Harnsäureabbau und Biosynthese der Enzyme Uricase,Glyoxylatcarboligase und Urease bei Hydrogenomonas H 16

Zusammenfassung Die Veränderungen der Uricase-, Glyoxylatcarboligaseund Ureaseaktivität wurden in Hydrogenomonas H 16 Zellen während der Inkubation mit Harnsäure, Allantoin und in stickstoffreiem Fructosemedium untersucht.Übertragen in ein harnsäurehaltiges Medium stiegen die spezifische Aktivität von Uricase und Glyoxylatcarboligase innerhalb von 2–3 Std auf 35 bzw. 1000 mole Substrat/min/g Protein an und sanken wieder ab, nachdem das Substrat verbraucht worden war. Der Harnsäureabbau wurde durch Fructose schwach gefördert und durch zusätzlich verabreichte Ammoniumionen nicht beeinflußt.In Gegenwart von Allantoin wurde nur Glyoxylatcarboligase mit voller Syntheserate gebildet, während Uricase nur einen vorübergehenden schwachen Anstieg zeigte.Beim Harnsäureabbau wurden Harnstoff und Ammoniak im molaren Verhältnis von etwa 1:2 freigesetzt und im Medium angehäuft. Dabei wurde die Ammoniakbildung offenbar nicht durch Urease katalysiert.Urease wurde während des Wachstums mit Harnsäure und mit Allantoin nicht gebildet, bedingt durch die Anhäufung von Ammoniumionen. Eine ausgeprägte Ureasesynthese erfolgte dagegen unter N-Mangelbedingungen, ohne daß hierbei die Uricase-oder Glyoxylatcarboligaseaktivität anstieg. Diese Beobachtungen lassen erkennen, daß Urease im Gegensatz zu anderen am Harnsäureabbau beteiligten Enzymen nicht durch ihr Substrat induziert wird. Vielmehr unterliegt die Ureasebildung bei Hydrogenomonas H 16 ausschließlich der Kontrolle durch Repression, wobei Ammoniumionen als reprimierendes Substrat wirksam sind.

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Uric acid degradation and biosynthesis of the enzymes uricase, glyoxylate carboligase and urease in Hydrogenomonas H 16 II. Effect of uric acid, fructose and nitrogen deficiency on enzyme formation

Summary Changes in uricase, glyoxylate carboligase and urease activity were determined in fructose grown Hydrogenomonas H 16 cells during subsequent incubation with uric acid, allantoin and in nitrogen-deficient fructose media.The specific activities of uricase and glyoxylate carboligase increased within 2–3 hours up to levels of 35 and 1000 moles of substrate/min/g protein respectively, when the cells were transferred to an uric acid containing medium. These enzyme levels decreased after the substrate was consumed. Uric acid degradation was slightly stimulated by fructose, but was not affected by the addition of ammonia.In the presence of allantoin only glyoxylate carboligase was synthesized at a full rate, while uricase exhibited only a slight, temporary increase.Urea and ammonia were liberated from uric acid and accumulated in the suspension at a molar ratio of approximately 1:2. This ammonia formation was apparently not catalyzed by urease.Urease was not formed during growth with uric acid and allantoin, presumably due to the accumulation of ammonia. A pronounced synthesis of urease was observed under nitrogen deficiency, under which conditions uricase and glyoxylate carboligase were not formed. These data indicate, that urease, unlike other enzymes of the uric acid degrading pathway, is not induced by its substrate. It is considered, that urease formation in Hydrogenomonas H 16 is controlled by repression only, with ammonium ions serving as the repressing substrate.

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Auszug aus der gleichlautenden Habilitationsschrift zur Erlangung der venia legendi der Landwirtschaftlichen Fakultät der Georg-August-Universität Göttingen.     

Identification of uric acid as the redox molecule secreted by the yeast Arxula adeninivorans

The yeast Arxula adeninivorans has been previously shown to secrete a large amount of an electro-active molecule. The molecule was produced by cells that had been cultivated in a rich medium, harvested, washed and then suspended in phosphate-buffered saline (PBS). The molecule was easily detectable after 60 min of incubation in PBS, and the cells continued to produce the molecule in these conditions for up to 3 days. The peak anodic potential of the oxidation peak was 0.42 V, and it was shown to be a solution species rather than a cell-attached species. We have optimised the production of the molecule, identified it by high-pressure liquid chromatography (HPLC) fractionation and high-resolution mass spectrometric analysis and determined the pathway involved in its synthesis. It has a mass/charge ratio that corresponds to uric acid, and this identification was supported by comparing UV spectra and cyclic voltammograms of the samples to those of uric acid. An A. adeninivorans xanthine oxidase gene disruption mutant failed to produce uric acid, which added further validity to this identification. It also demonstrated that the purine catabolism pathway is involved in its production. A transgenic A. adeninivorans strain with a switchable urate oxidase gene (AUOX) accumulated uric acid when the gene was switched off but did not when the gene was switched on. Cultivation of cells on amino acid and purine-free minimal media with an inorganic nitrogen source suggests that the cells synthesise purines from inorganic nitrogen and proceed to degrade them via the normal purine degradation pathway.     

Evaluation of purine utilization by Lactobacillus gasseri strains with potential to decrease the absorption of food-derived purines in the human intestine

Abstract

It is well accepted that frequent and heavy intake of purine-rich foods causes elevation of serum uric acid levels, which is a risk factor of hyperuricemia. Reducing intestinal absorption of dietary purines may attenuate the elevation of serum uric acid levels and exacerbation of hyperuricemia. This reduction may be achieved by the ingestion of lactic acid bacteria that take up purines in the intestine. In this study, we investigated the degree of uptake and utilization of purines of three lactobacilli strains. Among them, Lactobacillus gasseri PA-3 (PA-3) showed the greatest incorporation of ¹⁴C-adenine. PA-3 also incorporated ¹⁴C-adenosine and ¹⁴C-AMP. Additionally, using defined growth medium, PA-3 demonstrated greater proliferation in the presence of these purines than in their absence. Although further investigation is required, ingestion of PA-3 may lower serum uric acid levels by reducing intestinal absorption of purines in humans.     

Utilization of organic compounds as the sole source of nitrogen by Thiobacillus thiooxidans

Thiobacillus thiooxidans DSM 504 was shown to grow with adenine, hypoxanthine, xanthine and uric acid as sole sources of nitrogen. Growth with these compounds was observed after lag periods of varying lengths, unless the cells had been previously grown with the same purine base. The disappearance of adenine was accompanied by a temporary accumulation of hypoxanthine in the medium. The utilization of purines was inhibited by ammonia (1 mM). Guanine, pyrimidines and some other organic compounds were not utilized.Non-standard abbreviation U-¹⁴C uniformly labeled by ¹⁴C     

Phenylpropanoid Metabolism in Suspension Cultures of Vanilla planifolia Andr. : IV. Induction of Vanillic Acid Formation

Kinetin is used as an elicitor to induce vanillic acid formation in cell suspension cultures of Vanilla planifolia. Maximal induction is observed at a kinetin concentration of 20 micrograms per gram of fresh weight of cells. Vanillic acid synthesis is observed a few hours after elicitation. The effects of kinetin on the activity of some enzymes of the phenylpropanoid pathway, i.e. phenylalanine ammonia-lyase, 4-hydroxycinnamate:coenzyme A ligase and uridine 5′-diphosphate-glucose:trans-cinnamic acid glucosyltransferase, are reported and compared to the effects of chitosan. The former two enzymes are induced by chitosan with a maximum activity of approximately 25 to 40 hours after elicitation. All three enzymes are induced by kinetin with maximum activities for phenylalanine ammonia lyase and 4-hydroxycinnamate:coenzyme A ligase at approximately 50 hours after induction, whereas maximum glucosyltransferase activity is seen already after 24 hours. Furthermore, both elicitors induced the formation of lignin-like material, whereas only kinetin induced vanillic acid biosynthesis. Finally, kinetin but not chitosan induces catechol-4-O-methyltransferase activity, catalyzing the formation of 4-methoxycinnamic acids, which were shown to be intermediates of hydroxybenzoic acid biosynthesis within cells of V. planifolia. It is suggested that this methyltransferase is directly involved in the biosynthesis of vanillic acid.     

Arginine Catabolism and the Arginine Succinyltransferase Pathway in Escherichia coli

Arginine catabolism produces ammonia without transferring nitrogen to another compound, yet the only known pathway of arginine catabolism in Escherichia coli (through arginine decarboxylase) does not produce ammonia. Our aims were to find the ammonia-producing pathway of arginine catabolism in E. coli and to examine its function. We showed that the only previously described pathway of arginine catabolism, which does not produce ammonia, accounted for only 3% of the arginine consumed. A search for another arginine catabolic pathway led to discovery of the ammonia-producing arginine succinyltransferase (AST) pathway in E. coli. Nitrogen limitation induced this pathway in both E. coli and Klebsiella aerogenes, but the mechanisms of activation clearly differed in these two organisms. We identified the E. coli gene for succinylornithine aminotransferase, the third enzyme of the AST pathway, which appears to be the first of an astCADBE operon. Its disruption prevented arginine catabolism, impaired ornithine utilization, and affected the synthesis of all the enzymes of the AST pathway. Disruption of astB eliminated succinylarginine dihydrolase activity and prevented arginine utilization but did not impair ornithine catabolism. Overproduction of AST enzymes resulted in faster growth with arginine and aspartate. We conclude that the AST pathway is necessary for aerobic arginine catabolism in E. coli and that at least one enzyme of this pathway contributes to ornithine catabolism.     

Do Uric Acid Deposits in Zooxanthellae Function as Eye-Spots?

The symbiosis between zooxanthellae (dinoflagellate genus Symbiodinium) and corals is a fundamental basis of tropical marine ecosystems. However the physiological interactions of the hosts and symbionts are poorly understood. Recently, intracellular crystalline deposits in Symbiodinium were revealed to be uric acid functioning for nutrient storage. This is the first exploration of these enigmatic crystalline materials that had previously been misidentified as oxalic acid, providing new insights into the nutritional strategies of Symbiodinium in oligotrophic tropical waters. However, we believe these deposits also function as eye-spots on the basis of light and electron microscopic observations of motile cells of cultured Symbiodinium. The cells possessed crystalline deposit clusters in rows with each row 100–150 nm thick corresponding to 1/4 the wavelength of light and making them suitable for maximum wave interference and reflection of light. Crystalline clusters in cells observed with a light microscope strongly refracted and polarized light, and reflected or absorbed short wavelength light. The facts that purines, including uric acid, have been identified as the main constituents of light reflectors in many organisms, and that the photoreceptor protein, opsin, was detected in our Symbiodinium strain, support the idea that uric acid deposits in Symbiodinium motile cells may function as a component of an eye-spot.     

Studies on the Physiology of Bacillus fastidiosus

Bacillus fastidiosus was grown in a minimal medium that contained uric acid or allantoin, aerated by vigorous stirring. A constant, optimum pH of 7.4 was maintained by controlled addition of sulfuric acid. Washed cells converted both urate and allantoin into carbon dioxide and ammonia, simultaneously assimilating part of the available carbon and nitrogen. Urate oxidase (formerly called uricase) was present in extracts from urate-grown but not allantoin-grown cells. The formation of urate oxidase was apparently induced by urate. Urea was detected as an intermediate in some but not all of these experiments. However, the high urease activity observed in cell-free extracts may have prevented accumulation of urea in many of the experiments. The presence of glyoxylate carboligase and tartronic semialdehyde reductase activities indicates that the glycerate pathway may be involved in urate and allantoin catabolism in this organism.     

Anaerobic degradation of uric acid via pyrimidine derivatives by selenium-starved cells of Clostridium purinolyticum

Clostridium purinolyticum decomposed uric acid via pyrimidine derivatives under selenium starvation conditions. Products were acetate, formate, glycine, ammonia, and CO₂. 4,5-Diaminouracil could be identified as an intermediate after converting the labile substance into 6,7-dimethyllumazine. The breakdown of uric acid was inhibited by EDTA. High-pressure liquid chromatography methods have been developed for the simultaneous determination of uric acid, 4,5-diaminouracil, and 6,7-dimethyllumazine. The significance of the new pathway is discussed.Abbreviation HPLC high-pressure liquid chromatography     

Oxidative degradation of purines by the faculative phototrophic bacterium Rhodopseudomonas capsulata

The mechanism of purine degradation was studied in the facultative phototrophic bacterium Rhodopseudomonas capsulata. Using tungstate as an inhibitor of synthesis of an active xanthine dehydrogenase it could be shown in growth experiments that purine compounds are transformed to uric acid as central purine intermediate prior to ring cleavage. Because of its rapid degradation, the mechanism of uric acid conversion was investigated using 1-methyluric acid as substrate. The analogue was partially degraded by whole cells yielding 3-methylallantoin and methylurea. This implicated an oxidative degradation of 1-methyluric acid analogous to oxidation of uric acid to allantoin suggesting uric acid degradation via allantoin. In cell-free extracts, allantoinase, allantoicase, ureidoglycolase and urease activities degrading allantoin to NH₃, CO₂ and glyoxylic acid were detected. Apparently, purine degradation in R. capsulata proceeds in a manner similar to many aerobic microorganisms. It is peculiar to this bacterium, however, that the pathway evidently operates also under anaerobic conditions. In cell extracts, oxidation of uric acid was observed which could be increased by addition of cytochrome c. The basis of this stimulation is still unknown.     

Nitrogen control in Pseudomonas aeruginosa: A role for glutamine in the regulation of the synthesis of NADP-dependent glutamate dehydrogenase,urease and histidase

In Pseudomonas aeruginosa the formation of urease, histidase and some other enzymes involved in nitrogen assimilation is repressed by ammonia in the growth medium. The key metabolite in this process appears to be glutamine or a product derived from it, since ammonia and glutamate did not repress urease and histidase synthesis in a mutant lacking glutamine synthetase activity when growth was limited for glutamine. The synthesis of these enzymes was repressed in cells growing in the presence of excess glutamine. High levels of glutamine were also required for the derepression of NADP-dependent glutamate dehydrogenase formation in the glutamine synthetase-negative mutant.     

Arginine catabolism in wine lactic acid bacteria: is it via the arginine deiminase pathway or the arginase-urease pathway?

The wine lactic acid bacteria Leuconostoc oenos OENO and Lactobacillus buchneri CUC-3 catabolize L-arginine to ornithine and ammonia as major end-products, with 1 mole of arginine converted into 2 moles of ammonia and 1 mole of ornithine. Some citrulline was also excreted into the medium. The excreted citrulline was reassimilated and catabolized by the lactobacillus strain, though not by the leuconostoc. Urea was not detected during arginine degradation. The activities of all three enzymes of the arginine deiminase pathway (arginine deiminase, ornithine transcarbamylase and carbamate kinase) increased significantly over time in the presence of arginine. On the other hand, arginase and urease activities were undetectable in cell extracts of cultures grown in the presence of arginine. The results show that the arginine deiminase pathway, and not the arginase-urease pathway, is the route for arginine degradation in wine lactic acid bacteria.     

Nitrogen Metabolism in the Terrestrial Isopod, Oniscus asellus

The total diffusible content of ammonia in Oniscus asellus wasmeasured as 1.20 mg % body fluid. Daily excretions of ammoniaexceeded the total diffusible content in measurements on specimenstaken directly from nature. The content of uric acid in a groupof 35 specimens was 11.3 mg % body weight and 93% of this materialwas in the body wall. Urea was not measurable under conditionsthat would have allowed detection of at least 0.26 mg % bodyfluid. Only arginase of the enzymes in the ornithine-urea cycle wasmeasurable. Labeled urea was not detectable in aqueous extractsunder conditions where approximately 50,300 CPM of labeled carbonwas incorporated into metabolites. Labeled arginine was notdetectable under conditions where 9,100 CPM of KHC¹⁴O₃ wereincorporated into the soluble protein fraction and more than3,340,000 CPM were incorporated into particulate protein andnon-protein fractions of the organism. All of the enzymes of uricolysis to ammonia were present, uricaseand urease being rate-limiting. Uricase was measurable as myeloperoxidase.Although high rates of glutamate-oxalacetate aminotransferaseand glutamate-pyruvate aminotransferase were readily measured,glutamic dehydrogenase activity proceeded slowly, suggestingsynthesis of needed amino acids as the key function of the transaminaseactivities. Amino acid oxidase activities, glutaminase, and asparaginaseactivities were low, too, whereas peroxidatic deaminase activitiesproceeded at rates sufficiently fast to account for the levelsof volatile ammonia emitted in vivo. Although molting may serve for excretion of end-products ofpurine catabolism, no central organ homologous to kidney orliver appears to be present with respect to detoxication orexcretion of ammonia. Inasmuch as O. asellus is not exposed to osmotic stresses, doesnot ordinarily face problems of dehydration, and tolerates levelsof ammonia generally ascribed as toxic to other organisms, itis proposed that retention of ammonotelism probably offers thermodynamicadvantages to the organism. The evolution of a system whereinammonia is excreted as a gas may have provided the organismwith an adaptive mechanism for colonizing land.     

Inducible Formation of Urease in Canavalia ensiformis

Increase in urease activity in leaves of Canavdlia ensifomis has been demonstrated. The activity of excised leaves increased by about 100 percent when 1.5 × 10^?1M urea was added externally as inducer. Glycine-1-¹⁴C was used to investigate whether the increase in activity was dependent on de novo protein synthesis. The Incorporation of labeled amino acid into urease was twofold higher in induced samples than in non-induced ones. This indicates that the increase in activity is connected with de nova protein synthesis. The once increased activity was always followed by a rapid decrease. The urease activity was lost constantly with time after the external addition of ammonia in vivo. The inhibitory action of ammonia on urease fa vitro was eliminated by dialysis. Accordingly it may be concluded that the loss of activity was dependent on the product repression by ammonia.     

Urease and Dental Plaque Microbial Profiles in Children

Objective

Urease enzymes produced by oral bacteria generate ammonia, which can have a significant impact on the oral ecology and, consequently, on oral health. To evaluate the relationship of urease with dental plaque microbial profiles in children as it relates to dental caries, and to identify the main contributors to this activity.

Methods

82 supragingival plaque samples were collected from 44 children at baseline and one year later, as part of a longitudinal study on urease and caries in children. DNA was extracted; the V3–V5 region of the 16S rRNA gene was amplified and sequenced using 454 pyrosequencing. Urease activity was measured using a spectrophotometric assay. Data were analyzed with Qiime.

Results

Plaque urease activity was significantly associated with the composition of the microbial communities of the dental plaque (Baseline P = 0.027, One Year P = 0.012). The bacterial taxa whose proportion in dental plaque exhibited significant variation by plaque urease levels in both visits were the family Pasteurellaceae (Baseline P<0.001; One Year P = 0.0148), especially Haemophilus parainfluenzae. No association was observed between these bacteria and dental caries. Bacteria in the genus Leptotrichia were negatively associated with urease and positively associated with dental caries (Bonferroni P<0.001).

Conclusions

Alkali production by urease enzymes primarily from species in the family Pasteurellaceae can be an important ecological determinant in children’s dental plaque. Further studies are needed to establish the role of urease-associated bacteria in the acid/base homeostasis of the dental plaque, and in the development and prediction of dental caries in children.     

Streamlined Regulation and Gene Loss as Adaptive Mechanisms in Prochlorococcus for Optimized Nitrogen Utilization in Oligotrophic Environments

Prochlorococcus is one of the dominant cyanobacteria and a key primary producer in oligotrophic intertropical oceans. Here we present an overview of the pathways of nitrogen assimilation in Prochlorococcus, which have been significantly modified in these microorganisms for adaptation to the natural limitations of their habitats, leading to the appearance of different ecotypes lacking key enzymes, such as nitrate reductase, nitrite reductase, or urease, and to the simplification of the metabolic regulation systems. The only nitrogen source utilizable by all studied isolates is ammonia, which is incorporated into glutamate by glutamine synthetase. However, this enzyme shows unusual regulatory features, although its structural and kinetic features are unchanged. Similarly, urease activities remain fairly constant under different conditions. The signal transduction protein P_II is apparently not phosphorylated in Prochlorococcus, despite its conserved amino acid sequence. The genes amt1 and ntcA (coding for an ammonium transporter and a global nitrogen regulator, respectively) show noncorrelated expression in Prochlorococcus under nitrogen stress; furthermore, high rates of organic nitrogen uptake have been observed. All of these unusual features could provide a physiological basis for the predominance of Prochlorococcus over Synechococcus in oligotrophic oceans.