthase as a molecular machine

The experimentally measured effect of the odd magnetic isotope ²⁵Mg on the rate of ATP synthesis by the mitochondrial F₀F₁ enzyme is used to compare the rates of the main reactions in the catalytic site, in the framework of a radical-ion process. The rate-limiting step of synthesis is the addition of the ADP oxyradical to the phosphate P=O bond. The relationship of the rate constants deduced from the magnetic isotope effect shows that the mechanochemical efficiency of ATP synthase operating with spinless ²⁴Mg or ²⁶Mg nuclei will not exceed 50%. The performance is nearly doubled with magnetic ²⁵Mg.