Nitrogen Redox Metabolism of a Heterotrophic, Nitrifying-Denitrifying Alcaligenes sp. from Soil

Metabolic characteristics of a heterotrophic, nitrifier-denitrifier Alcaligenes sp. isolated from soil were further characterized. Pyruvic oxime and hydroxylamine were oxidized to nitrite aerobically by nitrification-adapted cells with specific activities (V_max) of 0.066 and 0.003 μmol of N × min⁻¹ × mg of protein⁻¹, respectively, at 22°C. K_m values were 15 and 42 μM for pyruvic oxime and hydroxylamine, respectively. The greater pyruvic oxime oxidation activity relative to hydroxylamine oxidation activity indicates that pyruvic oxime was a specific substrate and was not oxidized appreciably via its hydrolysis product, hydroxylamine. When grown as a denitrifier on nitrate, the bacterium could not aerobically oxidize pyruvic oxime or hydroxylamine to nitrite. However, hydroxylamine was converted to nearly equimolar amounts of ammonium ion and nitrous oxide, and the nature of this reaction is discussed. Cells grown as heterotrophic nitrifiers on pyruvic oxime contained two enzymes of denitrification, nitrate reductase and nitric oxide reductase. The nitrate reductase was the dissimilatory type, as evidenced by its extreme sensitivity to inhibition by azide and by its ability to be reversibly inhibited by oxygen. Cells grown aerobically on organic carbon sources other than pyruvic oxime contained none of the denitrifying enzymes surveyed but were able to oxidize pyruvic oxime to nitrite and reduce hydroxylamine to ammonium ion.     

Oxidative and hydrolytic transformations of hydroxylamine compounds in cyanobacteria

Ammonia-incubated cyanobacteria liberated H₂O₂, accumulated hydroxylamine compounds and nitrite and catalyzed dismutation of hydroxylamine as well as oxidations of ammonia, glutamine, and oximes. Ethyl acetohydroximate-adapted Phormidium released excess H₂O₂ and phototrophically metabolized the oxime via hydrolysis and dismutation to nitrite and ammonia, which were consumed by nitrite reductase and glutamine synthetase. Added ammonia stimulated H₂O₂ production and oxime metabolism via glutamate dehydrogenase pathway.Abbreviations EAH Ethyl acetohydroximate - GDH glutamate dehydrogenase - GS glutamine synthetase - NiR nitrite reductase     

An iron dioxygenase from Alcaligenes faecalis catalyzing the oxidation of pyruvic oxime to nitrite

Abstract An enzyme which participated in the oxidation of hydroxylamine to nitrite from was partially purified Alcaligenes faecalis , and some of its properties were studied. The enzyme oxidized aerobically pyruvic oxime to nitrite in the presence of hydroxylamine or ascorbate. As molecular oxygen equimolar to nitrite formed was consumed in the enzymatic oxidation of pyruvic oxime to nitrite, the enzyme was thought to be a dioxygenase. It was an iron protein, and a reducing reagent was required to keep the iron in the ferrous state for the action of the enzyme.     

Studies on the oxidation of ammonia by Nitrosomonas

1. Free-energy calculations for pH7 showed that the oxidation of ammonia to hydroxylamine is endergonic and that the oxidations of hydroxylamine to nitrite and hydrazine to nitrogen are exergonic. It is suggested that the oxidation of ammonia requires the expenditure of energy. 2. The anaerobic dehydrogenation of hydrazine to nitrogen by extracts of the autotrophic nitrifying micro-organism, Nitrosomonas, in the presence of methylene blue as electron acceptor, was less rapid than the anaerobic dehydrogenation of hydroxylamine to nitric oxide. The inhibition by hydrazine of the dehydrogenation of hydroxylamine was attributed to substrate competition. 3. Whole cells in air did not produce nitrite from hydrazine. They produced nitrite from low concentrations of hydroxylamine more rapidly than from equimolar concentrations of ammonia; this result is consistent if hydroxylamine is an intermediate of the oxidation of ammonia. 4. The production of nitrite from hydroxylamine by whole cells was slightly inhibited by hydrazine, but the production of nitrite from ammonia was greatly inhibited and small amounts of hydroxylamine were formed. These results suggested that the dehydrogenation of hydroxylamine supplied energy required for the oxidation of ammonia and that hydroxylamine appeared because the energy production was replaced by that of the dehydrogenation of hydrazine. 5. The oxidation of hydroxylamine by whole cells was not inhibited by thiourea, but micromolar concentrations of the metal-binding agent markedly inhibited the oxidation of ammonia to hydroxylamine, suggesting that the oxidation of ammonia involved copper. A possible mechanism for the activation of ammonia is suggested.     

Heterotrophic nitrification among denitrifiers.

Twelve denitrifying bacteria representing six genera were tested for an ability to nitrify pyruvic oxime heterotrophically. Six of these bacteria exhibited appreciable nitrification activity, yielding as much as 5.8 mM nitrite and little or no nitrate when grown in a mineral salts medium containing 7 mM pyruvic oxime and 0.05% yeast extract. Of the six active bacteria, four (Pseudomonas denitrificans, Pseudomonas aeruginosa, and two strains of Pseudomonas fluorescens) could grow on yeast extract but not pyruvic oxime, one (Pseudomonas aureofaciens) could grow slowly on pyruvic oxime, and one (Alcaligenes faecalis) could apparently grow on pyruvic oxime in the presence of yeast extract but not in its absence. Eight of the twelve bacteria in the resting state could oxidize hydroxylamine to nitrite, and P. aureofaciens was remarkably active in this regard. In general, those denitrifiers active in the nitrification of pyruvic oxime or hydroxylamine or both are abundant in soils. A possible advantage of having nitrification and denitrification capabilities in the same organism is discussed.     

Heterotrophic Nitrification by Arthrobacter sp

Arthrobacter sp. isolated from sewage oxidized ammonium to hydroxylamine, a bound hydroxylamine compound, a hydroxamic acid, a substance presumed to be a primary nitro compound, nitrite, and nitrate. The concentration of free hydroxylamine-nitrogen reached 15 mug/ml. The identification of hydroxylamine was verified by mass spectrometric analysis of its benzophenone oxime derivative. The bound hydroxylamine was tentatively identified as 1-nitrosoethanol on the basis of its mass spectrum, chemical reactions, and infrared and ultraviolet spectra. Hydroxylamine formation by growing cells was relatively independent of pH, but the accumulation of nitrite was strongly favored in alkaline solutions. The formation of hydroxylamine but not nitrite was regulated by the carbon to nitrogen ratio of the medium. The hydroxamic acid was the dominant product of nitrification in iron-deficient media, but hydroxylamine, nitrite, and 1-nitrosoethanol formation was favored in iron-rich solutions. Heterotrophic nitrification by Arthrobacter sp. was not inhibited by several compounds at concentrations which totally inhibited autotrophic nitrification.     

Identification of the sources of nitrous oxide produced by oxidative and reductive processes in Nitrosomonas europaea

1. Cells of Nitrosomonas europaea produced N(2)O during the oxidation of ammonia and hydroxylamine. 2. The end-product of ammonia oxidation, nitrite, was the predominant source of N(2)O in cells. 3. Cells also produced N(2)O, but not N(2) gas, by the reduction of nitrite under anaerobic conditions. 4. Hydroxylamine was oxidized by cell-free extracts to yield nitrite and N(2)O aerobically, but to yield N(2)O and NO anaerobically. 5. Cell extracts reduced nitrite both aerobically and anaerobically to NO and N(2)O with hydroxylamine as an electron donor. 6. The relative amounts of NO and N(2)O produced during hydroxylamine oxidation and/or nitrite reduction are dependent on the type of artificial electron acceptor utilized. 7. Partially purified hydroxylamine oxidase retained nitrite reductase activity but cytochrome oxidase was absent. 8. There is a close association of hydroxylamine oxidase and nitrite reductase activities in purified preparations.     

Vigorous denitrification by a heterotrophic nitrifier of the genusAlcaligenes

A heterotrophic nitrifyingAlcaligenes sp., previously isolated from soil and shown to be very active in the aerobic oxidation of pyruvic oxime (and hydroxylamine) to nitrite, is now shown to be quite active as a denitrifier. The bacterium synthesized nitrite, nitrous oxide, and nitrogen gas from nitrate when grown anaerobically and could individually reduce nitrate, nitrite, nitric oxide, and nitrous oxide to nitrogen gas when these nitrogen-oxides were added to dense cell suspensions. No evidence was obtained for the release of nitric oxide during reduction of nitrate and nitrite. The specific rates of reduction of the nitrogen-oxide were similar to those of well-known laboratory strains of denitrifying bacteria. The induction of an entire set of denitrifying enzymes at normal levels in a heterotrophic nitrifier is novel. The nitrification-denitrification capability ofAlcaligenes sp. may confer certain advantages to this and analogous organisms in the environment.     

Heterotrophic nitrification by Pseudomonas aeruginosa

Summary Several soil bacteria and fungi produce nitrite when provided with acetaldoxime. Nitrite formation by one isolate, identified as a strain of Pseudomonas aeruginosa, is not directly linked to growth but rather proceeds mainly after the active growth period. The added oxime-nitrogen is converted completely to nitrite, and nitrate is not formed. Extracts of the bacterium generate nitrite, but not nitrate, more rapidly from nitroethane than from the added oxime. The enzyme system catalyzing the formation of nitrite in oxime solutions is soluble and inducible, whereas the enzyme catalyzing the release of equimolar quantities of nitrite and acetaldehyde from nitroethane is constitutive. The slow rate of nitrite production when the enzyme preparation is provided with acetaldoxime is not markedly increased by added cofactors. The soluble enzymes also generate nitrite when incubated with several aliphatic and alicyclic oximes and nitro compounds. Nitroethane is not formed from acetaldoxime. The possible mechanism of this nitrification reaction catalyzed by a heterotrophic microorganism is discussed.     

Hydroxylamine reductase enzymes from maize scutellum and their relationship to nitrite reductase

Three enzymes contribute to the total hydroxylamine reductase activity of corn (Zea mays L.) scutellum extracts. Two of these resemble enzymes previously prepared from leaves, while the third, which accounts for a major part of the activity, appears to have no counterpart in leaf tissue. One of the hydroxylamine reductases found only in small amounts is associated with nitrite reductase and is induced, together with nitrite reductase, by nitrite. The other two enzymes are noninducible by nitrite and can be totally separated from nitrite reductase, which subsequently remains capable of catalyzing the reduction of nitrite to ammonia. Possible causes of the decline of hydroxylamine reductase activity during the induction of nitrite reductase are discussed.     

Nitrite reductase of Escherichia coli specific for reduced nicotinamide adenine dinucleotide

Kemp, John D. (University of California, Los Angeles), and Daniel E. Atkinson. Nitrite reductase of Escherichia coli specific for reduced nicotinamide adenine dinucleotide. J. Bacteriol. 92:628-634. 1966.-A nitrite reductase specific for reduced nicotinamide adenine dinucleotide (NADH(2)) appears to be responsible for in vivo nitrite reduction by Escherichia coli strain Bn. In extracts, the reduction product is ammonium, and the ratio of NADH(2) oxidized to nitrite reduced or to ammonium produced is 3. The Michaelis constant for nitrite is 10 mum. The enzyme is induced by nitrite, and the ability of intact cells to reduce nitrite parallels the level of NADH(2)-specific nitrite reductase activity demonstrable in cell-free preparations. Crude extracts of strain Bn will also reduce hydroxylamine, but not nitrate or sulfite, at the expense of NADH(2). Kinetic observations indicate that hydroxylamine and nitrite may both be reduced at the same active site. The high apparent Michaelis constant for hydroxylamine (1.5 mm), however, seems to exclude hydroxylamine as an intermediate in nitrite reduction. In vitro activity is enhanced by preincubation with nitrite, and decreased by preincubation with NADH(2).     

Nitric Oxide Emissions from Soybean Leaves during in Vivo Nitrate Reductase Assays

Recent work identified acetaldehyde oxime as the predominant product purged by inert gases from anaerobic in vivo nitrate reductase (NR) assays of soybean (Glycine max [L.] Merr.) leaves. Another recent study supported earlier research findings which identified the primary product evolved from soybean leaves as nitric oxide (NO). This paper provides evidence that eliminates acetaldehyde oxime and confirms that NO is the primary nitrogenous product purged from the in vivo NR assay system. A portion of the evidence is based on the high water solubility of acetaldehyde oxime. Other evidence presented is the failure by chemical and spectrophotometric means to detect oximes in gases emitted in the purging of the reaction medium or in the leaf tissues. The gaseous product from the in vivo NR assay system reacted identically to NO standards and did not resemble acetaldehyde oxime standards. It was concluded that the predominant N product within the leaves was nitrite and that the predominant gaseous N product evolved from the assay was NO.     

Differentiation of c, d1 cytochrome and copper nitrite reductase production in denitrifiers

Abstract Gas chromatographic analyses revealed that rates of release of nitrous oxide from nitrite or nitric oxide in extracts of the c , d ₁ cytochrome nitrite reductase-producing denitrifiers, Paracoccus denitrificans and Pseudomonas perfectomarina , were unaffected by preincubation with the metal chelator, diethyldithiocarbamate (DDC). In contrast, preincubation with DDC completely inhibited generation of nitrous oxide from nitrite in extracts of copper protein nitrite reductase-producing denitrifiers, " Achromobacter cycloclastes " and Rhodopseudomonas sphaeroides forma species denitrificans . Pre-exposure to DDC lessened but did not completely inhibit nitric oxide reduction in extracts of the copper protein nitrite reductase-producing denitrifiers. Proton consumption values resulting from pulsing with nitrite were similarly completely inhibited by preincubation with DDC of extracts of the two copper protein-producing denitrifiers. Uptake values related to pulsing with nitric oxide were also lessened but not completely inhibited by prior exposure to DDC. As anticipated, proton consumption was not affected by preincubation with DDC in extracts of P. denitrificans pulsed with nitrite or nitric oxide. Differential sensitivity of copper protein nitrite reductase activity to DDC could provide the simple assay method needed for determination of the distribution of two types of nitrite reductase producers among populations of denitrifiers in nature.     

Application of the concept of oxime library screening by mass spectrometry (MS) binding assays to pyrrolidine-3-carboxylic acid derivatives as potential inhibitors of γ-aminobutyric acid transporter 1 (GAT1)

In the present study, the concept of oxime library screening by MS Binding Assays was successfully extended to N-substituted lipophilic pyrrolidine-3-carboxylic acid derivatives in the pursuit of varying the amino acid motif in order to identify new inhibitors for GAT1 and to broaden structure–activity-relationships for this target, the most abundant GABA transporter in the central nervous system. For the screening, 28 different oxime sub-libraries were employed that were generated by simple condensation reaction of an excess of pyrrolidine-3-carboxylic acid derivatives carrying a hydroxylamine functionality with various sub-libraries each assembled of eight aldehydes with broadly varying chemical structures and functionalities. The compounds responsible for the activity of an oxime sub-library were identified by deconvolution experiments performed by employing single oximes. Binding affinities of the oxime hits were confirmed in full-scale competitive MS Binding Assays. Thereby, oxime derivatives with a 1,1′-biphenyl moiety were found as the first inhibitors of mGAT1 comprising a pyrrolidine-3-carboxylic acid motif with affinities in the submicromolar range.     

The partial characterization of purified nitrite reductase and hydroxylamine oxidase from Nitrosomonas europaea

Nitrite reductase has been separated from cell-free extracts of Nitrosomonas and partially purified from hydroxylamine oxidase by polyacrylamide-gel electrophoresis. In its oxidized state the enzyme, which did not contain haem, had an extinction maximum at 590nm, which was abolished on reduction. Sodium diethyldithiocarbamate was a potent inhibitor of nitrite reductase. Enzyme activity was stimulated 2.5-fold when remixed with hydroxylamine oxidase, but was unaffected by mammalian cytochrome c. The enzyme also exhibited a low hydroxylamine-dependent nitrite reductase activity. The results suggest that this enzyme is similar to the copper-containing ;denitrifying enzyme' of Pseudomonas denitrificans. A dithionite-reduced, 465nm-absorbing haemoprotein was associated with homogeneous preparations of hydroxylamine oxidase. The band at 465nm maximum was not reduced during the oxidation of hydroxylamine although the extinction was abolished on addition of hydroxylamine, NO(2) (-) or CO. These last-named compounds when added to the oxidized enzyme precluded the appearance of the 465nm-absorption band on addition of dithionite. Several properties of 465nm-absorbing haemoprotein are described.     

Mechanism of Nitrification by Arthrobacter sp

Resting cells of Arthrobacter sp. excrete as much as 60 mug of hydroxylamine-nitrogen per ml when supplied with ammonium. An organic carbon source in abundant supply is necessary for the oxidation. Resting cells oxidize hydroxylamine to nitrite and 1-nitrosoethanol, the former accumulating only when an exogenous carbon source is available. Cell-free extracts contain an enzyme catalyzing the formation of hydroxylamine from acetohydroxamic acid, a hydroxylamine-nitrite oxido-reductase, and an enzyme producing nitrite and nitrate from various primary nitro compounds. Nitrite is not produced from hydroxylamine by the extracts, but 1-nitrosoethanol is formed from hydroxylamine in the presence of acetate. 1-Nitrosoethanol is also produced from acetohydroxamic acid by these preparations. Nitrite was formed from hydroxylamine, however, by extracellular enzymes excreted by the bacterium.     

Nitrite and hydroxylamine reduction in higher plants. Fractionation, electron donor and substrate specificity of leaf enzymes, principally from vegetable marrow (Cucurbita pepo L.)

Nitrite reductase was purified between 760- and 1300-fold from vegetable marrow (Cucurbita pepo L.) and residual hydroxylamine reductase activity was low or negligible by comparison. With ferredoxin as electron donor, nitrite loss and ammonia formation at pH7.5 were stoicheiometrically equivalent. Crude nitrite reductase preparations showed negligible activity with NADPH as electron donor maintained in the reduced state by glucose 6-phosphate, whereas by comparison, activity was high when either ferredoxin or benzyl viologen were also present and reduced by the NADPH-glucose 6-phosphate system, whereas FMNH(2) produced variable and relatively low activity under the same conditions. At pH values below 7, non-enzymic reactions occurred between reduced benzyl viologen and nitrite, and intermediate reduction products were inferred to be produced instead of ammonia. Activity with ferredoxin (0.1mm), reduced by chloroplast grana in the light, was 25 times that produced with ferredoxin (40mum) reduced with NADPH and glucose 6-phosphate. For an approximate molecular weight 61000-63000 derived by chromatography on Sephadex G-100 and G-200, and a specific activity of 46mumol of nitrite reduced/min per mg of protein with light and chloroplast grana, a minimum turnover number of 3x10(3)mol of nitrite reduced/min per mol of enzyme was found. Two hydroxylamine reductases were separated on Sephadex gels. One (HR1) was initially associated with nitrite reductase during gel filtration but disappeared during later fractionation. This HR1 fraction showed nearly comparable activity with reduced benzyl viologen, ferredoxin or FMNH(2). The other (HR2), of molecular weight approx. 35000, reacted with reduced benzyl viologen but showed negligible activity with ferredoxin or NADPH. Activity with FMNH(2) was associated with an irregular trailing boundary during gel filtration, with much diminished activity in the HR2 region. Activity with NADPH was about 30% of that with FMNH(2), reduced benzyl viologen or ferredoxin and was considered to reside in fraction HR1. Hydroxylamine yielded ammonia under all assay conditions. No activity with hyponitrite or sulphite was observed with reduced benzyl viologen as electron donor in either the nitrite reductase or the hydroxylamine reductase systems, but pyruvic oxime produced about 4% of the activity of hydroxylamine.     

SOME PROPERTIES OF SULFITE REDUCTASE FROM YEAST

The MVH- and NADPH-linked sulfite reducing activities were assayedin extracts obtained from a wild strain of Saccharomyces cerevisiaeand various auxotrophic mutants derived from it. All the extractspossessing the NADPH-linked activity also showed the MVH linkedactivity, and all those lacking the latter also lacked the former.However, extracts from several mutants had the MVH-linked activitywithout having the other. NADPH-sulfite reductase was purified nearly 200-fold from extractsof the wild strain. Throughout the purification steps, the MVH-linkedactivity was also purified in association with the NADPH-linkedactivity, and the ratio between the two remained essentiallyconstant. However, on exposure to heat, low ionic strengthsand PCMB, only the NADPH-linked activity was lost, leaving theMVH-linked activity almost unaffected. Both activities weresensitive to cyanide. The sulfite reductase could also reduce nitrite and hydroxylamine, and the nitrite- and hydroxylamine-reducing activitywas sensitive to the treatments which inhibited the NADPH-sulfitereducing activity. Addition of nitrite or hydroxylamine competitivelyinhibits the NADPH linked sulfite reduction. The enzyme alsoshowed diaphorase activity, reducing DPIP or MV. This activitywas inhibited by the treatments to which the NADPH-linked sulfitereduction was sensitive, but it was not sensitive to cyanide. A tentative schematic model for yeast sulfite reductase is presented. ( Accepted October 8, 1964)     

New nitric oxide donating 1,2,4-triazole/oxime hybrids: Synthesis,investigation of anti-inflammatory,ulceroginic liability and antiproliferative activities

A series of novel nitric oxide (NO) donating triazole/oxime hybrids was prepared and evaluated for their anti-inflammatory activity. Most of the tested compounds showed significant anti-inflammatory activity using carrageenan-induced rat paw edema method compared to indomethacin. Calculation of the ulcer indices and histopathological investigation indicated that the prepared NO-donating oximes exhibited less ulcerogenicity compared to their intermediate ketones and indomethacin. The NO-donating oxime 6i revealed significant activity against renal cancer A498 cell lines with 50.52 cell growth inhibition.     

Pathway of oxidation of pyruvic oxime by a heterotrophic nitrifier of the genus Alcaligenes: evidence against nitroethane as an intermediate

The role of nitroethane as an intermediate in the oxidation of pyruvic oxime to nitrate by an Alcaligenes sp. was examined. Unlike pyruvic oxime, which serves as a sole source of C and N for the bacterium, nitroethane was incapable of supporting the growth of the microbe. Nitroethane was metabolized and diauxic growth did occur, however, if the nitroethane medium was amended with yeast extract. Alcaligenes sp. resting cells and cell-free extracts were prepared from nitroethane-yeast extract grown cultures and the maximum rate of nitrite synthesis when nitroethane was the substrate was 6.8 nmol min-1 mg cell protein-1, a 10-fold lower rate than that previously noted for pyruvic oxime oxidation. These cell-free extracts were unable to metabolize pyruvic oxime. Resting cells and cell-free extracts prepared from Alcaligenes sp. cells grown in a pyruvic oxime medium were, conversely, incapable of metabolizing nitroethane. Collectively, these results indicate that nitroethane is not an intermediate in the pathway of pyruvic oxime oxidation and that two separate enzyme systems exist in the Alcaligenes sp. for the metabolism of pyruvic oxime and nitroethane.