Georg Kaim - Academia.edu (original) (raw)

Papers by Georg Kaim

The EMBO Journal, Feb 1, 1998

We have recently isolated a mutant (aK220R, aV264E, aI278N) of the Na ϩ-translocating Escherichia... more We have recently isolated a mutant (aK220R, aV264E, aI278N) of the Na ϩ-translocating Escherichia coli/ Propionigenium modestum ATPase hybrid with a Na ϩ-inhibited growth phenotype on succinate. ATP hydrolysis by the reconstituted mutant ATPase was inhibited by external (N side) NaCl but not by internal (P side) NaCl. In contrast, LiCl activated the ATPase from the N side and inhibited it from the P side. A similar pattern of activation and inhibition was observed with NaCl and the ATPase from the parent strain PEF42. We conclude from these results that the binding sites for the coupling ions on the c subunits are freely accessible from the N side. Upon occupation of these sites, the ATPase becomes more active, provided that the ions can be further translocated to the P side through a channel of the a subunit. If by mutation of the a subunit this channel becomes impermeable for Na ϩ , N side Na ϩ ions specifically inhibit the ATPase activity. These conclusions were corroborated by the observation that proton transport into proteoliposomes containing the mutant ATPase was abolished by N side but not by P side Na ϩ ions. In contrast, LiCl affected proton translocation from either side, similar to the sidedness effect of Na ϩ ions on H ϩ transport by the parent hybrid ATPase. If the ATPase carrying the mutated a subunit was incubated with 22 NaCl and ATP, 1 mol 22 Na ϩ /mol enzyme was occluded. With the parent hybrid ATPase, 22 Na ϩ occlusion was not observed. The occluded 22 Na ϩ could be removed from its tight binding site by 20 mM LiCl, while incubation with 20 mM NaCl was without effect. Li ϩ but not Na ϩ is therefore apparently able to pass through the mutated a subunit and make the entrapped Na ϩ ions accessible again to the aqueous environment. These results suggest an ion translocation mechanism through F 0 that in the ATP hydrolysis mode involves binding of the coupling ions from the cytoplasm to the multiple c subunits, ATP-driven rotation to bring a Na ϩ , Li ϩ , or H ϩ-loaded c subunit into a contact site with the a subunit and release of the coupling ions through the a subunit channel to the periplasmic surface of the membrane.

Journal of Bacteriology, Aug 1, 2003

We describe here purification and biochemical characterization of the F 1 F o-ATP synthase from t... more We describe here purification and biochemical characterization of the F 1 F o-ATP synthase from the thermoalkaliphilic organism Bacillus sp. strain TA2.A1. The purified enzyme produced the typical subunit pattern of an F 1 F o-ATP synthase on a sodium dodecyl sulfate-polyacrylamide gel, with F 1 subunits ␣, ␤, ␥, ␦, and and F o subunits a, b, and c. The subunits were identified by N-terminal protein sequencing and mass spectroscopy. A notable feature of the ATP synthase from strain TA2.A1 was its specific blockage in ATP hydrolysis activity. ATPase activity was unmasked by using the detergent lauryldimethylamine oxide (LDAO), which activated ATP hydrolysis >15-fold. This activation was the same for either the F 1 F o holoenzyme or the isolated F 1 moiety, and therefore latent ATP hydrolysis activity is an intrinsic property of F 1. After reconstitution into proteoliposomes, the enzyme catalyzed ATP synthesis driven by an artificially induced transmembrane electrical potential (⌬). A transmembrane proton gradient or sodium ion gradient in the absence of ⌬ was not sufficient to drive ATP synthesis. ATP synthesis was eliminated by the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone, while the electroneutral Na ؉ /H ؉ antiporter monensin had no effect. Neither ATP synthesis nor ATP hydrolysis was stimulated by Na ؉ ions, suggesting that protons are the coupling ions of the ATP synthase from strain TA2.A1, as documented previously for mesophilic alkaliphilic Bacillus species. The ATP synthase was specifically modified at its c subunits by N,N-dicyclohexylcarbodiimide, and this modification inhibited ATP synthesis.

International Journal of Systematic and Evolutionary Microbiology, Mar 1, 2002

The mesophilic, anaerobic bacterium strain VenChi2 T was isolated with quinic acid (1,3,4,5-tetra... more The mesophilic, anaerobic bacterium strain VenChi2 T was isolated with quinic acid (1,3,4,5-tetrahydroxy-cyclohexane-1-carboxylic acid) as the sole source of carbon and energy. Of more than 30 substrates tested, only quinic acid and shikimic acid (3,4,5-trihydroxy-1-cyclohexene-1-carboxylic acid) were utilized, yielding acetate, propionate, butyrate, H 2 and CO 2 as fermentation products. Sugars, alcohols, (di-)carboxylic acids, amino acids and aromatic compounds were not fermented and no external electron acceptors were used. Strain VenChi2 T is a Gram-negative, strictly anaerobic, coccoid rod ; it does not employ the classical hydroaromatic pathway of aerobic bacteria for the degradation of hydroaromatic compounds (no aromatic intermediates involved). Comparative 16S and 23S rDNA sequence analyses placed strain VenChi2 T in the fusobacteria phylum, with the closest relatives among species of the genera Ilyobacter and Propionigenium. The results indicate that, disregarding the taxonomically misplaced Ilyobacter delafieldii, which is a member of the clostridia, the validly described Ilyobacter and Propionigenium species are phylogenetically intermixed. Based on its phenotypic properties, strain VenChi2 T (l DSM 6831 T l ATCC BAA-291 T) is assigned to the genus Ilyobacter as the type strain of a novel species, Ilyobacter insuetus sp. nov.

Biochimica Et Biophysica Acta - Bioenergetics, Jul 1, 2010

dimers [9], complicating the preparation of such a heterodimer. The acidic domain lacking complex... more dimers [9], complicating the preparation of such a heterodimer. The acidic domain lacking complex, on the contrary, is a dimer (results confirmed from Mass Spectrometry experiments and structural observation). Further experiments are currently ongoing. On the kinetic side, the deletion mutant is functional and shows properties comparable to the wild type. Here we show by Stopped-flow kinetics that the catalytic efficiency of the heterodimer is not affected by the mutation, the affinity for quinol is not changed and under pre steadystate conditions only one cytochrome c 1 and two cytochrome b H are reduced per dimer. Moreover, it is not possible to see the antimycin stimulation observed in the WT [7] since the second quinol oxidation site is not functional.

European journal of biochemistry, Jul 1, 1992

A DNA fragment containing the genes encoding subunits of the Fo part of the sodium-translocating ... more A DNA fragment containing the genes encoding subunits of the Fo part of the sodium-translocating ATPase of Propionigenium modestum was cloned in Escherichia coli and sequenced. The predicted amino acid sequences of subunits a, b and c of the P. modestum ATPase were compared with those of the corresponding subunits of proton-translocating ATPases from other bacteria and chloroplasts. Deletion mutants of E. coli, lacking different genes for ATPase subunits, were transformed with a recombinant plasmid, containing the genes for the subunits a, c, b, 6 and part of CI of the ATPase of P. modestum. Functionally reconstituted ATPase activity could be demonstrated for the transformants. The identity of the vector containing P. rnodestum genes was verified by restriction analysis of plasmid DNA.

The EMBO Journal, Oct 15, 1998

The mechanism by which ion-flux through the membrane-bound motor module (F 0) induces rotational ... more The mechanism by which ion-flux through the membrane-bound motor module (F 0) induces rotational torque, driving the rotation of the γ subunit, was probed with a Na ⍣-translocating hybrid ATP synthase. The ATP-dependent occlusion of 1 22 Na ⍣ per ATP synthase persisted after modification of the c subunit ring with dicyclohexylcarbodiimide (DCCD), when 22 Na ⍣ was added first and ATP second, but not if the order of addition was reversed. These results support the model of ATP-driven rotation of the c subunit oligomer (rotor) versus subunit a (stator) that stops when either a 22 Na ⍣-loaded or a DCCD-modified rotor subunit reaches the Na ⍣-impermeable stator. The ATP synthase with a Na ⍣-permeable stator catalyzed 22 Na ⍣ out /Na ⍣ in-exchange after reconstitution into proteoliposomes, which was not significantly affected by DCCD modification of the c subunit oligomer, but was abolished by the additional presence of ATP or by a membrane potential (∆Ψ) of 90 mV. We propose that in the idling mode of the motor, Na ⍣ ions are shuttled across the membrane by limited back and forth movements of the rotor against the stator. This motional flexibility is arrested if either ATP or ∆Ψ induces the switch from idling into a directed rotation. The Propionigenium modestum ATP synthase catalyzed ATP formation with ∆Ψ of 60-125 mV but not with ∆pNa ⍣ of 195 mV. These results demonstrate that electric forces are essential for ATP synthesis and lead to a new concept of rotary-torque generation in the ATP synthase motor.

European Journal of Biochemistry, 1994

The Escherichia coli strain DK8, a deletion mutant lacking the complete unc operon, was transform... more 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, delta and part of the alpha subunit of the Na(+)-dependent ATPase of Propionigenium modestum and the genes encoding the alpha, gamma, beta and epsilon 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 (delta psi) and a Na+ concentration gradient (delta p Na+) were imposed.

bacterium specialized in the degradation of hydroaromatic compounds

European Journal of Biochemistry, 1992

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2010

The EMBO Journal, 1998

subunits). No sequence could be determined for the putative a subunit (ap-parent molecular mass, ... more subunits). No sequence could be determined for the putative a subunit (ap-parent molecular mass, 25 kDa). The c subunits formed a strong aggregate with the apparent molecular massof 50 kDa which required treatment with trichloroacetic acid for dissociation. The ATPase was inhibited bydicyclohexyl carbodiimide, and Na

Journal of Experimental Biology, 2000

ATP, the universal carrier of cell energy, is manufactured from ADP and phosphate by the enzyme A... more ATP, the universal carrier of cell energy, is manufactured from ADP and phosphate by the enzyme ATP synthase using the free energy of an electrochemical gradient of protons (or Na+). The proton-motive force consists of two components, the transmembrane proton concentration gradient (ΔpH) and the membrane potential. The two components were considered to be not only thermodynamically but also kinetically equivalent, since the chloroplast ATP synthase appeared to operate on ΔpH only. Recent experiments demonstrate, however, that the chloroplast ATP synthase, like those of mitochondria and bacteria, requires a membrane potential for ATP synthesis. Hence, the membrane potential and proton gradient are not equivalent under normal operating conditions far from equilibrium. These conclusions are corroborated by the finding that only the membrane potential induces a rotary torque that drives the counter-rotation of the a and c subunits in the Fo motor of Propionigenium modestum ATP synthase.

Journal of Bacteriology, 1998

The ATPase of Ilyobacter tartaricus was solubilized from the bacterial membranes and purified. So... more The ATPase of Ilyobacter tartaricus was solubilized from the bacterial membranes and purified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed the usual subunit pattern of a bacterial F 1 F o ATPase. The polypeptides with apparent molecular masses of 56, 52, 35, 16.5, and 6.5 kDa were identified as the α, β, γ, ɛ, and c subunits, respectively, by N-terminal protein sequencing and comparison with the sequences of the corresponding subunits from the Na + -translocating ATPase of Propionigenium modestum . Two overlapping sequences were obtained for the polypeptides moving with an apparent molecular mass of 22 kDa (tentatively assigned as b and δ subunits). No sequence could be determined for the putative a subunit (apparent molecular mass, 25 kDa). The c subunits formed a strong aggregate with the apparent molecular mass of 50 kDa which required treatment with trichloroacetic acid for dissociation. The ATPase was inhibited by dicyclohexyl carbo...

Journal of Bioenergetics and Biomembranes, 2000

In Propionigenium modestum, ATP is manufactured from ADP and phosphate by the enzyme ATP synthase... more 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+,

European Journal of Biochemistry, 1993

European Journal of Biochemistry, 1990

The 30 N‐terminal amino acid residues of the purified ATPase c subunit of Propionigenium modestum... more 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.