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P Dimroth

Publications and source records attributed to P Dimroth.

At least 55 records · Page 3Linked to original sources
PubMed1997

Molecular basis for the coupling ion selectivity of F1F0 ATP synthases: probing the liganding groups for Na+ and Li+ in the c subunit of the ATP synthase from Propionigenium modestum.

The conserved glutamate residue at position 65 of the Propionigenium modestum c subunit is directly involved in binding and translocation of Na+ across the membrane. The site-specific introduction of the cQ32I and cS66A substitutions in the putative vicinity to cE65 inhibited growth of the single-site mutants on succinate minimal agar, indicating that both amino acid residues are important for proper function of the oxidative phosphorylation system. This growth inhibition was abolished, however, if the cF84L/cL87V double mutation was additionally present in the P. modestum c subunit. The newly constructed Escherichia coli strain MPC848732I, harboring the cQ32I/cF84L/cL87V triple mutation, revealed a change in the coupling ion specificity from Na+ to H+. ATP hydrolysis by this enzyme was therefore not activated by NaCl, and ATP-driven H+ transport was not affected by this alkali salt. Both activities were influenced, however, by LiCl. These data demonstrate the loss of the Na+ binding site and retention of Li+ and H+ binding sites within this mutant ATPase. In the E. coli strain MPC848766A (cS66A/cF84L/cL87V), the specificity of the ATPase was further restricted to H+ as the exclusive coupling ion. Therefore, neither Na+ nor Li+ stimulated the ATPase activity, and no ATP-driven Li+ transport was observed. The ATPase of the E. coli mutant MPC32N (cQ32N) was activated by NaCl and LiCl. The mutant ATPase exhibited a 5-fold higher Km for NaCl but no change in the Km for LiCl in comparison to that of the parent strain. These results demonstrate that the binding of Na+ to the c subunit of P. modestum requires liganding groups provided by Q32, E65, and S66. For the coordination of Li+, two liganding partners, E65 and S66, are sufficient, and H+ translocation was mediated by E65 alone.

Escherichia coli↗
PubMed1997

In vitro binding of the response regulator CitB and of its carboxy-terminal domain to A + T-rich DNA target sequences in the control region of the divergent citC and citS operons of Klebsiella pneumoniae.

The genes specifically required for citrate fermentation in Klebsiella pneumoniae form a cluster on the chromosome consisting of two divergently transcribed groups, citCDEFG and citS-oadGAB-citAB. Northern blot analyses described here and elsewhere indicate that each group forms an operon. The transcriptional start sites of citC and citS, which were mapped in this work by primer extension, are separated by a stretch of 193 bp with an extraordinary high A + T content of 67%. Expression of the citrate fermentation genes was recently shown to be positively controlled by a two-component signal transduction system encoded by the promoter-distal genes of the citS operon, citA (sensor kinase) and citB (response regulator). As a first step towards the functional characterization of CitB, we analysed its DNA-binding properties. To this end, the entire CitB, its N-terminal receiver domain (CitBN), and its C-terminal output domain (CitBC), all modified by a (His)6-tag, were purified. CitB(His) and CitBN(His) could be phosphorylated either with acetylphosphate or with ATP plus MalE-CitAC. The latter protein contains the kinase domain of CitA fused to the C terminus of the maltose-binding protein. Upon phosphorylation, CitB(His) became more resistant towards limited proteolysis by trypsin, reflecting substantial changes in tertiary structure. In gel retardation assays, CitB(His) bound specifically to the citC-citS intergenic region. The retardation pattern changed significantly upon phosphorylation and the apparent binding affinity increased 10 to 100-fold. Depending on the protein concentration, four different phospho-CitB(His)-DNA complexes could be resolved, suggesting the presence of multiple binding sites between citC and citS. DNase I footprints revealed two protected regions extending maximally from -55 to -89 relative to the citS transcription start and from -50 to -96 relative to the citC transcription start. Gel retardation and DNase I footprint assays with CitBC(His) showed that the C-terminal domain is sufficient for specific DNA binding. Since its properties were similar to that of unphosphorylated CitB(His), an essential role of the N-terminal receiver domain in high-affinity DNA binding was indicated. The positions of the binding sites for CitB and of putative recognition sequences for the cAMP receptor protein suggested a model for the interaction of these activators with RNA polymerase.

Bacterial Proteins↗
PubMed1997

Sequence of a gene cluster from Klebsiella pneumoniae encoding malonate decarboxylase and expression of the enzyme in Escherichia coli.

Malonate decarboxylase of Klebsiella pneumoniae consists of four different subunits and catalyzes the conversion of malonate plus H+ to acetate and CO2. The catalysis proceeds via acetyl and malonyl thioester residues with the phosphribosyl-dephospho-CoA prosthetic group of the acyl carrier protein (ACP) subunit. From a cosmid library of K. pneumoniae, a gene cluster of 9 kb has been isolated and sequenced that included the structural genes for the malonate decarboxylase. The cluster consisted of the eight consecutive genes mdcABCDEFGH and the divergently oriented mdcR gene. The intergenic regions were short (usually < 17 bp, 136 bp between mdcE and mdcF) and ribosome binding sites were found 4-10 bp before each gene. According to N-terminal protein sequencing, the mdcA, C, D and E genes encoded subunits alpha, delta, beta and gamma of malonate decarboxylase. Data bank searches for related proteins with known function revealed that MdcA represents the ACP-transferase and that MdcD and E together probably function as malonyl-S-ACP decarboxylase. MdcC is the (apo) ACP subunit. MdcB and MdcG could be involved in the synthesis and attachment of the prosthetic group. MdcH is similar to various malonyl-CoA:ACP-SH transacylases and therefore probably involved in the initial activation of the enzyme by malonylation. MdcF is a membrane protein that could function as a malonate carrier. The mdcR gene encodes a protein of the LysR regulator family. Malonate decarboxylase was functionally expressed in Escherichia coli from plasmids harbouring the entire gene cluster including mdcR. As partial deletion of the mdcR gene impaired growth of the transformants on malonate, MdcR is probably a transcriptional regulator of the mdc genes.

Amino Acid Sequence↗
PubMed1997

Sequence of a gene cluster from Malonomonas rubra encoding components of the malonate decarboxylase Na+ pump and evidence for their function.

Malonate decarboxylation in Malonomonas rubra involves the formation of malonyl-S-[acyl-carrier protein] from acetyl-S-[acyl-carrier protein] and malonate, carboxyltransfer to a biotin protein and its decarboxylation that is coupled to delta mu Na+ generation. The genes encoding components of the malonate decarboxylase enzyme system have been cloned and sequenced. These are located within a gene cluster of approximately 11 kb comprising 14 genes that have been termed madYZGBAECDHKFLMN in the given order. Upstream of madY an open reading frame pointing into the opposite direction of the mad genes was found with structural similarities to insertion-sequence elements. The upstream region also contains DNA regions which are typical for an Escherichia coli sigma 70 promoter. Within 950 bp downstream of madN no other open reading frame was found. This region contains a putative terminator sequence. The intergenic regions within the mad gene cluster are short (usually < 70 bp, maximum 302 bp) and ribosome binding sites were defined before all 14 genes. Thus, this DNA region could form a transcriptional unit and all 14 genes could be translated into proteins. The genes madABCDEF encode the structural proteins of the malonate decarboxylase as yet identified. By comparing protein and DNA sequences and by data bank searches for related proteins with known function the following assignments could be made: MadA represents the acyl-carrier-protein-transferase component. MadB is the integral membrane-bound carboxybiotin protein decarboxylase, MadC and MadD are the two subunits of the carboxyltransferase, MadE is the acyl carrier protein and MadF is the biotin protein. Sequence comparison further indicates that MadH could be involved in the acetylation of the phosphoribosyl-dephospho-CoA prosthetic group and MadG could be involved in its biosynthesis. MadL and MadM are membrane proteins that could function as malonate carrier. The function of the madY,Z,K and N gene products is as yet unknown.

Amino Acid Sequence↗
PubMed1997

Enzymic and genetic basis for bacterial growth on malonate.

Various bacteria are able to grow aerobically or anaerobically on malonate as sole source of carbon and energy. Independent of the mechanism for energy conservation, the decarboxylation of malonate is the key reaction in the decomposition of this compound. To achieve malonate decarboxylation under physiological conditions, the substrate must be converted into an activated (thioester) derivative. We report here on the malonate decarboxylases of Malonomonas rubra and Klebsiella pneumoniae. These enzymes perform an interesting substrate activation mechanism by generating a malonyl thioester with the enzyme. Formation of the malonyl-S-enzyme involves an 'activation module' that comprises the acetylation of a specific thiol group of an acyl carrier protein (ACP) and the transfer of the ACP moiety to malonate, yielding malonyl-S-ACP and acetate. The malonyl-S-ACP is subsequently decarboxylated with regeneration of the acetyl-ACP. The malonate activation mechanism is related to the activation of citrate by citrate lyase. The relationship extends to the identical 2'-(5''-phosphoribosyl)-3'-dephospho-CoA thiol cofactor that is bound covalently to the corresponding ACP subunit. In Klebsiella pneumoniae, malonate is decarboxylated by a water-soluble enzyme complex. In the anaerobic bacterium Malonomonas rubra, malonate decarboxylation is catalysed by a set of water-soluble as well as membrane-bound enzymes that function together in converting the free energy of the decarboxylation reaction into delta muNa+. Therefore, this malonate decarboxylase includes a biotin carrier protein that accepts the CO2 moiety from malonyl-S-ACP and delivers it to a membrane-bound decarboxylase acting as a Na+ pump. Genes encoding the individual protein components that perform the decarboxylation of malonate in K. pneumoniae or M. rubra have been identified within the mdc and mad gene clusters respectively. The function of most of the derived proteins could be envisaged from sequence similarities with proteins of known functions. The genetic evidence firmly supports the idea that malonate decarboxylation is carried out by the two different decarboxylases, as deduced from the biochemical studies of the enzymes.

Bacterial Proteins↗
PubMed1996

An oxaloacetate decarboxylase homologue protein influences the intracellular survival of Legionella pneumophila.

Legionella pneumophila is a facultative intracellular parasite which is able to survive in various eukaryotic cells. We characterised a Tn5-mutant of the L. pneumophila Corby strain and were able to identify the insertion site of the transposon. It is localised within an open reading frame which shows high homology to the alpha-subunit of the oxaloacetate decarboxylase (OadA) of Klebsiella pneumoniae. The OadA homologous protein of L. pneumophila was detected in the wild-type strain by Western blotting. Since the intracellular multiplication of the oadA- mutant strain is reduced in guinea pig alveolar macrophages and human monocytes, it is concluded that the oadA gene product has an effect on the intracellular survival of L. pneumophila.

Animals↗
PubMed1996

NADH:ubiquinone oxidoreductase of Vibrio alginolyticus: purification, properties, and reconstitution of the Na+ pump.

The Na+-activated NADH:ubiquinone oxidoreductase of Vibrio alginolyticus was extracted from the membranes with lauryldimethylamine-N-oxide and purified by two successive anion exchange columns. This preparation, yielding four major and several minor stained bands after SDS-PAGE, retained the NADH-dehydrogenase activity (with menadione as an artificial electron acceptor) and ubiquinone-1 (Q) reductase activity. On further fractionation of the enzyme, the Q-reductase activity essentially disappeared. Chemical analyses revealed the presence of FAD but not FMN, of non-heme iron and of acid-labile sulfur and tightly-bound ubiquinone-8 in the purified Q-reductase preparation. The participation of an iron-sulfur cluster of the [2Fe-2S] type in the electron translocation was demonstrated by the appearance of a typical EPR signal for this prosthetic group after the reduction of Q-reductase with NADH. A strong EPR signal typical for a radical observed upon reduction of the enzyme might arise from the formation of quinone radicals. In the absence of Na+, the path of the electrons apparently ends with the reduction of ubiquinone-1 to the semiquinone derivative which in the presence of O2 becomes reoxidized with concomitant formation of superoxide radicals. In the presence of Na+, these oxygen radicals are not formed and the semiquinone is further reduced to the quinol derivative. These results indicate that the Na+-dependent step in the electron transfer catalyzed by NADH:ubiquinone oxidoreductase is the reduction of ubisemiquinone to ubiquinol. After reconstitution of the purified Q-reductase into proteoliposomes, NADH oxidation by ubiquinone-1 was coupled to Na+ transport with an apparent stoichiometry of 0.5 Na+ per NADH oxidized. The transport was stimulated by valinomycin (+ K+) or by the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). The transport of Na+ is therefore a primary event and does not involve the intermediate formation of a proton gradient.

Biological Transport, Active↗
PubMed1996

The acyl carrier protein of malonate decarboxylase of Malonomonas rubra contains 2'-(5"-phosphoribosyl)-3'-dephosphocoenzyme A as a prosthetic group.

Malonate decarboxylase of Malonomonas rubra is composed of soluble and membrane-bound components and contains an acetyl residue that is essential for catalytic activity. Upon incubation with hydroxylamine, the acetyl residue is removed, forming an inactive thiol enzyme, which is reactivated by acetylation with ATP, acetate, and a specific ligase. After incubation of the thiol enzyme with iodoacetate in the presence of excess dithioerythritol, the prosthetic group thiol residue was carboxymethylated and reactivation by acetylation was impaired. Radioactive labeling with [1-14C] iodoacetate revealed the site of carboxymethyation on a distinct cytoplasmic protein with the apparent molecular mass of 14 000 Da. The same protein was specifically labeled by enzymic acetylation of the thiol enzyme with [1-14C]acetate and ATP. Malonate decarboxlyation by [14C]acetyl malonate decarboxlyation resulted in the release of the radioactive acetyl residue from the enzyme,indicating that this acetyl residue is exchanged for a malonyl residue during catalysis. The acyl carrier protein has been purified as its [14C]carboxymethylated derivative to apparent homogeneity. The prosthetic group of the acyl carrier protein was isolated after alkaline hydrolysis, and its chemical structure was identified by high-performance liquid chromatography (HPLC) with the corresponding compound from citrate lyase from Klebsiella pneumoniae as reference and by mass spectrometry. Malonate decarboxylase was found to carry the same prosthetic group as citrate lyase, i.e. 2'-(5"-phosphoribosyl)-3'-dephospho-CoA.

Acyl Carrier Protein↗
PubMed1996

Aspartate 203 of the oxaloacetate decarboxylase beta-subunit catalyses both the chemical and vectorial reaction of the Na+ pump.

We report here a new mode of coupling between the chemical and vectorial reaction explored for the oxaloacetate decarboxylase Na+ pump from Klebsiella pneumoniae. The membrane-bound beta-subunit is responsible for the decarboxylation of carboxybiotin and the coupled translocation of Na+ ions across the membrane. The biotin prosthetic group which is attached to the alpha-subunit becomes carboxylated by carboxyltransfer from oxaloacetate. The two conserved aspartic acid residues within putative membrane-spanning domains of the beta-subunit (Asp149 and Asp203) were exchanged by site-directed mutagenesis. Mutants D149Q and D149E retained oxaloacetate decarboxylase and Na+ transport activities. Mutants D203N and D203E, however, had lost these two activities, but retained the ability to form the carboxybiotin enzyme. Direct participation of Asp203 in the catalysis of the decarboxylation reaction is therefore indicated. In addition, all previous and present data on the enzyme support a model in which the same aspartic acid residue provides a binding site for the metal ion catalysing its movement across the membrane. The model predicts that asp203 in its dissociated form binds Na+ and promotes its translocation, while the protonated residue transfers the proton to the acid-labile carboxybiotin which initiates its decarboxylation. Strong support for the model comes from the observation that Na+ transport by oxaloacetate decarboxylation is accompanied by H+ transport in the opposite direction. The inhibition of oxaloacetate decarboxylation by high Na+ concentrations in a pH-dependent manner is also in agreement with the model.

Aspartic Acid↗
PubMed1996

Malonate decarboxylase of Klebsiella pneumoniae catalyses the turnover of acetyl and malonyl thioester residues on a coenzyme-A-like prosthetic group.

During aerobic growth of Klebsiella pneumoniae on malonate, a soluble malonate decarboxylase is induced. Malonate decarboxylation consumes a proton (not H2O) and forms acetate and CO2 (not HCO3-) as products. The enzyme was purified 56-fold to apparent homogeneity. It has a native molecular mass of 142 kDa and consists of four subunits alpha, beta, gamma and delta with molecular masses of 65, 34, 30, and 12 kDa, respectively. Two different forms of the enzyme were recognised: a catalytically inactive SH-enzyme and the catalytically active acetyl-S-enzyme which is formed by post-translational acetylation of the SH-enzyme with ATP, acetate and a specific ligase. The acetyl-S-enzyme was converted into the SH-enzyme by incubation with hydroxylamine or dithioerythritol. Chemical reacylation of the SH-enzyme, which restores catalytic activity, was achieved with acetic anhydride or more efficiently with malonyl-CoA. This acylation of the SH group was prevented after incubation with various thiol-specific reagents. After incubation of the SH-enzyme with iodo[1-14C]acetate, the delta subunit became specifically labelled. This subunit was also labelled after incubation of the acetyl-S-enzyme with [2-14C]malonate. The radioactivity was completely liberated from the protein upon malonate addition. These results indicate that the delta subunit is the acyl-carrier protein of the complex and that malonate decarboxylation proceeds in two steps: the acetyl residue on the ACP is first replaced by a malonyl residue which subsequently undergoes decarboxylation thereby regenerating the acetyl-S-ACP. The binding site for the acyl residues on the acyl-carrier protein was shown to be 2'-(5"-phosphoribosyl)-3'-dephospho-CoA after alkaline cleavage of this prosthetic group from the enzyme and chromatographic as well as mass spectroscopic analyses.

Bicarbonates↗
PubMed1996

Cellular localisation by immunolabelling and transmission electron microscopy of oxaloacetate decarboxylase or its individual subunits synthesised in Escherichia coli.

The genes oadGAB encoding the oxaloacetate decarboxylase gamma, alpha and beta-subunits from Klebsiella pneumoniae were expressed in Escherichia coli. Using different expression vectors, the entire enzyme or its individual subunits were synthesised. The expression was evidenced immunologically in whole cells with polyclonal antibodies raised against the purified oxaloacetate decarboxylase. The expressed alpha-subunit or a combination of alpha and beta-subunits were shown to reside in the cytoplasm, while the entire oxaloacetate decarboxylase or a gammaalpha-complex were located mostly in the cytoplasmic membrane. Interestingly, overexpression of the gammaalpha-complex or the entire oxaloacetate decarboxylase in E. coli led to a significant immunogold labelling in the cytoplasm, indicating that the alpha-subunit was not completely complexed to the membrane-bound gamma or betagamma-subunits.

Carboxy-Lyases↗
PubMed1996

Functional properties of the purified Na(+)-dependent citrate carrier of Klebsiella pneumoniae: evidence for asymmetric orientation of the carrier protein in proteoliposomes.

The sodium-ion-dependent citrate carrier of Klebsiella pneumoniae (CitS) was purified and reconstituted into liposomes to investigate the properties of this transport system without interference from other proteins. Citrate uptake was an electroneutral process, where delta pNa+ and/or delta pH are driving forces. Delta psi was unable to stimulate citrate transport, either alone or in addition to the other driving forces. Sodium ions on the inside of the proteoliposomes stimulated the uptake of citrate, indicating that Na+ ions recycle during the transport of citrate. CitS also performed Na+ counterflow in the absence of citrate. The citrate carrier performed citrate/citrate counterflow but no heterologous antiport of citrate with one of the end products arising from the anaerobic citrate fermentation pathway (acetate, formate, or bicarbonate) in K. pneumoniae. Citrate counterflow kinetics revealed that CitS transports citrate according to a simultaneous type of mechanism. The Km and Ki values revealed two binding sites for citrate: one with low and one with high affinity. This transport mode is in accord with an asymmetric organization of the carrier protein in proteoliposomes.

Antiporters↗
PubMed1995

A double mutation in subunit c of the Na(+)-specific F1F0-ATPase of Propionigenium modestum results in a switch from Na+ to H(+)-coupled ATP synthesis in the Escherichia coli host cells.

The in vivo synthesis of an F1F0-ATPase hybrid in Escherichia coli strain PEF42 which harbours the genes for the Propionigenium modestum subunits a, b, c, and delta, a gene for hybrid alpha subunit with the N-terminal portion (amino acids 1 to 173) of P. modestum and the C-terminal region (amino acids 176 to 513) from E. coli, and the genes for the E. coli subunits beta, gamma and epsilon, yielded a functional enzyme complex. This hybrid ATPase coupled ATP synthesis to Na+ transport and required Na+ for growth on succinate. After random mutagenesis of the P. modestum genes of strain PEF42, clones were selected that grew on succinate in the absence of Na+. A double-mutation cPhe84Leu, cLeu87Val that was found in several of these clones, was introduced by site specific mutagenesis into the parent strain PEF42. The resulting strain E. coli MPC8487 also exhibited Na(+)-independent growth on succinate, showing that the double mutation is the only reason for the new phenotype. The mutation causes a change of the coupling ions of the hybrid ATPase from Na+ in strain PEF42 to H+ in strain MPC8487. This conclusion was supported by the biochemical properties of the ATPase from strain MPC8487. Unlike the parent enzyme, the mutated ATPase was not activated by Na+, but retained activation by Li+. The pH optimum of the mutated ATPase (in the absence of Na+ or Li+) was shifted from pH 6.5 to pH 7.5, and the specific ATPase activity of the cell membranes increased about fourfold over that found in membranes of the parent cells. The mutated ATPase pumped protons or Li+ after reconstitution into proteoliposomes, and the transport of both cations was not affected by Na+. The double mutation in the c subunit thus results in the loss of Na+ binding, retention of Li+ binding and an improvement of H+ binding.

Adenosine Triphosphate↗
PubMed1995

In vivo synthesis of ATPase complexes of Propionigenium modestum and Escherichia coli and analysis of their function.

Expression studies of Propionigenium modestum ATPase genes in various combinations with Escherichia coli ATPase genes were performed in the unc deletion mutant strain E. coli DK8. Plasmids containing the whole unc operon from P. modestum were unable to complement the E. coli unc deletion mutant. Although all ATPase subunits were expressed from the plasmids, there was no detectable ATP hydrolysing activity, indicating that the F1 part was not functional. Transformants expressing an E. coli F1-P. modestum F0 hybrid exhibited considerable ATPase activities. Binding of the F1 part to the membrane was very weak, however, and the coupling between ATP hydrolysis and Na+ transport was impaired. After combining the genes for E. coli ATPase subunits alpha, beta, gamma, delta and epsilon and the hydrophilic part of subunit b with P. modestum ATPase subunits a and c and the hydrophobic part of subunit b on a plasmid, a non-functional hybrid ATPase was expressed in E. coli. The ATPase was only loosely bound to the membrane, from which it was solubilized with Triton X-100 and purified. Subunit b and a proteolytic degradation product were the only F0 subunits detectable in the purified enzyme. A stable F0 complex is thus not formed with the hybrid b subunit. The absence of a functional F0 complex was in accord with proton-conduction measurements with bacterial vesicles. The only functional Na(+)-translocating ATPase expressed in E. coli thus far consists of E. coli subunits alpha, beta, gamma and epsilon, and P. modestum subunits delta, a, b and c [Kaim, G. & Dimroth, P. (1993) Eur. J. Biochem. 218, 937-944]. During the cloning conducted in our present study, errors in the sequence entry into the EMBL data bank (accession no. X58461) for the P. modestum ATPase alpha and beta subunits became evident, which are corrected in this paper.

Adenosine Triphosphatases↗
PubMed1995

The Na(+)-translocating NADH:ubiquinone oxidoreductase from the marine bacterium Vibrio alginolyticus contains FAD but not FMN.

The Na(+)-translocating NADH:ubiquinone oxidoreductase from Vibrio alginolyticus was extracted from the bacterial membranes and purified by ion exchange chromatographic procedures. The enzyme catalyzed NADH oxidation by suitable electron acceptors, e.g. menadione, and the Na+ and NADH-dependent reduction of ubiquinone-1. Four dominant bands and a number of minor bands were visible on SDS-PAGE that could be part of the enzyme complex. Flavin analyses indicated the presence of FAD but no FMN in the purified enzyme. FAD but no FMN were also present in V. alginolyticus membranes. FAD is therefore a prosthetic group of the Na(+)-translocating NADH:ubiquinone oxidoreductase and FMN is not present in the enzyme. The FAD was copurified with the NADH dehydrogenase. The purified enzyme exhibited an absorption spectrum with a maximum at 450 nm that is typical for a flavoprotein. Upon incubation with NADH this absorption disappeared indicating reduction of the enzyme-bound FAD.

Cell Membrane↗
PubMed1995

Synthesis of the oxaloacetate decarboxylase Na+ pump and its individual subunits in Escherichia coli and analysis of their function.

The oadGAB genes encoding the gamma, alpha and beta-subunits of the oxaloacetate decarboxylase Na+ pump in Klebsiella pneumoniae have been cloned on plasmid pSK-GAB and expressed in Escherichia coli. The membranes of the recombinant E. coli clone contained about three times as much catalytically active oxaloacetate decarboxylase (3 mg protein/2 g wet cells) as those of the K. pneumoniae strain from which the genes were derived. The enzyme was solubilised from the membranes with Triton X-100 and purified. Its Na+ transport function was demonstrated after reconstitution into proteoliposomes. Proteoliposomes containing only the membrane-bound subunits beta and gamma (not the peripheral alpha-subunit) were unable to catalyse Na+ translocation in response to a transmembrane Na+ (delta pNa+) or electrical gradient (delta psi). Individual subunits of oxaloacetate decarboxylase and combinations of two subunits were expressed from appropriate derivatives of plasmid pSK-GAB. The hydrophobic subunits beta and beta gamma were membrane-bound as expected. Interestingly, the alpha-subunit was located in the cytoplasm if expressed separately or together with beta, but became membrane-bound if expressed together with gamma. A gamma alpha complex was isolated from such membranes by avidin-Sepharose affinity chromatography. Interactions of the gamma-subunit with the water-soluble alpha-subunit and with the membrane-bound beta-subunit are therefore required to form the oxaloacetate decarboxylase complex. The combinations of separately expressed subunits gamma alpha + beta and beta gamma+alpha were shown to yield the catalytically active enzyme. The alpha or the beta-subunit and the combinations of these subunits with the gamma-subunit were therefore expressed in E. coli in a catalytically competent state. Functional expression of the separate gamma-subunit, however, could not be demonstrated. The alpha-subunit was strongly overexpressed from a pT7-7 derived plasmid, but was only partially biotinylated under these conditions. On coexpression of the birA gene encoding biotin ligase the major part (80-100%) of the overexpressed alpha-subunit was biotinylated. Highly purified alpha-subunit was obtained by fractionated precipitation of the soluble cell fraction with ammonium sulfate. Incubation of the alpha-subunit with oxaloacetate led to a CO2 transfer to its prosthetic biotin group with the formation of stoichiometric amounts of pyruvate. The velocity of the CO2 transfer to the biotin on the alpha-subunit was about three orders of magnitude too low to account for the rate of the overall reaction. The carboxyltransfer reaction was significantly accelerated if the gamma-subunit was additionally present.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗