Utilization of amino acids by Frankia sp. strain CpI1

The utilization of some amino acids, added at 1 mM and 10 mM concentrations, as the sole combined nitrogen sources by Frankia sp. strain CpI1, has been investigated. Glutamine, like NH ₄ ⁺ , provided rapid growth without N₂ fixation. Histidine at 1 mM yielded poor N₂-fixing activity but better cell growth than N₂. Aspartate, glutamate, alanine, proline, each at 1 mM concentration, supported similar levels of N₂ fixation and growth. Growth on 10 mM glutamate, proline, or histidine resulted in poor N₂-fixing activity and poor cell growth. Cells grown on 10 mM alanine had about half the N₂-fixing activity of cells grown on N₂ but growth was good. Aspartate at 10 mM concentration, however, stimulated N₂-fixing activity dramatically and promoted faster growth. Enzyme analysis suggested that asparate is catabolized by glutamate-oxaloacetate transaminase (GOT), since GOT specific activity was induced, and aspartase activity was not detected, in cells grown on aspartate as the sole combined nitrogen source. Thinlayer chromatography (TLC) of metabolites extracted from N₂-grown cells fed with [¹⁴C]-aspartate showed that label was rapidly accumulated mainly on aspartate and/or glutamate, depending on the cells' physiological state, without detectable labeling on fumarate or oxaloacetate (OAA). These findings provide evidence that aspartate is catabolized by GOT to OAA which, in turn, is rapidly converted to -ketoglutarate through the TCA cycle and then to glutamate by GOT or by glutamate synthase (GOGAT). The stimulation of N₂ fixation and growth by aspartate is probably caused by an increased intracellular glutamate pool.     

Analysis of energetic amino acid metabolism in Acyrthosiphon pisum: A multidimensional approach to amino acid metabolism in aphids

Aphids are highly specialized insects that feed on the phloem-sap of plants, the amino acid composition of which is very unbalanced. Amino acid metabolism is thus crucial in aphids, and we describe a novel investigation method based on the use of ¹⁴C-labeled amino acids added in an artificial diet. A metabolism cage for aphids was constructed, allowing for the collection and analysis of the radioactivity incorporated into the aphid body, expired as CO₂, and rejected in the honeydew and exuviae. This method was applied to the study of the metabolism of eight energetic amino acids (aspartate, glutamate, glutamine, glycine, serine, alanine, proline, and threonine) in the pea aphid, Acyrthosiphon pisum. All these amino acids except threonine were subject to substantial catabolism as measured by high ¹⁴CO₂ production. The highest turnover was displayed by aspartate, with 60% of its carbons expired as CO₂. For the first time in an aphid, we directly demonstrated the synthesis of three essential amino acids (threonine, isoleucine, and lysine) from carbons of common amino acids. The synthesis of these three compounds was only observed from amino acids that were previously converted into glutamate. This conversion was important for aspartate, and lower for alanine and proline. To explain the quantitative results of interconversion between amino acids, we propose a compartmentation model with the intervention of bacterial endosymbiotes for the synthesis of essential amino acids and with glutamate as the only amino acid supplied by the insect to the symbiotes. Moreover, proline exhibited partial conversion into arginine, and it is suggested that proline is probably indirectly involved in excretory nitrogen metabolism. © 1995 Wiley-Liss, Inc.     

A mechanism for accelerated degradation of intracellular proteins after limited damage by free radicals

I propose that limited free radical attack upon proteins, occurring continuously in cells, creates new N-termini (notably aspartate and glutamate) which render the proteins more susceptible to proteolysis by the ubiquitin conjugation system. I suggest that these reactions are a significant part of the previously described ‘N-end’ and ‘PEST’ rules, which indicate amino acid termini or sequences which tend to dictate short protein half-lives. I also argue that the N-end rule may apply to sequestered intracellular sites, such as mitochondria, these also being sites of radical generation.     

The metabolism of amino acids in the bovine lens. Their oxidation as a source of energy

1. The metabolism by the bovine lens of nine (14)C-labelled l-amino acids was studied. These were: alanine, aspartate, glutamate, leucine, lysine, proline, serine, tyrosine and tryptophan. 2. All were taken up by the tissue and incorporated into protein. 3. Aspartate and glutamate, although poorly taken up, were readily metabolized to CO(2). Radioactivity from glutamate was also found in glutathione, glutamine, proline and ophthalmic acid. Aspartate was converted into glutamate, glutathione, proline, alanine and lactate. 4. Alanine was largely converted into lactate, which was released into the medium, but incorporation of radioactivity into CO(2), glutamate, glutathione, aspartate and lipids also occurred. 5. Radioactivity from leucine was detected in CO(2), lipids, glutamate, glutathione, proline and glutamine. 6. Lysine was only slightly broken down by the bovine lens; radioactivity was observed in CO(2), glutamate, glutathione, proline and two unidentified compounds. 7. Proline was metabolized to glutamate from which CO(2), glutathione and glutamine were formed. Hydroxyproline in the capsule collagen was labelled. 8. Radioactivity from serine was found in CO(2), lipids, glutathione, glycine, cystine, ATP, lactate and three unidentified compounds, one of which was probably taurine. 9. Neither tyrosine nor tryptophan were metabolized by the bovine lens. 10. The ability of the lens to metabolize amino acids was also shown by measurement of NH(3) production: more NH(3) was formed when glucose was absent from the incubation medium. 11. These experiments suggest that oxidation of amino acids is a source of energy for the lens.     

Proline metabolism in isolated rat liver cells

The metabolism of proline was studied in liver cells isolated from starved rats. The following observations were made. 1. Consumption of proline could be largely accounted for by production of glucose, urea, glutamate and glutamine. 2. At least 50% of the total consumption of oxygen was used for proline catabolism. 3. Ureogenesis and gluconeogenesis from proline could be stimulated by partial uncoupling of oxidative phosphorylation. 4. Addition of ethanol had little effect on either proline uptake or oxygen consumption, but strongly inhibited the production of both urea and glucose and caused further accumulation of glutamate and lactate. Accumulation of glutamine was not affected by ethanol. 5. The effects of ethanol could be overcome by partial uncoupling of oxidative phosphorylation. 6. The apparent K_m values of argininosuccinate synthetase (EC 6.3.4.5) for aspartate and citrulline in the intact hepatocyte are higher than those reported for the isolated enzyme. 7. 3-Mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase (EC 4.1.1.32), greatly enhanced cytosolic aspartate accumulation during proline metabolism, but inhibited urea synthesis. 8. It is concluded that when proline is provided as a source of nitrogen to liver cells, production of ammonia by oxidative deamination of glutamate is inhibited by the highly reduced state of the nicotinamide nucleotides within the mitochondria. 9. Conversion of proline into glucose and urea is a net-energy-yielding process, and the high state of reduction of the nicotinamide nucleotides is presumably maintained by a high phosphorylation potential. Thus when proline is present as sole substrate, the further oxidation of glutamate by glutamate dehydrogenase (EC 1.4.1.3) is limited by the rate of energy expenditure of the cell.     

A theoretical study of zinc(II) interactions with amino acid models and peptide fragments

Density functional theory calculations have been employed to study the interaction between the Zn²⁺ ion and some standard amino acid models. The highest affinities towards the Zn²⁺ ion are predicted for serine, cysteine, and histidine. Relatively high affinities are reported also for proline and glutamate/aspartate residues. It was found that the zinc complexes with cysteine adopt a tetrahedral conformation. Conversely, complexes with one or two histidine moieties remain in an octahedral geometry, while those with three or more histidine groups adopt a square-planar geometry. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Amino Acid Metabolism of Lemna minor L. : I. Responses to Methionine Sulfoximine

When Lemna minor L. is supplied with the potent inhibitor of glutamine synthetase, methionine sulfoximine, rapid changes in free amino acid levels occur. Glutamine, glutamate, asparagine, aspartate, alanine, and serine levels decline concomitantly with ammonia accumulation. However, not all free amino acid pools deplete in response to this inhibitor. Several free amino acids including proline, valine, leucine, isoleucine, threonine, lysine, phenylalanine, tyrosine, histidine, and methionine exhibit severalfold accumulations within 24 hours of methionine sulfoximine treatment. To investigate whether these latter amino acid accumulations result from de novo synthesis via a methionine sulfoximine insensitive pathway of ammonia assimilation (e.g. glutamate dehydrogenase) or from protein turnover, fronds of Lemna minor were prelabeled with [¹⁵N]H₄⁺ prior to supplying the inhibitor. Analyses of the ¹⁵N abundance of free amino acids suggest that protein turnover is the major source of these methionine sulfoximine induced amino acid accumulations. Thus, the pools of valine, leucine, isoleucine, proline, and threonine accumulated in response to the inhibitor in the presence of [¹⁵N]H₄⁺, are ¹⁴N enriched and are not apparently derived from ¹⁵N-labeled precursors. To account for the selective accumulation of amino acids, such as valine, leucine, isoleucine, proline, and threonine, it is necessary to envisage that these free amino acids are relatively poorly catabolized in vivo. The amino acids which deplete in response to methionine sulfoximine (i.e. glutamate, glutamine, alanine, aspartate, asparagine, and serine) are all presumably rapidly catabolized to ammonia, either in the photorespiratory pathway or by alternative routes.     

Influence of amino acids and organic antimetabolites on growth and biosynthesis of the chemoautotroph Thiobacillus neapolitanus strain C

Summary The growth of Thiobacillus neapolitanus strain C in liquid cultures was depressed by phenylalanine, p-fluorophenylalanine, cysteine, methionine, nor-leucine, azetidine-2-carboxylic acid, and chloramphenicol, but was little affected by glutamic acid, glycine, proline, azathymine, or oligomycin.Growing cultures assimilated ¹⁴C-labelled glycine, glutamic acid, phenylalanine, and tyrosine into protein. Tyrosine and phenylalamine were incorporated unchanged, but glutamate was used also for synthesis of arginine and proline. Glycine-¹⁴C contributed also to adenine and guanine synthesis. The extremely large amounts of phenylalanine incorporated into protein could indicate its toxicity to depend on its producing abnormal protein synthesis. Azetidine-2-carboxylic acid appeared to lower the amount of proline in the protein.Assimilation of glutamate and glycine by non-growing organisms was almost entirely dependent on energy from thiosulphate oxidation, thus suggesting a cause of obligate chemoautotrophy. Chloramphenicol specifically inhibited this thiosulphate-dependent incorporation of glutamate, glycine or CO₂ into protein at concentrations which did not affect total CO₂-fixation. Provided that energy is available from thiosulphate-oxidation this Thiobacillus is thus able to (a) activate exogenous amino acids; (b) incorporate them and CO₂ into protein by a chloramphenicol sensitive mechanism; (c) synthesise proline and arginine from glutamate; or adenine and guanine from glycine. Its biosynthesis thus depends on mechanisms like those of heterotrophs but requires to be driven by a chemolithotrophic energy supply.     

Computational Analysis of Cysteine Substitutions Modelled on the α- and β-domains of Cd5,Zn2-Metallothionein 2

Several mutant forms of rat liver Cd₅,Zn₂-metallothionein 2 (Cd₅,Zn₂-MT 2) [1] have been computationally modelled and analysed. All terminal cysteines (5, 13, 19, 21, 26, 29, 33, 36, 41, 48, 57 and 59, Figure 1) have been independently substituted by three other co-ordinating amino-acids (aspartate, glutamate and histidine), and the side-chains of the mutated residues have been modelled to co-ordinate the seven metal ions while minimizing the conformational variations with respect to the wild type protein. We have compared the ability of the putative mutant forms to maintain the MT binding properties. Substitution by aspartate residue best preserves the 3D MT structure. In addition, the mutations C5H plus C21H/E/D show neighbouring impairments that prevent their simultaneous substitution. Although replacement of cysteine by aspartate is feasible in all cases, to our knowledge there is no example of aspartate and cysteine residues co-ordinating to the same zinc atom. Accordingly, the use of histidine or glutamate instead of aspartate cannot be ruled out. The mutant forms in the -domain of Cd₅,Zn₂-MT 2 have yielded more neighbouring contacts than those in the -domain, which is corroborated by the accessible surface areas [2] of the sulfur atoms [3] in the native form.Abbreviations MT metallothionein - CD₅,Zn₂-MT Cadmium, Zinc-metallothionein - RMSD Root Mean Square Deviation - PDB Protein Data Bank - FEP Free Energy Perturbation - CnX mutant form of cysteine n (n = residue number) substituted by X (X = H, E or D, with H = histidine, E = glutamate, D = aspartate) - CnX/Y mutant forms CnX and CnY     

Light stimulation of proline synthesis in water-stressed barley leaves

The effect of light on [¹⁴C]glutamate conversion to free proline during water stress was studied in attached barley (Hordeum vulgare L.) leaves which had been trimmed to 10 cm in length. Plants at the three-leaf stage were stressed by flooding the rooting medium with polyethylene glycol 6000 (osmotic potential-19 bars) for up to 3 d. During this time the free proline content of 10-cm second leaves rose from about 0.02 to 2 mol/leaf while free glutamate content remained steady at about 0.6 mol/leaf. In stressed leaves, the amount of [¹⁴C]glutamate converted to proline in a 3-h period of light or darkness was taken to reflect the in-vivo rate of proline biosynthesis because the following conditions were met: (a) free-glutamate levels were not significantly different in light and darkness; (b) both tracer [¹⁴C]-glutamate and [¹⁴C]proline were rapidly absorbed; (c) rates of [¹⁴C]proline oxidation and incorporation into protein were very slow. As leaf water potential fell, more [¹⁴C]glutamate was converted to proline in both light and darkness, but at any given water potential in the range-12 to-20 bars, illuminated leaves converted twice as much [¹⁴C]glutamate to proline.     

Metabolic conversion of exogenous14C-aspartate and14C-gIutamate in the dark byFucus serratus L

The rates of uptake of exogenous L[U-¹⁴C] aspartate and glutamate into tissues of vegetative growing tips ofFucus serratus and their metabolism were studied in the dark. In these non-photosynthetic conditions, aspartate was fixed and metabolically converted more rapidly than glutamate. Radioactivity from¹⁴C-aspartate was principally transferred into glutamate. On the other hand, metabolism of absorbed¹⁴C-glutamate was very slow and its rate did not increase during incubation time, but produced more diversified soluble radioactive compounds. Thus inF. serratus, glutamate principally seems to be in the dark more a temporary¹⁴CO₂ storage product coming from β-carboxylation than a rapidly turned over intermediate.     

Changes in protein profile and amino acids in <Emphasis Type="Italic">Cladophora vagabunda</Emphasis> (Chlorophyceae) in response to salinity stress

The aim of the present study was to detect organic substances functioning as osmoticants that are used by the intertidal alga, Cladophora vagabunda (L.) Hoek (Chlorophyceae), to adapt to a wide range of salinity. The major constituents of the amino acid pool were aspartate, glutamate, glycine, valine, lysine, histidine, arginine, and proline. There were concomitant increases in the acidic amino acids: aspartate and the glutamate and the basic amino acids: lysine, histidine and arginine in response to salinity stress. The appearance of proline at hypersalinity alone showed that it acts as an osmoticant. As salinity increased, there was a progressive shift in the electrophoretic pattern of protein bands. New peptide bands appeared under hyposalinity (10‰) and hypersalinity (65‰) stress conditions in addition to the usual bands which appeared in the control (35‰). Glycine betaine, which has been considered a novel organic osmolyte in a number of organisms, has also been observed in C. vagabunda in response to salinity stress. The synthesis of the compatible solute glycine betaine and the amino acid proline with increasing salinity illustrates the contention that marine algae establish an osmotic equilibrium primarily by the synthesis of organic compounds. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Metabolism of amino acids in Ascaridia galli: decarboxylation reactions

Production of ¹⁴CO₂ from 12 carbon-labelled amino acids by Ascaridia galli was studied. Appreciable amounts of CO₂ were evolved from alanine, aspartate, glutamate, serine, leucine and valine by intestines, ovaries, cuticle and intact worms, in that order, but not from lysine, proline and tyrosine. Maximum CO₂ produced by whole worms was from serine, while with isolated organs it was from alanine. For cuticle, the decarboxylations of alanine, aspartate and glutamate were found to be associated with the mitochondrial fraction.     

The metabolism of proline in cotyledons of pumpkin (Cucurbita moschata)

Exogenous proline-U-₁₄C is readily metabolized to glutamate,ornithine, sugars, CO₂, and organic acids, and is incorporatedinto protein by etiolated and green pumpkin cotyledons. As littletranslocation of proline from the cotyledons occur, it was proposedthat in young tissue proline is converted to glutamate, ornithineor sugar which are then readily translocated from the cotyledons.In older tissue some glutamate carbon derived from proline isalso used as an energy source and metabolized to CO₂. As proteinsynthesis is occurring rapidly in these cotyledons, considerableproline is incorporated into new protein. After 10-hr, 15% ofthe absorbed radioactivity still remained as free proline. ¹Present address: Instituto de Ciencias Biologicas, UniversidadeFederal de Vicosa, Vicosa, Minas Gerais, Brasil. (Received February 1, 1974; )     

Analysis of glutamate homeostasis by overexpression of Fd-GOGAT gene in Arabidopsis thaliana

Glutamate plays a central role in nitrogen flow and serves as a nitrogen donor for the production of amino acids. In plants, some amino acids work as buffers: during photorespiration, ammonium derived from the conversion of glycine to serine is promptly reassimilated into glutamate by the glutamine synthetase (GS-2)/ferredoxin-dependent glutamate synthase (Fd-GOGAT) cycle. The glutamate concentration is relatively stable compared with those of other amino acids under environmental changes. The few studies dealing with glutamate homeostasis have but all used knockouts or mutants of these enzymes. Here, we generated Fd-GOGAT (GLU1)-overexpressing Arabidopsis plants to analyze changes in the amino acid pool caused by glutamate overproduction under different ammonium conditions controlled by CO₂ concentration, light intensity and nitrate concentration. Under photorespiratory conditions with sufficient ammonium supply, aspartate increased and glutamine and glycine decreased, but glutamate barely changed. Under non-photorespiratory conditions, however, glutamate and most other amino acids increased. These results suggest that the synthesized glutamate is promptly converted into other amino acids, especially aspartate. In addition, ammonium supply by photorespiration does not limit glutamate biosynthesis, but glutamine and glycine are important. This study will contribute to the understanding of glutamate homeostasis in plants.     

α-Ketoisocaproate Alters the Production of Both Lactate and Aspartate from [U-13C]Glutamate in Astrocytes: A 13C NMR Study

Abstract: The present study determined the metabolic fate of [U-¹³C]glutamate in primary cultures of cerebral cortical astrocytes from rat brain and also in cultures incubated in the presence of 1 or 5 mMα-ketoisocaproate (α-KIC). When astrocytes were incubated with 0.2 mM [U-¹³C]glutamate, 64.1% of the ¹³C metabolized was converted to glutamine, and the remainder was metabolized via the tricarboxylic acid (TCA) cycle. The formation of [1,2,3-¹³C₃]glutamate demonstrated metabolism of the labeled glutamate via the TCA cycle. In control astrocytes, 8.0% of the [¹³C]glutamate metabolized was incorporated into intracellular aspartate, and 17.2% was incorporated into lactate that was released into the medium. In contrast, there was no detectable incorporation of [¹³C]glutamate into aspartate in astrocytes incubated in the presence of α-KIC. In addition, the intracellular aspartate concentration was decreased 50% in these cells. However, there was increased incorporation of [¹³C]glutamate into the 1,2,3-¹³C₃-isotopomer of lactate in cells incubated in the presence of α-KIC versus controls, with formation of lactate accounting for 34.8% of the glutamate metabolized in astrocytes incubated in the presence of α-KIC. Altogether more of the [¹³C]glutamate was metabolized via the TCA cycle, and less was converted to glutamine in astrocytes incubated in the presence of α-KIC than in control cells. Overall, the results demonstrate that the presence of α-KIC profoundly influences the metabolic disposition of glutamate by astrocytes and leads to altered concentrations of other metabolites, including aspartate, lactate, and leucine. The decrease in formation of aspartate from glutamate and in total concentration of aspartate may impair the activity of the malate-aspartate shuttle and the ability of astrocytes to transfer reducing equivalents into the mitochondria and thus compromise overall energy metabolism in astrocytes.     

Biosynthesis of amino acids in Clostridium pasteurianum

1. Clostridium pasteurianum was grown on a synthetic medium with the following carbon sources: (a) (14)C-labelled glucose, alone or with unlabelled aspartate or glutamate, or (b) unlabelled glucose plus (14)C-labelled aspartate, glutamate, threonine, serine or glycine. The incorporation of (14)C into the amino acids of the cell protein was examined. 2. In both series of experiments carbon from exogenous glutamate was incorporated into proline and arginine; carbon from aspartate was incorporated into glutamate, proline, arginine, lysine, methionine, threonine, isoleucine, glycine and serine. Incorporations from the other exogenous amino acids indicated the metabolic sequence: aspartate --> threonine --> glycine right harpoon over left harpoon serine. 3. The following activities were demonstrated in cell-free extracts of the organism: (a) the formation of aspartate by carboxylation of phosphoenolpyruvate or pyruvate, followed by transamination; (b) the individual reactions of the tricarboxylic acid route to 2-oxoglutarate from oxaloacetate; glutamate dehydrogenase was not detected; (c) the conversion of aspartate into threonine via homoserine; (d) the conversion of threonine into glycine by a constitutive threonine aldolase; (e) serine transaminase, phosphoserine transaminase, glycerate dehydrogenase and phosphoglycerate dehydrogenase. This last activity was abnormally high. 4. The combined evidence indicates that in C. pasteurianum the biosynthetic role of aspartate and glutamate is generally similar to that in aerobic and facultatively aerobic organisms, but that glycine is synthesized from glucose via aspartate and threonine.     

Metabolism of absorbed aspartate,asparagine, and arginine by rat small intestine in vivo

l-[U-¹⁴C]aspartate, l-[U-¹⁴C]asparagine, and l-[U-¹⁴C]arginine were administered luminally into isolated segments of rat jejunum in situ, and the radioactive products appearing in venous blood from the segment were identified and quantified, in a continuation of similar studies with l-glutamate and l-glutamine (Windmueller H.G. and Spaeth, A. E. (1975) Arch. Biochem. Biophys. 171, 662–672). Aspartate, administered alone (6 mm) or with 18 other amino acids plus glucose, was absorbed more rapidly than glutamate, but, as with glutamate, less than 1% was recovered intact in intestinal venous blood. More than 50% of aspartate carbon was recovered in CO₂, 24% in organic acids, mostly lactate, 12% in other amino acids (alanine, glutamate, proline, ornithine, and citrulline), and 10% in glucose, apparently the first demonstration of gluconeogenesis by intestine in vivo. In contrast to aspartate and glutamine, nearly all asparagine was absorbed intact, less than 1% being catabolized. About 4% of the absorbed dose was incorporated into the acid-insoluble fraction of intestine, as was the case with all the amino acids studied. In conventional or germ-free rats, only 60% of arginine was absorbed intact, while 33% was hydrolyzed to ornithine and urea. The urea and 38% of the ornithine were released into the blood; the remaining ornithine was metabolized further by intestine to citrulline, proline, glutamate, organic acids, and CO₂. Catabolism of several amino acids from the lumen plus glutamine from arterial blood may provide an important energy source in small intestine.     

Stable isotope peptide mass spectrometry to decipher amino acid metabolism in Dehalococcoides strain CBDB1

Dehalococcoides species are key players in the anaerobic transformation of halogenated solvents at contaminated sites. Here, we analyze isotopologue distributions in amino acid pools from peptides of Dehalococcoides strain CBDB1 after incubation with (13)C-labeled acetate or bicarbonate as a carbon source. The resulting data were interpreted with regard to genome annotations to identify amino acid biosynthesis pathways. In addition to using gas chromatography-mass spectrometry (GC-MS) for analyzing derivatized amino acids after protein hydrolysis, we introduce a second, much milder method, in which we directly analyze peptide masses after tryptic digest and peptide fragments by nano-liquid chromatography-electrospray ionization-tandem mass spectrometry (nano-LC-ESI-MS/MS). With this method, we identify isotope incorporation patterns for 17 proteinaceous amino acids, including proline, cysteine, lysine, and arginine, which escaped previous analyses in Dehalococcoides. Our results confirmed lysine biosynthesis via the α-aminoadipate pathway, precluding lysine formation from aspartate. Similarly, the isotopologue pattern obtained for arginine provided biochemical evidence of its synthesis from glutamate. Direct peptide MS/MS analysis of the labeling patterns of glutamine and asparagine, which were converted to glutamate and aspartate during protein hydrolysis, gave biochemical evidence of their precursors and confirmed glutamate biosynthesis via a Re-specific citrate synthase. By addition of unlabeled free amino acids to labeled cells, we show that in strain CBDB1 none of the 17 tested amino acids was incorporated into cell mass, indicating that they are all synthesized de novo. Our approach is widely applicable and provides a means to analyze amino acid metabolism by studying specific proteins even in mixed consortia.     

Radical scavenging and electron-transfer reactions in Polyporus Versicolor laccase: A pulse radiolysis study

The interaction of the radicals OH^?, t-BuO^?, e_aq^?, CO₂^XXX and O₂^XXX with the copper oxidase. laccase. from Polyporus, has been studied by the pulse-radiolysis technique. Each of these radicals formed transient adducts with a broad absorption maximum around 310 nm. Analysis of the optical properties and of the very fast rates of formation of these compounds shows that each radical interacts with a limited number of sites on the polypeplide part of the protein amongst R-S-S-R. histidine and aromatic residues. Interaction with the carbonyl group of some of the peptide bonds is also possible. The few target sites are probably hit simultaneously and electron transfer between these sites may also occur. In all cases, in a subsequent step, intramolecular electron transfer from the polypeptide radical adducts leads to a partial reduction of the blue type-1 Cu²⁺ with rates varying between 10³ and 10⁴ s^?1. Further reduction of the type-1 Cu²⁺ occurs through a slow intermolecular reaction between two laccase radical transient adducts. In the case of CO^XXX₂ and O^XXX₂, this slow reduction could alternatively be due to an intermolecular reaction between laccase and CO^XXX₂ or O^XXX₂. The oxidant radicals OH^?. Br^XXX₂ and (SCN)^XXX₂, which formed radical adducts with fully ascorbate-reduced laccase, did not induce any type-1 copper reoxidation.