Formic Dehydrogenase Research Articles

Janus Reactors with Highly Efficient Enzymatic CO2 Nanocascade at Air-Liquid Interface.

Though enzymatic cascade reactors have been the subject of intense research over the past few years, their application is still limited by the complicated fabrication protocols, unsatisfactory stability and lack of effective reactor designs. In addition, the spatial positioning of the cascade reactor has so far not been investigated, which is of significant importance for biphase catalytic reaction systems. Inspired by the Janus properties of the lipid cellular membrane, here we show a highly efficient Janus gas-liquid reactor for CO2 hydration and conversion. Within the Janus reactor, nanocascades containing the nanoscale compartmentalized carbonic anhydrase and formic dehydrogenase were positioned at a well-defined gas-liquid interface, with a high substrate concentration gradient. The Janus reactor exhibited 2.5 times higher CO2 hydration efficiency compared with the conventional gas-liquid contactor with pristine membranes, and the formic acid conversion rate can reach approximately 90%. Through this work, we provide evidence that the spatial arrangement of the nanocascade is also crucial to efficient reactions, and the Janus reactor can be a promising candidate for the biphase catalytic reactions in environmental, biological and energy aspects.

Enzymatic assay of formic acid and gas chromatography of methanol for urinary biological monitoring of exposure to methanol

An enzymatic assay method for the determination of urinary formic acid is described. Formic acid in urine was cleaved to carbon dioxide and water by formic acid dehydrogenase, whereby NAD+ was converted to NADH, which reacted with INT (p-iodonitrotetrazolium violet) in the presence of NAD-diaphorase. The color thus produced was determined at 500 nm. In addition, a simple gas chromatographic method of urinary formic acid is described, in which head space gas of formic acid methylester was applied into the wide bore column. The urinary formic acid concentrations by the enzymatic method agreed well with that by the gas chromatographic method. A simple gas chromatographic method for urinary methanol assay is also described. Acetonitrile was added to an equal volume of urine containing methanol. After centrifugation, the supernatant was injected into gas chromatography (GC). The peaks of urinary methanol and ethanol were separated by GC. Formic acid and methanol in urine of unexposed healthy subjects and workers exposed to methanol were analyzed by the colorimetric and gas chromatographic methods. Geometric mean concentrations of urinary formic acid and methanol in the healthy subjects were 7.82 mg/g creatinine and 1.34 mg/l, respectively. The concentration ratio of formic acid to methanol in the urine of the workers exposed to methanol was calculated to be 3.67 +/- 2.10, which agreed with the ratio under a controlled exposure experiment. A slower excretion of formic acid than that of methanol in the urine of a volunteer was also observed.

The seleno-polypeptide of formic dehydrogenase (formate hydrogen-lyase linked) from Escherichia coli: genetic analysis.

The site of integration of phage M mu d (Ap lac) in mutant M9s which leads to deficiency of formic dehydrogenase (benzylviologen-linked) activity was determined. It was shown that the phage had inserted into the gene for the seleno-polypeptide of the enzyme (80 kd) leading to the formation of a truncated peptide (60 kd) still able to incorporate Se. Synthesis of the truncated polypeptide is subject to the same regulatory signals as that of the wild-type enzyme. The formation of the 110 kd seleno-polypeptide, which is a constituent component of the formic dehydrogenase from the formate-nitrate respiratory pathway, is unimpaired in mutant M9s. The location of the gene for the 80 kd seleno-polypeptide was mapped at 92.4 min of the Escherichia coli chromosome.

Mapping of chlorate-resistant mutants of Citrobacter freundii by deletion and complementation analysis

From Citrobacter freundii mutants have been isolated, with deletions extending chl genes. 53% of these mutants mapped in the gal-bio region of the chromosome. Genetic mapping by three methods—deletion analysis, linkage analysis in crosses with C. freundii Hfr donors and complementation with Escherichia coli F′ factors—establishes the gene order: chl H-gal-chl D-hut-bio-uvr B-chl A-chl I-chl E In an other segment a gene order ilv-chl-pdx was found. Furthermore chl loci were found adjacent to 7 different chromosomal markers. C. freundii can form nitrate reductase A, thiosulfate reductase, tetrathionate reductase and formic dehydrogenase. These enzymes are not formed in most of the chlorate-resistant mutants. In some of these mutants the enzyme activities can be restored by complementation with F′ factors of E. coli.

Isolation and phenotypes of mutants from Salmonella typhimurium defective in formate hydrogenlyase activity.

A new method is described for the isolation of different mutants defective in formic dehydrogenase activity measured with benzyl viologen as electron acceptor.

Effects of Selenium Compounds on Formate Metabolism and Coincidence of Selenium-75 Incorporation and Formic Dehydrogenase Activity in Cell-Free Preparations of Escherchia coli

The effects of selenite, selenocystine, and selenomethionine in a defined growth medium on formic dehydrogenase biosynthesis in aerobically and anaerobically grown Escherichia coli have been studied. Sucrose gradient centrifugation of a partially purified enzyme demonstrated a coincidence of (75)Se incorporation and formic dehydrogenase activity.

Alterations in the Cytoplasmic Membrane Proteins of Various Chlorate-Resistant Mutants of Escherichia coli

Chlorate-resistant mutants corresponding to each known genetic locus (chlA, chlB, chlC, chlD, chlE) were isolated from Escherichia coli K-12. All these mutants showed decreased amounts of membrane-bound nitrate reductase, cytochrome b, and formic dehydrogenase, but all had normal succinic dehydrogenase activity. Proteins from the cytoplasmic membranes of these mutants were compared to those of the wild type-on polyacrylamide gels. The addition of nitrate to wild-type anaerobic cultures caused increased formation of three membrane proteins. These same proteins, along with one other, were missing in varying patterns in mutants altered at the different genetic loci. One of the missing proteins was found to be the enzyme nitrate reductase, although this protein was present in some mutants lacking nitrate reductase activity. None of the others has been identified.

Isolation of plaque forming λ phages which carry bacterial genes involved in the functional organization of the cell membrane

Mutants of the bacteriophage lambda have been isolated which can form plaques as well as transduce a series of bacterial loci. Included amongst the loci carried by the phages are the chlA genes which control membrane components involved in the nitrate respiration complex of E. coli . By using the appropriate chlA deletion mutants and λpchlA phage, it was possible to transduce control of formic dehydrogenase activity with the chlA genetic locus.

The hydrogen lyase multienzyme system of Escherichia coli K 12 and of its chlorate-resistant mutants

The hydrogen lyase system of Escherichia coli K12 which decomposed formate to hydrogen and carbon dioxide involves two particulate enzymes, formate dehydrogenase and hydrogenase, and a soluble component.Formate dehydrogenase, as measured using phenazine methosulfate as electron acceptor, has been localized in particles bearing nitrate reductase activity. Formate dehydrogenase is synthesized throughout anaerobic growth and is not repressed by KNO3. This behavior is unlike that of the soluble fraction which does not appear until the end of exponential growth and which can only be considered as an electron carrier.Particulate preparations from cells of the E. coli K12 pleotropic mutants chl A− and chl B− do not have formic dehydrogenase activity, but if the supernatants of these same cells are added to particulate fractions of the wild type, the mixture produces H2 from formate. These results agree with our earlier observations suggesting that these pleiotropic mutations affect particle structure.

A genetical and biochemical study of chlorate-resistant mutants of Salmonella typhimurium.

S.typhimurium can form nitrate reductase A, chlorate reductase C, thiosulfate reductase, tetrathionate reductase and formic dehydrogenase. None of these enzymes are formed in chlorate-resistant mutants. Conjugation experiments showed the presence of a strong linkage between thechl andgal markers of the bacterial chromosome. By deletion mapping the gene ordernic A aro G gal bio chl D uvr B chl A was found. Strains with deletions terminating betweenbio anduvr B or betweenuvr B andchl A have a number of aberrant properties. Though resistant against chlorate they reduce nitrate and form gas. After growth with nitrate they form less nitrate reductase than the wild type which may explain the resistance against chlorate. After growth with thiosulfate they form small amounts of thiosulfate reductase and chlorate reductase C. In crosses between anE.coli Hfrchl + strain and aS.typhimurium chl A strain recombinants were obtained, forming nitrate reductase A and chlorate reductase C. These recombinants do not form gas, which indicates that thechl + gene fromE.coli does not function normally inS.typhimurium.

Formic hydrogenlyase and the photoassimilation of formate by a strain of Rhodopseudomonas palustris.

A photosynthetic bacterium isolated by enrichment on media containing formate as major source of cell carbon was identified as a strain of Rhodopseudomonas palustris. It grew on a wide range of simple organic compounds including alcohols, fatty acids, and hydroxyacids, on a chemically defined medium with biotin and p-aminobenzoic acid as essential growth factors. The organism grew on formate or photoautotrophically with molecular hydrogen or thiosulfate only in the presence of yeast extract. Ability to photoassimilate formate could be shown only in organisms grown in the presence of formate. The organism contained an inducible formic hydrogenlyase consisting of a soluble formic dehydrogenase, a particulate hydrogenase, and one or more intermediate, but as yet unidentified, electron carriers. The formic hydrogenlyase could be reconstituted from a particulate hydrogenase and a partially purified soluble formic dehydrogenase. Some properties of the formic dehydrogenase and hydrogenase have been compared with that of the formic hydrogenlyase system.

ELECTROPHORETIC MOBILITIES OF FORMIC AND GLUTAMIC DEHYDROGENASES IN THE FABACEAE: A SYSTEMATIC SURVEY

SummaryThe albumins extracted from the seeds of a number of different species of the Fabaceae have been subjected to disc electrophoresis on acrylamide gel. Both formic and glutamic dehydrogenase activities were readily detectable on the gels for ninety of the 103 species examined and one or other of the dehydrogenases on gels of the remaining species.The mobility values of the dehydrogenases varied among the different taxa examined. The more morphologically homogeneous the taxon the less variable were the enzymic data of the species within it, thus within the Vicieae, Trifolieae, Loteae and tribal groupings of Bentham's Phaseoleae species had more nearly similar formic dehydrogenase mobilities than did species of the Podalyrieae and Genisteae (sensu lato).Before any taxonomic conclusions can be drawn a much more extensive survey is necessary; however, sufficient correlations were observed between formic dehydrogenase mobilities and the globulin data of the previous paper to indicate the potential usefulness of this method in systematic studies.

The inter-relationships of low redox potential cytochrome and hydrogenase in facultative anaerobes

1. 1. Cytochrome c552 and hydrogenase (EC 1.12.1.1) activities have been compared in Escherichia coli and Escherichia aurescens cells grown aerobically and anaerobically in both complex and defined media. 2. 2. Sodium formate added to the growth media stimulates synthesis of increased amounts of cytochrome c552. 3. 3. Extensive washing in 0.1 M phosphate buffer (pH 7.4), extensive sonication, and washing with 0.2% w/v sodium deoxycholate all solubilise cytochrome c from hydrogenase-active membranes. 4. 4. Residues from the above procedures still show hydrogenase activity. 5. 5. Cells grown anaerobically with nitrate s terminal electron acceptor contain greatly enhanced amounts of cytochrome: these cells do not have hydrogenase, hydrogenlyase or soluble viologen-linked formic dehydrogenase activities. The relationship of cytochrome c552 to hydrogenase is discussed.

Pigment production from tryptophan by an Achromobacter species.

Duerre, John A. (University of North Dakota, Grand Forks), and Patrick J. Buckley. Pigment production from tryptophan by an Achromobacter species. J. Bacteriol. 90:1686-1691. 1965.-A microorganism was isolated from the soil near the University of North Dakota. Biochemical and morphological characteristics indicated that this organism would best be classified as a member of the family Achromobacteraceae, genus Achromobacter, species unknown. The organism produced a red pigment when grown in a medium containing yeast extract and tryptophan. The pH optimum for pigment production was about 8.0 and the optimal temperature was 25 C. During a study of the nutritional requirements for growth and pigment production, it was found that the organism would grow and produce pigment in a medium containing tryptophan and nucleosides, but the rate of both growth and pigment formation in this medium was slower than that observed with tryptophan and yeast extract. The organism grew well in the presence of acid-hydrolyzed casein and nucleosides without producing pigment, indicating that the pigment is not necessary for growth. Resting-cell experiments definitely established tryptophan as the sole exogenous requirement for pigment production. The pigment was extracted from yeast extract-tryptophan medium with chloroform. Thin layer chromatographic analysis of the crude pigment extracted from this medium revealed the presence of two other pigments in addition to the major red pigment. One of these was a highly fluorescent orange pigment and the other a pink pigment. Only the red pigment was produced by resting cells in the presence of tryptophan alone. This pigment served as an electron acceptor when coupled with formic dehydrogenase, indicating its possible function as an oxidation-reduction pigment. The oxidized pigment had absorption peaks at 506 and 304 mmu. The peak at 506 mmu disappeared upon reduction with sodium sulfite. Shaking the reduced pigment in air proved to be an unsatisfactory method for returning the reduced pigment to the oxidized, colored state.

Induced Biosynthesis of Formic Hydrogenlyase in Iron-Deficient Cells of Escherichia coli

Fukuyama, T. (University of Washington, Seattle), and E. J. Ordal. Induced biosynthesis of formic hydrogenlyase in iron-deficient cells of Escherichia coli. J. Bacteriol. 90:673-680. 1965.-Escherichia coli cells were grown aerobically on a lactate-mineral salts medium from which iron had been removed by extraction with 8-hydroxyquinoline and chloroform. These cells carried out induced biosynthesis of formic hydrogenlyase in a reaction mixture containing glucose, formate, and phosphate without the addition of amino acids, providing adequate amounts of iron salts were present. In the absence of iron, glucose was fermented and acids were produced, but no formic hydrogenlyase developed. When iron-deficient E. coli cells were repeatedly washed, the property of carrying out induced biosynthesis of formic hydrogenlyase with glucose, formate, phosphate, and iron was lost, but was restored on addition of acid-hydrolyzed casein to the reaction mixture. An energy source (provided as glucose) was necessary for enzyme production. Iron-deficient cells were devoid of hydrogenase and formic hydrogenlyase but showed formic dehydrogenase activity when adequate amounts of selenium and molybdenum were present in the growth medium. Hydrogenase was consistently absent in iron-deficient cells but appeared concomitantly with formic hydrogenlyase during induced biosynthesis of the latter in iron-deficient cells of E. coli.

The utilization of carbon-1 compounds by plants. II. The formation and metabolism of formate by higher plant tissues.

Tissue slices of carrot root and sunflower cotyledons readily produce formate-C14when incubated with glyoxylate-1,2-C14and glycine-2-C14solutions. Formate-C14feeding experiments using a wide variety of higher plant tissues have shown that this compound is readily oxidized to carbon dioxide. This oxidation probably involves a formic dehydrogenase system which has been demonstrated to occur in several of the tissues. Large amounts of formate-C14were also incorporated into serine, methionine, and methionine sulfoxide. Degradations of the serine produced from formate-C14showed that the bulk of the C14was present in the 3 position. The incorporation of formate into serine by radish cotyledons was stimulated under anaerobic conditions and by additions of glycine. Synthesis of methionine and methionine sulfoxide from formate-C14was stimulated by additions of homocysteine. The results are interpreted as an indication that formate-C14can be oxidized to carbon dioxide by formic dehydrogenase and can serve as a precursor of carbon-1 units that are to be utilized for the biosynthesis of serine and methionine.

Electron microscopy of the localization of formic dehydrogenase in regenerating forms ofProteus vulgaris

The localization of the formic dehydrogenase system was studied by means of nitro-blue tetrazolium, using the ultrathin section technique, in large long bodies ofProteus vulgaris, which form the intermediate stage in regeneration of penicillin-induced spheroplasts. In the electron microscope, empty areas, numbering on an average four per cell section, were found at the site of formazan deposits, while in the controls (without tetrazolium salts) the average number was 0.05. From these findings, and from the findings of other authors showing that formic dehydrogenase is bound to cell structures, it can be concluded that inProteus cells this enzyme is bound to the cytoplasmic membrane.

Electron-transport system of Vibrio succinogenes: I. Enzymes and cytochromes of the electron-transport system

Enzyme systems responsible for the oxidation of formate and H2 by fumarate and O2 have been demonstrated and partially characterized in cell-free extracts of the cytochrome containing anaerobic vibrio, Vibrio succinogenes. The formic dehydrogenase and hydrogenase are similar to those found in Escherichia coli. Oxidase and peroxidase systems involving formate and H2 were demonstrated. Pyridine nucleotides are not involved in any of these systems. During the oxidation of formate by O2, small amounts of H2O2 are formed.Cytochromes of the b and c types are shown to be present in cells grown with formate as an energy source and either fumarate or nitrate as an electron acceptor. Some of the chemical characteristics of the cytochromes were determined. The protoheme of the cytochrome of the b type was isolated and identified. The cytochrome of the c type was partially reduced by ascorbate. Both cytochromes could be reduced by H2 or formate and the cytochrome of the b type could be oxidized by fumarate suggesting a catalytic role for this cytochrome in fumarate reduction. Both cytochromes were partially reduced by succinate.

Electron-transport system of Vibrio succinogenes: II. Inhibition of electron transport by 2-heptyl-4-hydroxyquinoline N-oxide

The effect of the inhibitor 2-heptyl-4-hydroxyquinoline N-oxide on the reduction by formate of fumarate, O2 and cytochromes was studied in extracts of Vibrio succinogenes. The inhibitor causes 90% inhibition of the couple between formate and fumarate but less than 10% inhibition of the formic dehydrogenase or fumarate reductase activity. Spectrophotometric studies showed that 2-heptyl-4-hydroxyquinoline N-oxide causes an appreciable delay in the reduction of both cytochromes b and c by formate. No effect of the inhibitor on the oxidation of reduced cytochrome b by fumarate could be detected. With succinate as substrate, the inhibitor causes a delay in the reduction of cytochrome c but no delay in the reduction of cytochrome b was detected. The effect of other inhibitory agents on the reduction of fumarate by formate and H2 was also studied.Oxygen consumption with formate as substrate is not inhibited by 2-heptyl-4-hydroxyquinoline N-oxide, but a marked increase in H2O2 formation occurs in the presence of this inhibitor. The formate peroxidase system of this extract is partially inhibited by 2-heptyl-4-hydroxyquinoline N-oxide.The implications of these findings with regard to the role of cytochrome b as an electron carrier between the formic dehydrogenase and the fumarate reductase, and the role of the cytochromes in the oxidation of formate by O2 are discussed. It is suggested that the presence of the H2O2-producing formate oxidase system and the formate peroxidase system explains the microaerophilic nature of this organism when grown with O2 as an electron acceptor.

Carbohydrate Metabolism of Trichomonads: Growth, Respiration, and Enzyme Activity in Four Species*

Tritrichomonas foetus, Pentatrichomonas gallinarum, and nasal and fecal forms of Trichomonas suis grew well in NIH Thioglycollate Broth with 1% beef serum, and produced acid end‐products. Succinic acid was the major acid produced by T. foetus P. gallinarum, and the nasal T. suis, accounting for over 50% of the total acid for each. The fecal T. suis produced more lactic than succinic acid; about 45% of the total acid was not accounted for. Pyruvic acid was found in small amounts in all cultures. The lactic/pyruvic ratio was about 2.5 for the two swine forms, and about 1.0 for the other two. The nasal form of T. suis produced much more total acid than the others.O2 uptake, CO2 production, H2 evolution, and anaerobic acid formation varied with age of organism. Respiratory activity was highest at 12 hours but varied considerably thereafter. The nature of the buffer used in manometric experiments also affected the results.Glucose, mannose, fructose and galactose most stimulated respiration. Disaccharides were slowly utilized (lactose and sucrose were not used by P. gallinarum). In a few cases lactate, pyruvate, formate, and malate stimulated O2 uptake.Evidence was obtained for hexokinase, phosphohexoisomer‐ase, phosphofructokinase, aldolase, and glucose‐6‐phcsphate dehydrogenase in all 4 organisms. Phosphoglucomutase was demonstrated in T. foetus and the fecal T. suis. T. foetus and P. gallinarum showed evidence of phosphoglyceromutase and enolase. T. suis (fecal) and P. gallinarum had malic dehydrogenase, and P. gallinarum gave evidence of “malic enzyme” activity. Formic dehydrogenase activity was marked in all and presumptive evidence for a formic hydrogenlyase system was obtained for T. foetus and P. gallinarum.Study of reactions involving pyruvate and malate (with a possible linkage to a hexose monophosphate shunt) might perhaps provide a key to a better understanding of trichmonad metabolism.