Resolution of distinct selenium-containing formate dehydrogenases from Escherichia coli.

Formate dehydrogenase, a component activity of two alternative electron transport pathways in anaerobic Escherichia coli, has been resolved as two distinguishable enzymes. One, which was induced with nitrate reductase as a component of the formate-nitrate reductase pathway, utilized phenazine methosulfate (PMS) in preference to benzyl viologen (BV) as an artificial electron acceptor and appeared to be exclusively membrane-bound. A second formate dehydrogenase, which was induced as a component of the formate hydrogenlyase pathway, appeared to exist both as a membrane-bound form and as a cytoplasmic enzyme; the cytoplasmic activity was resolved completely from the PMS-linked activity on a sucrose gradient. When E. coli was grown in the presence of 75Se-selenite, a 110,000-dalton selenopeptide, previously shown to be a component of the PMS-linked enzyme, was induced and repressed with this activity. In contrast, an 80,000-dalton selenopeptide was induced and repressed with the BV-linked activity and exhibited a distribution similar to the BV-linked formate dehydrogenase in cell fractions and in sucrose gradients. The results indicate that the two formate dehydrogenases are distinguishable on the basis of their artificial electron acceptor specificity, their cellular localization, and the size of their respective selenoprotein components.     

Formate dehydrogenases for CO2 utilization

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Aerobic and anaerobic alcohol dehydrogenases in Escherichia coli

Abstract Mutants unable to use ethanol for carbon and energy were counterselected from an ethanolutilizing mutant of Escherichia coli K12 derepressed for alcohol dehydrogenase (ADH). Mutants of one class were devoid of ADH activity under anaerobic conditions but exhibited aerobic activities comparable to those of wild-type E. coli. Mutants of a second class exhibited ADH activity levels intermediate between those of the wild-type and derepressed parent. Immunological studies showed that mutants of the former class synthesized far less ADH protein than did the derepressed parent while mutants of the latter class synthesized about the same amount. The ADH mutations in both classes were located within the previously described adh region which contains the structural gene for the activity that is derepressed in the parent. An Eth⁻ adh-lac fusion mutant with an insertion in the structural gene was also isolated and characterized. It exhibited no ADH activity under anaerobic conditions and wild-type levels under aerobic conditions. These data are consistent with the existence in E. coli of distinct aerobic and anaerobic ADH enzymes and a derepression of the anaerobic but not the aerobic enzyme in the ethanol utilizing strain.     

Topological Analysis of the Aerobic Membrane-Bound Formate Dehydrogenase of Escherichia coli

Besides formate dehydrogenase N (FDH-N), which is involved in the major anaerobic respiratory pathway in the presence of nitrate, Escherichia coli synthesizes a second isoenzyme, called FDH-O, whose physiological role is to ensure rapid adaptation during a shift from aerobiosis to anaerobiosis. FDH-O is a membrane-bound enzyme complex composed of three subunits, α (FdoG), β (FdoH), and γ (FdoI), which exhibit high sequence similarity to the equivalent polypeptides of FDH-N. The topology of these three subunits has been studied by using blaM (β-lactamase) gene fusions. A collection of 47 different randomly generated Fdo-BlaM fusions, 4 site-specific fusions, and 3 sandwich fusions were isolated along the entire sequence of the three subunits. In contrast to previously reported predictions from sequence analysis, our data suggested that the αβ catalytic dimer is located in the cytoplasm, with a C-terminal anchor for β protruding into the periplasm. As expected, the γ subunit, which specifies cytochrome b, was shown to cross the cytoplasmic membrane four times, with the N and C termini exposed to the cytoplasm. Protease digestion studies of the ³⁵S-labelled FDH-O heterotrimer in spheroplasts add further support to this model. Consistently, prior studies regarding the bioenergetic function of formate dehydrogenase provided evidence for a mechanism in which formate is oxidized in the cytoplasm.     

Formate protects stationary-phase Escherichia coli and Salmonella cells from killing by a cationic antimicrobial peptide

For a sustained infection, enteric bacterial pathogens must evade, resist or tolerate a variety of antimicrobial host defence peptides and proteins. We report here that specific organic acids protect stationary-phase Escherichia coli and Salmonella cells from killing by a potent antimicrobial peptide derived from the human bactericidal/permeability-increasing protein (BPI). BPI-derived peptide P2 rapidly halted oxygen consumption by stationary-phase cells preincubated with glucose, pyruvate or malate and caused a 109-fold drop in cell viability within 90 min of addition. In marked contrast, O2 consumption and viability were not significantly affected in stationary-phase cells preincubated with formate or succinate. Experiments with fdhH, fdoG, fdnG, selC and sdhO mutants indicate that protection by formate and succinate requires their oxidation by the Fdh-N formate dehydrogenase and succinate dehydrogenase respectively. Protection was also dependent on the BipA GTPase but did not require the RpoS sigma factor. We conclude that the primary lesion caused by this cationic peptide is not gross permeabilization of the bacterial cytoplasmic membrane but may involve specific disruption of the respiratory chain. Because P2 shares sequence similarity with a range of other antimicrobial peptides, its cytotoxic mechanism has broader significance. Additionally, protective quantities of formate are secreted by E. coli and Salmonella during growth suggesting that such compounds are important determinants of bacterial survival in the host.     

Properties of recombinant NADP-malate dehydrogenases from Sorghum vulgare leaves expressed in Escherichia coli cells

In this study, a cDNA clone coding for sorghum leaf NADP-malate dehydrogenase [Crétin, C., Luchetta, P., Joly, C., Decottignies, P., Lepiniec, L., Gadal, P., Sallantin, M., Huet, J. C. & Pernollet, J. C. (1990) Eur. J. Biochem. 192, 299-303] was used either in the full-length form or in a shorter form deprived of the 5' end coding for the transit peptide. Both cDNA fragments were cloned into the expression vector pKK233-2 and the resulting constructions were used to transform E. coli cells. The bacterial cells which do not contain any NADP-dependent malate dehydrogenase before transformation were able to express the protein after transformation and induction, as detected both by activity measurements and by immunoblot. The recombinant proteins could be purified to homogeneity and their biochemical characteristics studied. They were identical to those of the enzyme isolated from corn or sorghum leaves, including the well known redox regulatory properties. The NADP-malate dehydrogenases derived from both constructions had a similar subunit size and the analysis of their N-terminal sequences revealed that E. coli cells were able to recognize the processing signal of the precursor polypeptide and to mature and assemble the protein in a manner similar to higher plants.     

Novel beta -hydroxyacid dehydrogenases in Escherichia coli and Haemophilus influenzae

Our laboratory has previously reported a structurally and mechanistically related family of beta-hydroxyacid dehydrogenases with significant homology to beta-hydroxyisobutyrate dehydrogenase. A large number of the members of this family are hypothetical proteins of bacterial origin with unknown identity in terms of their substrate specificities and metabolic roles. The Escherichia coli beta-hydroxyacid dehydrogenase homologue corresponding to the locus was cloned and expressed with a 6-histidine tag for specific purification. The purified recombinant protein very specifically catalyzed the NAD(+)-dependent oxidation of d-glycerate and the NADH-dependent reduction of tartronate semialdehyde, identifying this protein as a tartronate semialdehyde reductase. Further evidence for identification as tartronate semialdehyde reductase is the observation that the coding region for this protein is directly preceded by genes coding for hydroxypyruvate isomerase and glyoxylate carboligase, two enzymes that synthesize tartronate semialdehyde, producing an operon clearly designed for d-glycerate biosynthesis from tartronate semialdehyde. The single beta-hydroxyacid dehydrogenase homologue from Haemophilus influenzae was also cloned, expressed, and purified with a 6-histidine tag. This protein also catalyzed the NAD(+)-dependent oxidation of d-glycerate but was significantly more efficient in the oxidation of four-carbon beta-hydroxyacids like d-hydroxybutyrate and d-threonine. This enzyme differs from all the presently known beta-hydroxybutyrate dehydrogenases which are well established members of the short chain dehydrogenase/reductase superfamily.     

Chromogenic assessment of the three molybdo-selenoprotein formate dehydrogenases in Escherichia coli

Escherichia coli synthesizes three selenocysteine-dependent formate dehydrogenases (Fdh) that also have a molybdenum cofactor. Fdh-H couples formate oxidation with proton reduction in the formate hydrogenlyase (FHL) complex. The activity of Fdh-H in solution can be measured with artificial redox dyes but, unlike Fdh-O and Fdh-N, it has never been observed by chromogenic activity staining after non-denaturing polyacrylamide gel electrophoresis (PAGE). Here, we demonstrate that Fdh-H activity is present in extracts of cells from stationary phase cultures and forms a single, fast-migrating species. The activity is oxygen labile during electrophoresis explaining why it has not been previously observed as a discreet activity band. The appearance of Fdh-H activity was dependent on an active selenocysteine incorporation system, but was independent of the [NiFe]-hydrogenases (Hyd), 1, 2 or 3. We also identified new active complexes of Fdh-N and Fdh-O during fermentative growth. The findings of this study indicate that Fdh-H does not form a strong complex with other Fdh or Hyd enzymes, which is in line with it being able to deliver electrons to more than one redox-active enzyme complex.     

Immunochemical properties of NAD+-linked glycerol dehydrogenases from Escherichia coli and Klebsiella pneumoniae.

An NAD+-linked glycerol dehydrogenase hyperproduced by a mutant of Escherichia coli K-12 was found to be immunochemically homologous to a minor glycerol dehydrogenase of unknown physiological function in Klebsiella pneumoniae 1033, but not to the glycerol dehydrogenase of the dha system responsible for anaerobic dissimilation of glycerol or to the 2,3-butanediol dehydrogenase of K. pneumoniae.     

Oxidation-reduction properties of two engineered redox-sensitive mutant Escherichia coli malate dehydrogenases

Redox potentials for two inactivating intrasubunit disulfides that link helix-5 and helix-9 in mutant Escherichia coli malate dehydrogenases have been determined. The Em is -285 mV when cysteines are at positions 121 and 305 and -295 mV when the cysteines are at positions 122 and 305. Oxidation to the disulfide affects kcat but not Km values. In the single V121C and N122C mutants, the Cys in helix-5 affects the Km for oxalacetate. The pH optimum in the direction of malate formation is affected by the redox state of the enzyme. Clearly, a disulfide bond can and does form between Cys residues substituted into positions 121 or 122 in the nucleotide binding domain and 305 in the carbon substrate binding domain of this NAD-dependent malate dehydrogenase. Apparently, crosslinking the domains interferes with catalysis.     

Structural and Functional Features of Formate Hydrogen Lyase, an Enzyme of Mixed-Acid Fermentation from Escherichia coli

Formate hydrogen lyase from Escherichia coli is a membrane-bound complex that oxidizes formic acid to carbon dioxide and molecular hydrogen. Under anaerobic growth conditions and fermentation of sugars (glucose), it exists in two forms. One form is constituted by formate dehydrogenase H and hydrogenase 3, and the other one is the same formate dehydrogenase and hydrogenase 4; the presence of small protein subunits, carriers of electrons, is also probable. Other proteins may also be involved in formation of the enzyme complex, which requires the presence of metal (nickel-cobalt). Its formation also depends on the external pH and the presence of formate. Activity of both forms requires F(0)F(1)-ATPase; this explains dependence of the complex functioning on proton-motive force. It is also possible that the formate hydrogen lyase complex will exhibit its own proton-translocating function.     

Formate accumulation due to DNA release in aerobic cultivations of Escherichia coli.

Three different aerobic fed-batch processes of Escherichia coli were studied, two for the production of a recombinant protein and one process with a wild-type E. coli strain. In all three processes, an accumulation of formate could be observed in the latter part of the process. Analysis of the concentration of DNA in the medium revealed that the release of DNA coincided with the accumulation of formate. It was found that increasing concentrations of DNA correlated in almost linearly increasing concentrations of formate. Formate accumulation is caused by mixed acid fermentation, although no oxygen limitation was measured with the DO electrode. It is proposed that extracellular DNA restrained mass transfer between the bulk medium and the cell. To investigate if the DNA accumulation caused formate production, DNA was removed by continuous feeding of a DNA binding polymer to the medium. The addition of the polymer decreased the content of free DNA in the broth and the formate was reassimilated. Furthermore, additional DNA early in the process resulted in early formate accumulation.     

Effects of Molybdate and Selenite on Formate and Nitrate Metabolism in Escherichia coli

The effects of adding molybdate and selenite to a glucose-minimal salts medium on the formation of enzymes involved in the anaerobic metabolism of formate and nitrate in Escherichia coli have been studied. When cells were grown anaerobically in the presence of nitrate, molybdate stimulated the formation of nitrate reductase and a b-type cytochrome, resulting in cells that had the capacity for active nitrate reduction in the absence of formate dehydrogenase. Under the same conditions, selenite in addition to molybdate was required for forming the enzyme system which permits formate to serve as an effective electron donor for nitrate reduction. When cells were grown anaerobically on a glucose-minimal salts medium without nitrate, active hydrogen production from formate as well as formate dehydrogenase activity depended on the presence of both selenite and molybdate. The effects of these metals on the formation of formate dehydrogenase was blocked by chloramphenicol, suggesting that protein synthesis is required for the increases observed. It is proposed that the same formate dehydrogenase is involved in nitrate reduction, hydrogen production, and in aerobic formate oxidation.     

A sulfurtransferase is essential for activity of formate dehydrogenases in Escherichia coli

l-Cysteine desulfurases provide sulfur to several metabolic pathways in the form of persulfides on specific cysteine residues of an acceptor protein for the eventual incorporation of sulfur into an end product. IscS is one of the three Escherichia coli l-cysteine desulfurases. It interacts with FdhD, a protein essential for the activity of formate dehydrogenases (FDHs), which are iron/molybdenum/selenium-containing enzymes. Here, we address the role played by this interaction in the activity of FDH-H (FdhF) in E. coli. The interaction of IscS with FdhD results in a sulfur transfer between IscS and FdhD in the form of persulfides. Substitution of the strictly conserved residue Cys-121 of FdhD impairs both sulfur transfer from IscS to FdhD and FdhF activity. Furthermore, inactive FdhF produced in the absence of FdhD contains both metal centers, albeit the molybdenum cofactor is at a reduced level. Finally, FdhF activity is sulfur-dependent, as it shows reversible sensitivity to cyanide treatment. Conclusively, FdhD is a sulfurtransferase between IscS and FdhF and is thereby essential to yield FDH activity.     

Comparison of the fermentative alcohol dehydrogenases of Salmonella typhimurium and Escherichia coli

The adhE gene, encoding the fermentative alcohol dehydrogenase, from Salmonella typhimurium (Genbank accession number U68173) was cloned and sequenced. The Salmonella AdhE protein has 619/878 (70%) amino acid residues identical to the AdhE protein of Escherichia coli. Salmonella AdhE was synthesized only anaerobically. It was present in higher amounts when cells were grown on reduced substrates such as sorbitol, instead of glucose. Growth on glucuronate, which generated no net nicotinamide-adenine dinucleotide reduced (NADH) during metabolism, showed the lowest AdhE levels. Analysis of fermentation products by in vivo nuclear magenetic resonance showed that the proportion of ethanol was highest with sorbitol, intermediate with glucose and negligible with glucuronate. The Salmonella enzyme had a lower Michaelis-Menten constant (Km) for alcohol substrates than AdhE of E. coli although both enzymes displayed a similar Km for nicotinamide-adenine dinucleotide (NAD+). Although AdhE of E. coli was inactive with alcohols longer than four carbons, the Salmonella enzyme was still active with alcohols up to eight carbons.     

Simultaneous reconstitution of Escherichia coli membrane vesicles with D-lactate and D-amino acid dehydrogenases

Purified preparations of D-amino acid dehydrogenase [Olsiewski, P.J., Kaczorowski, G. J., & Walsh, C. T. (1980) J. Biol. Chem. 225, 4487] and D-lactate dehydrogenase [Kohn, L.D., & Kaback, H.R. (1973) J. Biol. Chem. 248, 7012] bind independently to right-side-out and inverted Escherichia coli vesicles and to phosphatidylcholine liposomes without detectable competition. The reconstituted vesicles catalyze D-lactate- and D-alanine-dependent respiration (O2 uptake), proton translocation, and proton/lactose symport. The enzymes do not share common sites of association on either face of the E. coli membrane, and binding of both enzymes to the bilayer appears to be due to nonspecific affinity for the surface rather than specific binding to proteinaceous receptors. Each enzyme, however, appears to reduce a common proton translocating step in the membrane-bound respiratory chain, and substrate-derived electrons are transferred through a common rate-determining redox component that precedes the site of proton translocation. The results suggest that although binding is nonspecific, there is a common site for proton translocation in the membrane between the flavin-linked dehydrogenases and the cytochromes and that this site is accessible by distinct routes of electron transfer from primary dehydrogenases on either surface of the membrane.