Biomedical subjects

P Dimroth

Publications and source records attributed to P Dimroth.

At least 19 recordsLinked to original sources

PubMed2001

The central plug in the reconstituted undecameric c cylinder of a bacterial ATP synthase consists of phospholipids.

The isolated rotor cylinder of the ATP synthase from Ilyobacter tartaricus was reconstituted into two-dimensional crystalline arrays. Atomic force microscopy imaging indicated a central cavity on one side of the rotor and a central plug protruding from the other side. Upon incubation with phospholipase C, the plug disappeared, but the appearance of the surrounding c subunit oligomer was not affected. This indicates that the plug consists of phospholipids. As the detergent-purified c cylinder is completely devoid of phospholipids, these are incorporated into the central hole from one side of the cylinder during the reconstitution procedure.

Fusobacterium↗

PubMed2001

ATP synthase: constrained stoichiometry of the transmembrane rotor.

Recent structural data suggest that the number of identical subunits (c or III) assembled into the cation-powered rotor of F1F0 ATP synthase depends on the biological origin. Atomic force microscopy allowed individual subunits of the cylindrical transmembrane rotors from spinach chloroplast and from Ilyobacter tartaricus ATP synthase to be directly visualized in their native-like environment. Occasionally, individual rotors exhibit structural gaps of the size of one or more subunits. Complete rotors and arch-shaped fragments of incomplete rotors revealed the same diameter within one ATP synthase species. These results suggest the rotor diameter and stoichiometry to be determined by the shape of the subunits and their nearest neighbor interactions.

ATP Synthetase Complexes↗

PubMed2001

Coupling mechanism of the oxaloacetate decarboxylase Na(+) pump.

The oxaloacetate decarboxylase Na(+) pump consists of subunits alpha, beta and gamma, and contains biotin as the prosthetic group. The peripheral alpha subunit catalyzes the carboxyltransfer from oxaloacetate to the prosthetic biotin group to yield the carboxybiotin enzyme. Subsequently, this is decarboxylated in a Na(+)-dependent reaction by the membrane-bound beta subunit. The decarboxylation is coupled to Na(+) translocation from the cytoplasm into the periplasm, and consumes a periplasmically derived proton. The gamma subunit contains a Zn(2+) metal ion which may be involved in the carboxyltransfer reaction. It is proposed to insert with its N-terminal alpha-helix into the membrane and to form a complex with the alpha subunit with its water-soluble C-terminal domain. The beta subunit consists of nine transmembrane alpha-helices, a segment (IIIa) which inserts from the periplasm into the membrane but does not penetrate it, and connecting hydrophilic loops. The most highly conserved regions of the molecule are segment IIIa and transmembrane helix VIII. Functionally important residues are D203 (segment IIIa), Y229 (helix IV) and N373, G377, S382 and R389 (helix VIII). The polar of these amino acids may constitute a network of ionizable groups which promotes the translocation of Na(+) and the oppositely oriented translocation of H(+) across the membrane. Evidence indicates that two Na(+) ions are bound simultaneously to subunit beta with D203 and S382 acting as binding sites. Sodium ion binding from the cytoplasm to both sites elicits decarboxylation of carboxybiotin possibly with the consumption of the proton extracted from S382 and delivered via Y229 to the carboxylated prosthetic group. A conformational change exposes the bound Na(+) ions toward the periplasm. With H(+) entering from the periplasm, the hydroxyl group of S382 is regenerated, and as a consequence, the Na(+) ions are released into this compartment. After switching back to the original conformation, Na(+) pumping continues.

Amino Acid Sequence↗

PubMed2001

Bacterial Na(+)-ATP synthase has an undecameric rotor.

Synthesis of adenosine triphosphate (ATP) by the F(1)F(0) ATP synthase involves a membrane-embedded rotary engine, the F(0) domain, which drives the extra-membranous catalytic F(1) domain. The F(0) domain consists of subunits a(1)b(2) and a cylindrical rotor assembled from 9-14 alpha-helical hairpin-shaped c-subunits. According to structural analyses, rotors contain 10 c-subunits in yeast and 14 in chloroplast ATP synthases. We determined the rotor stoichiometry of Ilyobacter tartaricus ATP synthase by atomic force microscopy and cryo-electron microscopy, and show the cylindrical sodium-driven rotor to comprise 11 c-subunits.

Bacteria, Anaerobic↗

PubMed2001

Catabolite repression of the citrate fermentation genes in Klebsiella pneumoniae: evidence for involvement of the cyclic AMP receptor protein.

Klebsiella pneumoniae is able to grow anaerobically with citrate as a sole carbon and energy source by a fermentative pathway involving the Na(+)-dependent citrate carrier CitS, citrate lyase, and oxaloacetate decarboxylase. The corresponding genes are organized in the divergent citC and citS operons, whose expression is strictly dependent on the citrate-sensing CitA-CitB two-component system. Evidence is provided here that the citrate fermentation genes are subject to catabolite repression, since anaerobic cultivation with a mixture of citrate and glucose or citrate and gluconate resulted in diauxic growth. Glucose, gluconate, and also glycerol decreased the expression of a chromosomal citS-lacZ fusion by 60 to 75%, whereas a direct inhibition of the citrate fermentation enzymes was not observed. The purified cyclic AMP (cAMP) receptor protein (CRP) of K. pneumoniae bound to two sites in the citC-citS intergenic region, which were centered at position -41.5 upstream of the citC and citS transcriptional start sites. Binding was apparently stimulated by the response regulator CitB. These data indicate that catabolite repression of the citrate fermentation genes is exerted by CRP and that in the absence of repressing carbon sources the cAMP-CRP complex serves to enhance the basal, CitB-dependent transcription level.

Anaerobiosis↗

PubMed2000

Biosynthesis of triphosphoribosyl-dephospho-coenzyme A, the precursor of the prosthetic group of malonate decarboxylase.

Malonate decarboxylase from Klebsiella pneumoniae consists of four subunits MdcA, D, E, and C and catalyzes the cleavage of malonate to acetate and CO(2). The smallest subunit MdcC is an acyl carrier protein to which acetyl and malonyl thioester residues are bound via a 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA prosthetic group and turn over during the catalytic mechanism. We report here on the biosynthesis of holo acyl carrier protein from the unmodified apoprotein. The prosthetic group biosynthesis starts with the MdcB-catalyzed condensation of dephospho-CoA with ATP to 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA. In this reaction, a new alpha (1' ' --> 2') glycosidic bond between the two ribosyl moieties is formed, and thereby, the adenine moiety of ATP is displaced. MdcB therefore is an ATP:dephospho-CoA 5'-triphosphoribosyl transferase. The second protein involved in holo ACP synthesis is MdcG. This enzyme forms a strong complex with the 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA prosthetic group precursor. This complex, called MdcG(i), is readily separated from free MdcG by native polyacrylamide gel electrophoresis. Upon incubation of MdcG(i) with apo acyl carrier protein, holo acyl carrier protein is synthesized by forming the phosphodiester bond between the 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA prosthetic group and serine 25 of the protein. MdcG corresponds to a 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA:apo ACP 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA transferase. In absence of the prosthetic group precursor, MdcG catalyzes at a low rate the adenylylation of apo acyl carrier protein using ATP as substrate. The adenylyl ACP thus formed is an unphysiological side product and is not involved in the biosynthesis of holo ACP. The 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA precursor of the prosthetic group has been purified and its identity confirmed by mass spectrometry and enzymatic analysis.

Acyl Carrier Protein↗

PubMed2000

Identification of the active site of phosphoribosyl-dephospho-coenzyme A transferase and relationship of the enzyme to an ancient class of nucleotidyltransferases.

Malonate decarboxylase from Klebsiella pneumoniae contains an acyl carrier protein (MdcC) to which a 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA prosthetic group is attached via phosphodiester linkage to serine 25. We have shown in the preceding paper in this issue that the formation of this phosphodiester bond is catalyzed by a phosphoribosyl-dephospho-coenzyme A transferase MdcG with the substrate 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA that is synthesized from ATP and dephospho-coenzyme A by the triphosphoribosyl transferase MdcB. The reaction catalyzed by MdcG is related to nucleotidyltransfer reactions, and the enzyme indeed catalyzes unphysiological nucleotidyltransfer, e.g., adenylyltransfer from ATP to apo acyl carrier protein (ACP). These unspecific side reactions are favored at high Mg(2+) concentrations. A sequence motif including D134 and D136 of MdcG is a signature of all nucleotidyltransferases. It is known from the well-characterized mammalian DNA polymerase beta that this motif is at the active site of the enzyme. Site-directed mutagenesis of D134 and/or D136 of MdcG to alanine abolished the transfer of the prosthetic group to apo ACP, but the binding of triphosphoribosyl-dephospho-CoA to MdcG was not affected. Evidence is presented that similar to MdcG, MadK encoded by the malonate decarboxylase operon of Malonomonas rubra and CitX from the operon encoding citrate lyase in Escherichia coli are phosphoribosyl-dephospho-CoA transferases catalyzing the attachment of the phosphoribosyl-dephospho-CoA prosthetic group to their specific apo ACPs.

Acyl Carrier Protein↗

PubMed2000

Identification of triphosphoribosyl-dephospho-CoA as precursor of the citrate lyase prosthetic group.

The gamma-subunit of citrate lyase (EC 4.1.3.6) contains the prosthetic group 2'-(5"-phosphoribosyl)-3'-dephospho-CoA and serves as an acyl carrier protein (ACP). We recently showed that in Escherichia coli the proteins CitG and CitX are essential for holo-ACP synthesis and provided evidence that CitG catalyzes the formation of a prosthetic group precursor from ATP and dephospho-CoA, which is subsequently attached via phosphodiester linkage to apo-ACP by CitX. Here we prove that CitG indeed catalyzes the conversion of ATP and dephospho-CoA to adenine and 2'-(5"-triphosphoribosyl)-3'-dephospho-CoA, the predicted precursor of the prosthetic group. Furthermore, this precursor was transferred by CitX to apo-ACP, yielding holo-ACP. Thus, our proposed mechanism for holo-ACP synthesis could be verified.

ATP Citrate (pro-S)-Lyase↗

PubMed2000

Critical evaluation of the one- versus the two-channel model for the operation of the ATP synthase's F(o) motor.

The mechanism of converting an electrochemical gradient of protons or Na(+) ions across the membrane into rotational torque by the F(o) motor of the ATP synthase has been described by a two-channel model or by a one-channel model. Experimental evidence obtained with the F(o) motor from the Propionigenium modestum ATP synthase is described which is in accordance with the one-channel model, but not with the two-channel model. This evidence includes the ATP-dependent occlusion of one (22)Na(+) per ATP synthase with a mutated Na(+)-impermeable a subunit or the Na(+)(in)/(22)Na(+)(out) exchange which is not affected by modifying part of the c subunit sites with dicyclohexylcarbodiimide.

Adenosine Triphosphate↗

PubMed2000

Biosynthesis of the prosthetic group of citrate lyase.

Citrate lyase (EC 4.1.3.6) catalyzes the cleavage of citrate to acetate and oxaloacetate and is composed of three subunits (alpha, beta, and gamma). The gamma-subunit serves as an acyl carrier protein (ACP) and contains the prosthetic group 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA, which is attached via a phosphodiester linkage to serine-14 in the enzyme from Klebsiella pneumoniae. In this work, we demonstrate by genetic and biochemical studies with citrate lyase of Escherichia coli and K. pneumoniae that the conversion of apo-ACP into holo-ACP is dependent on the two proteins, CitX (20 kDa) and CitG (33 kDa). In the absence of CitX, only apo-ACP was synthesized in vivo, whereas in the absence of CitG, an adenylylated ACP was produced, with the AMP residue attached to serine-14. The adenylyltransferase activity of CitX could be verified in vitro with purified CitX and apo-ACP plus ATP as substrates. Besides ATP, CTP, GTP, and UTP also served as nucleotidyl donors in vitro, showing that CitX functions as a nucleotidyltransferase. The conversion of apo-ACP into holo-ACP was achieved in vitro by incubation of apo-ACP with CitX, CitG, ATP, and dephospho-CoA. ATP could not be substituted with GTP, CTP, UTP, ADP, or AMP. In the absence of CitG or dephospho-CoA, AMP-ACP was formed. Remarkably, it was not possible to further convert AMP-ACP to holo-ACP by subsequent incubation with CitG and dephospho-CoA. This demonstrates that AMP-ACP is not an intermediate during the conversion of apo- into holo-ACP, but results from a side activity of CitX that becomes effective in the absence of its natural substrate. Our results indicate that holo-ACP formation proceeds as follows. First, a prosthetic group precursor [presumably 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA] is formed from ATP and dephospho-CoA in a reaction catalyzed by CitG. Second, holo-ACP is formed from apo-ACP and the prosthetic group precursor in a reaction catalyzed by CitX.

Acyl Carrier Protein↗

PubMed2000

Operation of the F(0) motor of the ATP synthase.

ATP, the universal carrier of cell energy is manufactured from ADP and phosphate by the enzyme ATP synthase using the energy stored in a transmembrane ion gradient. The two components of the ion gradient (DeltapH or DeltapNa(+)) and the electrical potential difference Deltapsi are thermodynamically but not kinetically equivalent. In contrast to accepted wisdom, the electrical component is kinetically indispensable not only for bacterial ATP synthases but also for that from chloroplasts. Recent biochemical studies with the Na(+)-translocating ATP synthase of Propionigenium modestum have given a good idea of the ion translocation pathway in the F(0) motor. Taken together with biophysical data, the operating principles of the motor have been delineated.

ATP Synthetase Complexes↗

PubMed2000

Essential role of tyrosine 229 of the oxaloacetate decarboxylase beta-subunit in the energy coupling mechanism of the Na(+) pump.

The membrane-bound beta-subunit of oxaloacetate decarboxylase from Klebsiella pneumoniae catalyzes the decarboxylation of carboxybiotin, which is coupled to Na(+) translocation and consumes a periplasmically derived proton. Upon site-directed mutagenesis of 20 polar and/or conserved residues within putative membrane-integral regions, the specific oxaloacetate decarboxylase activities were reduced to various extents, but only the enzyme with a Y229F mutation was completely inactive. We propose that Y229 is part of the network by which the proton of S382 is delivered to carboxybiotin, where it is consumed upon catalyzing the immediate decarboxylation of this acid-labile compound. Unlike S382 or D203, Y229 appears to be not involved in Na(+) binding, because in the Y229F orY229A mutants, the beta-subunit was protected from tryptic digestion by 50 mM NaCl like in the wild-type enzyme. Oxaloacetate decarboxylase with a betaC291E mutation was unstable in the absence of Na(+) and dissociated into an alpha-gamma subcomplex and the beta-subunit. The enzyme could only be isolated in the presence of 0. 5 M NaCl. These results are consistent with the notion that the beta-subunit changes its conformation upon Na(+) binding.

Amino Acid Substitution↗

PubMed2000

A molecular coupling mechanism for the oxaloacetate decarboxylase Na+ pump as inferred from mutational analysis.

The oxaloacetate decarboxylase Na+ pump consists of subunits alpha, beta, and gamma, and contains biotin as the prosthetic group. Membrane-bound subunit beta catalyzes the decarboxylation of carboxybiotin coupled to Na+ translocation, and consumes a periplasmically derived proton. Site-directed mutagenesis of conserved amino acids of transmembrane helix VIII indicated that residues N373, G377, S382, and R389 are functionally important. The polar side groups of these amino acids may constitute together with D203 a network of ionizable groups which promotes the translocation of Na+ and the oppositely oriented H+ across the membrane. Evidence is presented that two Na+ ions are bound simultaneously to subunit beta during transport with D203 and S382 acting as binding sites. Sodium ion binding from the cytoplasm to both sites elicits decarboxylation of carboxybiotin, and a conformational switch exposes the bound Na+ ions toward the periplasm. After dissociation of Na+ and binding of H+, the cytoplasmically exposed conformation is regained.

Amino Acid Sequence↗

PubMed2000

Osmomechanics of the Propionigenium modestum F(o) 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 gamma subunit, thereby releasing tightly bound ATP from catalytic sites in F(1). 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.

Adenosine Triphosphate↗

PubMed2000

Na+ translocation by complex I (NADH:quinone oxidoreductase) of Escherichia coli.

Following on from our previous discovery of Na+ pumping by the NADH:ubiquinone oxidoreductase (complex I) of Klebsiella pneumoniae, we show here that complex I from Escherichia coli is a Na+ pump as well. Our study object was the Escherichia coli mutant EP432, which lacks the Na+/H+ antiporter genes nhaA and nhaB and is therefore unable to grow on LB medium at elevated Na+ concentrations. During growth on mineral medium, the Na+ tolerance of E. coli EP432 was influenced by the organic substrate. NaCl up to 450 mM did not affect growth on glycerol and fumarate, but growth on glucose was inhibited. Correlated to the Na+ tolerance was an increased synthesis of complex I in the glycerol/fumarate medium. Inverted membrane vesicles catalysed respiratory Na+ uptake with NADH as electron donor. The sodium ion transport activity of vesicles from glycerol/fumarate-grown cells was 40 nmol mg-1 min-1 and was resistant to the uncoupler carbonyl-cyanide m-chlorophenylhydrazone (CCCP), but was inhibited by the complex I-specific inhibitor rotenone. With an E. coli mutant deficient in complex I, the Na+ transport activity was low (1-3 nmol mg-1 min-1), and rotenone was without effect.

Cell Membrane↗

PubMed2000

Crucial role of the membrane potential for ATP synthesis by F(1)F(o) ATP synthases.

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 (delta pH) 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 delta pH 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 F(o) motor of Propionigenium modestum ATP synthase.

Adenosine Triphosphate↗

PubMed1999

Membrane topology of the beta-subunit of the oxaloacetate decarboxylase Na+ pump from Klebsiella pneumoniae.

The topology of the beta-subunit of the oxaloacetate Na+ pump (OadB) was probed with the alkaline phosphatase (PhoA) and beta-galactosidase (lacZ) fusion technique. Additional evidence for the topology was derived from amino acid alignments and comparative hydropathy profiles of OadB with related proteins. Consistent results were obtained for the three N-terminal and the six C-terminal membrane-spanning alpha-helices. However, the two additional helices that were predicted by hydropathy analyses between the N-terminal and C-terminal blocks did not conform with the fusion results. The analyses were therefore extended by probing the sideness of various engineered cysteine residues with the membrane-impermeant reagent 4-acetamido-4'-maleimidylstilbene-2, 2'-disulfonate. The results were in accord with those of the fusion analyses, suggesting that the protein folds within the membrane by a block of three N-terminal transmembrane segments and another one with six C-terminal transmembrane segments. The mainly hydrophobic connecting segment is predicted not to traverse the membrane fully, but to insert in an undefined manner from the periplasmic face. According to our model, the N-terminus is at the cytoplasmic face and the C-terminus is at the periplasmic face of the membrane.

Alkaline Phosphatase↗

PubMed1999

ATP synthesis by F-type ATP synthase is obligatorily dependent on the transmembrane voltage.

ATP synthase is the universal enzyme that manufactures cellular ATP using the energy stored in a transmembrane ion gradient. This energy gradient has two components: the concentration difference (DeltapH or DeltapNa(+)) and the electrical potential difference DeltaPsi, which are thermodynamically equivalent. However, they are not kinetically equivalent, as the mitochondrial and bacterial ATP synthases require a transmembrane potential, DeltaPsi, but the chloroplast enzyme has appeared to operate on DeltapH alone. Here we show that, contrary to the accepted wisdom, the 'acid bath' procedure used to study the chloroplast enzyme develops not only a DeltapH but also a membrane potential, and that this potential is essential for ATP synthesis. Thus, for the chloroplast and other ATP synthases, the membrane potential is the fundamental driving force for their normal operation. We discuss the biochemical reasons for this phenomenon and a model that is consistent with these new experimental facts.

Adenosine Triphosphate↗