Amino Acid Metabolism of Lemna minor L. : IV. N-Labeling Kinetics of the Amide and Amino Groups of Glutamine and Asparagine

A serious limitation to the use of N(O,S)-heptafluorobutyryl isobutyl amino acid derivatives in the analysis of ¹⁵N-labeling kinetics of amino acids in plant tissues, is that the amides glutamine and asparagine undergo acid hydrolysis to glutamate and aspartate, respectively, during derivatization. This led us to consider an alternative procedure (G Fortier et al. [1986] J Chromatogr 361: 253-261) for derivatization of glutamine and asparagine with N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide in pyridine. Gas chromatography-mass spectrometry (electron ionization) yielded fragment ions (M-57) of mass 417 and 431 for the [¹⁴N]asparagine and [¹⁴N]glutamine derivatives, respectively, suitable for monitoring unlabeled, single-¹⁵N- and double-¹⁵N-labeled amide species from the ion clusters at mass to charge ratio (m/z) 415 to 423 for asparagine, and m/z 429 to 437 for glutamine. From separate analyses of the specific isotope abundance of the amino-N groups of asparagine and glutamine as their N-heptafluorobutyryl isobutyl derivatives, the specific amide-[¹⁵N] abundance of these amino acids was determined. We demonstrate that this approach to ¹⁵N analysis of the amides can yield unique insights as to the compartmentation of asparagine and glutamine in vivo. The ratios of unlabeled:single-¹⁵N:double-¹⁵N-labeled species are highly diagnostic of the relative sizes and turnover of metabolically active and inactive pools of the amides and their precursors. Kinetic evidence is presented to indicate that a significant proportion (approximately 10%) of the free asparagine pool may be metabolically inactive (vacuolar). If the amide group of asparagine is derived exclusively from glutamine-amide, then asparagine must be synthesized in a compartment of the cell in which both glutamine-amide and aspartate are more heavily labeled with ¹⁵N than the bulk pools of these amino acids. This compartment is presumably the chloroplast. The transaminase inhibitor aminooxyacetate is shown to markedly inhibit amino acid synthesis; several amino acid pools accumulated in the presence of aminooxyacetate and [¹⁵N]H₄⁺ are ¹⁴N-enriched and must be derived primarily from protein turnover.     

Utilization of the amide groups of asparagine and 2-hydroxysuccinamic Acid by young pea leaves

The fate of nitrogen originating from the amide group of asparagine in young pea leaves (Pisum sativum) has been studied by supplying [¹⁵N-amide]asparagine and its metabolic product, 2-hydroxysuccinamate (HSA) via the transpiration stream. Amide nitrogen from asparagine accumulated predominantly in the amide group of glutamine and HSA, and to a lesser extent in glutamate and a range of other amino acids. Treatment with 5-diazo,4-oxo-L-norvaline (DONV) a deamidase inhibitor, caused a decrease in transfer of label to glutamine-amide. Virtually no ¹⁵N was detected in HSA of leaves supplied with asparagine and the transaminase inhibitor aminooxyacetate. When [¹⁵N]HSA was supplied to pea leaves, most of the label was also found in the amide group of glutamine and this transfer was blocked by the addition of methionine sulfoximine, which caused a large increase in NH₃ accumulation. DONV was not specific for asparaginase, and inhibited the deamidation of HSA, causing a decrease in transfer of ¹⁵N into glutamine-amide, NH₃, and other amino acids. It is concluded from these results that use of the amide group of asparagine as a nitrogen source for young pea leaves involves deamidation of both asparagine and its transamination product HSA (possibly also oxosuccinamate). The amide group, released as ammonia, is then reassimilated via the glutamine synthetase/glutamate synthase system.     

Asparagine formation in soybean nodules

¹⁵NH₄⁺ and [¹⁵N](amide)-glutamine externally supplied to detached nodules from soybean plants (cv. Tamanishiki) were incorporated within nodule tissues by vacuum infiltration and metabolized to various nitrogen compounds during 60 minutes of incubation time. In the case of ¹⁵NH₄⁺ - feeding, the ¹⁵N abundance ratio was highest in the amide nitrogen of glutamine, followed by glutamate and the amide nitrogen of asparagine. In ¹⁵N content (micrograms excess ¹⁵N), the amide nitrogen of asparagine was most highly enriched after 60 minutes. ¹⁵NH₄⁺ was also appreciably assimilated into alanine.     

N]NMR determination of asparagine and glutamine nitrogen utilization for synthesis of storage protein in developing cotyledons of soybean in culture

Solid-state [¹⁵N]NMR was used to measure the use of the amide and amino nitrogens of glutamine and asparagine for synthesis of storage protein in cotyledons of soybean (Glycine max L. cv. Elf) in culture. No major discrimination in the incorporation of the amide or amino nitrogens of glutamine into protein is apparent, but the same nitrogens of asparagine are used with a degree of specificity. During the first seven days in culture with asparagine as the sole nitrogen source, the amino nitrogen donates approximately twice as much nitrogen to protein as does the amide nitrogen. The use of the amide nitrogen increases with longer periods of culture. The reduced use of the amide nitrogen was confirmed by its early appearance as ammonium in the culture medium. The amide nitrogen of asparagine was found at all times to be an essential precursor for protein because of its appearance in protein in residues whose nitrogens were not supplied by the amino nitrogen. In addition, methionine sulfoximine inhibited growth completely on asparagine, indicating that some ammonium assimilation is essential for storage protein synthesis. These results indicate that in a developing cotyledon, a transaminase reaction is of major importance in the utilization of asparagine for synthesis of storage protein and that, at least in the early stages of cotyledon development, reduced activities of ammonium-assimilating enzymes in the cotyledon tissue or in other tissues of the seed or pod may be a limiting factor in the use of asparagine-amide nitrogen.     

Estimation of amino acid residue side-chain absorption in the infrared spectra of protein solutions in heavy water.

The values of maximum frequencies, intensities, and other spectral parameters of the main absorption bands of amino acid residue side-chain groups have been obtained in the 1500–1800-cm^?1 region for solutions in heavy water at pD 1–12. It is shown that absorption of residues of asparagine, glutamine, aspartic and glutamic acids, arginine, and tyrosine must be taken into account in quantitative studies of the infrared spectra of polypeptide and protein solutions in heavy water. Examples of separating out the amide I band for ribonuclease A in heavy water are given.     

Evidence for the Glutamine Synthetase/Glutamate Synthase Pathway during the Photorespiratory Nitrogen Cycle in Spinach Leaves

Spinach leaf (Spinacia oleracea L.) discs infiltrated with [¹⁵N]glycine were incubated at 25°C in the light and in darkness for 0, 30, 60 and 90 minutes. The kinetics of ¹⁵N-incorporation into glutamine, glutamate, asparagine, aspartate, and serine from [¹⁵N]glycine was determined. At the beginning of the experiment, just after infiltration (0 min incubation) serine, and the amido-N of glutamine and asparagine were the only compounds significantly labeled in both light- and dark-treated leaf discs. Incorporation of ¹⁵N-label into the other amino acids was observed at longer incubation time. The per cent ¹⁵N-enrichment in all amino acids was found to increase with incubation. However, serine and the amido-N of glutamine remained the most highly labeled products in all treatments. The above pattern of ¹⁵N-labeling suggests that glutamine synthetase was involved in the initial refixation of ¹⁵NH₃ derived from [¹⁵N]glycine oxidation in spinach leaf discs.

The ¹⁵N-enrichment of the amino-N of glutamine was found to increase rapidly from 0 to 19% during incubation in the light. There was a comparatively smaller increase (4-9%) in the ¹⁵N-label of the amino-N of glutamine in tissue incubated in darkness. Furthermore the total flux of ¹⁵N-label into each of the amino acids examined was found to be greater in tissue incubated in the light than those in the dark. The above evidence indicates the involvement of the glutamine synthetase/glutamate synthase pathway in the recycling of photorespiratory NH₃ during glycine oxidation in spinach leaves.

     

Nitrogen metabolism of asparagine and glutamate in Vero cells studied by <Superscript>1</Superscript>H/<Superscript>15</Superscript>N NMR spectroscopy

Glutamine-free culture of Vero cells has previously been shown to cause higher cell yield and lower ammonia accumulation than that in glutamine-containing culture. Nitrogen metabolism of asparagine and glutamate as glutamine replacer was studied here using nuclear magnetic resonance (NMR) spectroscopy. ¹⁵N-labelled glutamate or asparagine was added and their incorporation into nitrogenous metabolites was monitored by heteronuclear multiple bond coherence (HMBC) NMR spectroscopy. In cells incubated with l-[¹⁵N]glutamate, the ¹⁵N label was subsequently found in a number of metabolites including alanine, aspartate, proline, and an unidentified compound. No detectable signal occurred, indicating that glutamate was utilized by transamination rather than by oxidative deamination. In cells incubated with l-[2-¹⁵N]asparagine, the ¹⁵N label was subsequently found in aspartate, the amine group of glutamate/glutamine, and in two unidentified compounds. Incubation of cells with l-[4-¹⁵N]asparagine showed that the amide nitrogen of asparagine was predominantly transferred to glutamine amide. There was no detectable production of , showing that most of the asparagine amide was transaminated by asparagine synthetase rather than deaminated by asparaginase. Comparing with a glutamine-containing culture, the activities of phosphate-activated glutaminase (PAG), glutamate dehydrogenase (GDH) and alanine aminotransferase (ALT) decreased significantly and the activity of aspartate aminotransferase (AST) decreased slightly.     

Efficient isotopic tryptophan labeling of membrane proteins by an indole controlled process conduct

A protocol for the efficient isotopic labeling of large G protein‐coupled receptors with tryptophan in Escherichia coli as expression host was developed that sufficiently suppressed the naturally occurring L‐tryptophan indole lyase, which cleaves tryptophan into indole, pyruvate, and ammonia resulting in scrambling of the isotopic label in the protein. Indole produced by the tryptophanase is naturally used as messenger for cell–cell communication. Detailed analysis of different process conducts led to the optimal expression strategy, which mimicked cell–cell communication by the addition of indole during expression. Discrete concentrations of indole and ¹⁵N₂‐L‐tryptophan at dedicated time points in the fermentation drastically increased the isotopic labeling efficiency. Isotope scrambling was only observed in glutamine, asparagine, and arginine side chains but not in the backbone. This strategy allows producing specifically tryptophan labeled membrane proteins at high concentrations avoiding the disadvantages of the often low yields of auxotrophic E. coli strains. In the fermentation process carried out according to this protocol, we produced ～15 mg of tryptophan labeled neuropeptide Y receptor type 2 per liter medium. Biotechnol. Bioeng. 2013; 110: 1681–1690. © 2013 Wiley Periodicals, Inc.     

Pathways of Nitrogen Metabolism in Nodules of Alfalfa (Medicago sativa L.)

Exposure of intact alfalfa nodules to ¹⁵N₂ showed that in bacteroids the greatest flow of ¹⁵N was to NH₃. Label was also detected in glutamic acid, aspartic acid, and asparagine (Glu, Asp and Asn), but at far lower levels. In the host plant cytosols, more ¹⁵N was incorporated into Asn than into other compounds. Detached nodules were also used to study the metabolic pathway of N assimilation after exposure to ¹⁵N₂ or vacuum infiltration with (¹⁵NH₄)₂SO₄ in the presence or absence of different inhibitors of nitrogen assimilation: methionine sulfoximine (MSO), azaserine (AZA), or amino-oxyacetate (AOA). Treatment with MSO, an inhibitor of glutamine synthetase (GS), inhibited the flow of the label to glutamine (Gln)-amide, resulting in subsequently decreased label in Asnamide. Aza, which inhibits the formation of Glu from Gln by glutamate synthase (GOGAT), enhanced the labeling of the amide groups of both Gln and Asn, while that of Asn-amino decreased. When AOA was used to block the transamination reaction very little label was found in Asp and Asn-amino. The results are consistent with the role of GS/GOGAT in the cytosol for the assimilation of NH₃ produced by N₂ fixation in the bacteroids of alfalfa nodules. Asn, a major nitrogen transport compound in alfalfa, is mainly synthesized by a Gln-dependent amidation of Asp, according to feeding experiments using the ¹⁵N-labeled amide group of glutamine. Data from ¹⁵NH₄⁺ feeding support some direct amidation of Asp to form Asn.     

Pathways of Nitrogen Assimilation in Cowpea Nodules Studied using N(2) and Allopurinol

In the presence of 0.5 millimolar allopurinol (4-hydroxypyrazolo [3,4-d]pyrimidine), an inhibitor of NAD:xanthine oxidoreductase (EC 1.2.3.2), intact attached nodules of cowpea (Vigna unguiculata L. Walp. cv Vita 3) formed [¹⁵N]xanthine from ¹⁵N₂ at rates equivalent to those of ureide synthesis, confirming the direct assimilation of fixed nitrogen into purines. Xanthine accumulated in nodules and was exported in increasing amounts in xylem of allopurinol-treated plants. Other intermediates of purine oxidation, de novo purine synthesis, and ammonia assimilation did not increase and, over the time course of experiments (4 hours), allopurinol had no effect on nitrogenase (EC 1.7.99.2) activity. Negligible ¹⁵N-labeling of asparagine from ¹⁵N₂ was observed, suggesting that the significant pool (up to 14 micromoles per gram of nodule fresh weight) of this amide in cowpea nodules was not formed directly from fixation but may have accumulated as a consequence of phloem delivery.     

Amino Acid metabolism in pea leaves : utilization of nitrogen from amide and amino groups of [N]asparagine

The flow of nitrogen from the amino and amide groups of asparagine has been followed in young pea (Pisum sativum CV Little Marvel) leaves, supplied through the xylem with ¹⁵N-labeled asparagine. The results confirm that there are two main routes for asparagine metabolism: deamidation and transamination.

Nitrogen from the amide group is found predominantly in 2-hydroxy-succinamic acid (derived from transamination of asparagine) and in the amide group of glutamine. The amide nitrogen is also found in glutamate and dispersed through a range of amino acids. Transfer to glutamineamide results from assimilation of ammonia produced by deamidation of both asparagine and its transamination products: this assimilation is blocked by methionine sulfoximine. The release of amide nitrogen as ammonia is greatly reduced by aminooxyacetate, suggesting that, for much of the metabolized asparagine, transamination precedes deamidation.

The amino group of asparagine is widely distributed in amino acids, especially aspartate, glutamate, alanine, and homoserine. For homoserine, a comparison of N and C labeling, and use of a transaminase inhibitor, suggests that it is not produced from the main pool of aspartate, and transamination may play a role in the accumulation of homoserine in peas.

     

The impact of mycorrhizal colonization upon nitrogen source utilization and metabolism in seedlings of Eucalyptus grandis Hill ex Maiden and Eucalyptus maculata Hook

Analysis of soil solution from forest sites dominated by Eucalyptus grandis and Eucalyptus maculata indicates that soluble forms of organic nitrogen (amino acids and protein) are present in concentrations similar to those of mineral nitrogen (nitrate and ammonium). Experiments were conducted to determine the extent to which mycorrhizal associations might broaden nitrogen source utilization in Eucalyptus seedlings to include organic nitrogen. In isolation, species of ectomycorrhizal fungi from northern Australia show varying abilities to utilize mineral and organic forms of nitrogen as sole sources. Pisolithus sp. displayed strongest growth on NH₄⁺, glutamine and asparagine, but grew poorly on protein, while Amanita sp. grew well both on mineral sources and on a range of organic sources (e.g. arginine, asparagine, glutamine and protein). In sterile culture, non-mycorrhizal seedlings of Eucalyptus grandis and Eucalyptus maculata grew well on mineral sources of nitrogen, but showed no ability to grow on sources of organic nitrogen other than glutamine. In contrast, mycorrhizal seedlings grew well on a range of organic nitrogen sources. These observations indicate that mycorrhizal associations confer on species of Eucalyptus the ability to broaden their resource base substantially with respect to nitrogen. This ability to utilize organic nitrogen was not directly related to that of the fungal symbiont in isolation. Seedlings mycorrhizal with Pisolithus sp. were able to assimilate sources of nitrogen (in particular histidine and protein) on which the fungus in pure culture appeared to grow weakly. Experiments in which plants were fed ¹⁵N-labelled ammonium were undertaken in order to investigate the influence of mycorrhizal colonization on the pathway of nitrogen metabolism. In roots and shoots of all seedlings, ¹⁵N was incorporated into the amide group of glutamine, and label was also found in the amino groups of glutamine, glutamic acid, γ-aminobutyric acid and alanine. Mycorrhizal colonization appeared to have no effect on the assimilation pathway and metabolism of [¹⁵N]H₄⁺; labelling data were consistent with the operation of the glutamate synthase cycle in plants infected with either Pisolithus sp. (which in isolation assimilates via the glutamate synthase cycle) or Elaphomyces sp. (which assimilates via glutamate dehydrogenase). It is likely that the control of carbon supply to the mycorrhizal fungus from the host may have a profound effect on both the assimilatory pathway and the range of nitrogen sources that can be utilized by the association.     

Amino Acid metabolism of pea leaves: labeling studies on utilization of amides

Short term (2-hour) incorporation of nitrogen from nitrate, glutamine, or asparagine was studied by supplying them as unlabeled (¹⁴N) tracers to growing pea (Pisum sativum L.) leaves, which were previously labeled with ¹⁵N, and then following the elimination of ¹⁵N from various amino components of the tissue. Most components had active and inactive pools. Ammonia produced from nitrate was assimilated through the amide group of glutamine. When glutamine was supplied, its nitrogen was rapidly transferred to glutamic acid, asparagine, and other products, and there was some transfer to ammonia. Nitrogen from asparagine was widely distributed into ammonia and amino compounds. There was a rapid direct transfer to glutamine, which did not appear to involve free ammonia. Alanine nitrogen could be derived directly from asparagine, probably by transamination. Homoserine was synthesized in substantial amounts from all three nitrogen sources. Homoserine appears to derive nitrogen more readily from asparagine than from free aspartic acid. A large proportion of the pool of γ-aminobutyric acid turned over, and was replenished with nitrogen from all three supplied sources.     

Assignment of aliphatic side-chain 1HN/15N resonances in perdeuterated proteins

Summary The perdeuteration of aliphatic sites in large proteins has been shown to greatly facilitate the process of sequential backbone and side-chain ¹³C assignments and has also been utilized in obtaining long-range NOE distance restraints for structure calculations. To obtain the maximum information from a 4D ¹⁵N/¹⁵N-separated NOESY, as many main-chain and side-chain ¹H_N/¹⁵N resonances as possible must be assigned. Traditionally, only backbone amide ¹H_N/¹⁵N resonances are assigned by correlation experiments, whereas slowly exchanging side-chain amide, amino, and guanidino protons are assigned by NOEs to side-chain aliphatic protons. In a perdeuterated protein, however, there is a minimal number of such protons. We have therefore developed several gradient-enhanced and sensitivity-enhanced pulse sequences, containing water-flipback pulses, to provide through-bond correlations of the aliphatic side-chain ¹H_N/¹⁵N resonances to side-chain ¹³C resonances with high sensitivity: NH₂-filtered 2D ¹H-¹⁵N HSQC (H₂N-HSQC), 3D H₂N(CO)C_/ and 3D H₂N(COC_/)C_/ for glutamine and asparagine side-chain amide groups; 2D refocused H(N_/)C_/ and H(N_/C_/)C_/ for arginine side-chain amino groups and non-refocused versions for lysine side-chain amino groups; and 2D refocused H(N)C and nonrefocused H(N_.)C for arginine side-chain guanidino groups. These pulse sequences have been applied to perdeuterated ¹³C-/¹⁵N-labeled human carbonic anhydrase II (²H-HCA II). Because more than 95% of all side-chain ¹³C resonances in ²H-HCA II have already been assigned with the C(CC)(CO)NH experiment, the assignment of the side-chain ¹H_N/¹⁵N resonances has been straightforward using the pulse sequences mentioned above. The importance of assigning these side-chain H_N protons has been demonstrated by recent studies in which the calculation of protein global folds was simulated using only ¹H_N-¹H_N NOE restraints. In these studies, the inclusion of NOE restraints to side-chain H_N protons significantly improved the quality of the global fold that could be determined for a perdeuterated protein [R.A. Venters et al. (1995) J. Am. Chem. Soc., 117, 9592–9593].To whom correspondence should be addressed.     

Hydrogen-deuterium exchange of a primary amide as a model for asparagine and glutamine exchange in proteins

Hydrogen-deuterium exchange of the primary amide, isobutyramide, was investigated as a model for asparagine and glutamine-NH₂ exchange in a protein. A simple amide was chosen since the structures of several well-characterized proteins show most of these residues to be exposed to solvent. Isobutyramide-exchange data were obtained in 1:1 D₂O:dioxane solutions using a near-infrared method. The rate data were strictly pseudo-first order and yielded an average of 95% exchange of the primary amide hydrogens. In analogy with secondary amides, the pD dependence of the rate constants was characteristic of specific acid and base catalysis. In addition, analysis of the rate-pD profile for isobutyramide indicated a significant uncatalyzed exchange reaction. Temperature-dependence studies of the first-order rate constants at a fixed pD yielded an apparent activation energy of 19.3 kcal/mole. Predicted half-life times for the exposed primary amide hydrogens in proteins, based on these exchange parameters, indicate that asparagine and glutamine side chains generally would contribute to the overall rate data only below 15°C and then only for approximately 1 pD unit around the point of minimum reaction velocity.     

Amino acid inhibition of the autophagic/lysosomal pathway of protein degradation in isolated rat hepatocytes

Protein degradation in isolated rat hepatocytes, as measured by the release of [¹⁴C]valine from pre-labelled protein, is partly inhibited by a physiologically balanced mixture of amino acids. The inhibition is largely due to the seven amino acids leucine, phenylalanine, tyrosine, tryptophan, histidine, asparagine and glutamine.When the amino acids are tested individually at different concentrations, asparagine and glutamine are the strongest inhibitors. However, when various combinations are tested, a mixture of the first five amino acids as well as a combination of leucine and asparagine inhibit protein degradation particularly strongly.The inhibition brought about by asparagine plus leucine is not additive to the inhibition by propylamine, a lysosomotropic inhibitor; thus indicating that the amino acids act exclusively upon the lysosomal pathway of protein degradation.Following a lag of about 15 min the effect of asparagine plus leucine is maximal and equal to the effect of propylamine, suggesting that their inhibition of the lysosomal pathway is complete as well as specific.Degradation of endocytosed ¹²⁵I-labelled asialofetuin is not affected by asparagine plus leucine, indicating that the amino acids do not affect lysosomes directly, but rather inhibit autophagy at a step prior to the fusion of autophagic vacuoles with lysosomes.The aminotransferase inhibitor, aminooxyacetate, does not prevent the inhibitory effect of any of the amino acids, i.e. amino acid metabolites are apparently not involved.     

Stereospecific assignment of the NH2 resonances from the primary amides of asparagine and glutamine side chains in isotopically labeled proteins

An HMQC-based pulse scheme is presented for the stereospecific assignment of asparagineand glutamine side-chain amide protons. The approach makes use of the recently developedquantitative-J correlation spectroscopy [Bax, A. et al. (1994) Methods Enzymol., 239,79–105] to distinguish the E and Z primary amide protons and, as such, eliminates theneed for assignments derived from more time-consuming and potentially ambiguous NOEmethods. An application of this method to a uniformly 15N,13C-labeled cellulose-bindingdomain is presented. When used in combination with a NOESY-HSQC experiment, thepredominant 2 dihedral angles of two asparagine side chains in this protein can also bedefined.     

Protein <Superscript>19</Superscript>F-labeling using transglutaminase for the NMR study of intermolecular interactions

The preparation of stable isotope-labeled proteins is important for NMR studies, however, it is often hampered in the case of eukaryotic proteins which are not readily expressed in Escherichia coli. Such proteins are often conveniently investigated following post-expression chemical isotope tagging. Enzymatic ¹⁵N-labeling of glutamine side chains using transglutaminase (TGase) has been applied to several proteins for NMR studies. ¹⁹F-labeling is useful for interaction studies due to its high NMR sensitivity and susceptibility. Here, ¹⁹F-labeling of glutamine side chains using TGase and 2,2,2-trifluoroethylamine hydrochloride was established for use in an NMR study. This enzymatic ¹⁹F-labeling readily provided NMR detection of protein-drug and protein–protein interactions with complexes of about 100 kDa since the surface residues provided a good substrate for TGase. The ¹⁹F-labeling method was 3.5-fold more sensitive than ¹⁵N-labeling, and could be combined with other chemical modification techniques such as lysine ¹³C-methylation. ¹³C-dimethylated-¹⁹F-labeled FKBP12 provided more accurate information concerning the FK506 binding site.     

NQ-Flipper: validation and correction of asparagine/glutamine amide rotamers in protein crystal structures

The error rate of asparagine (Asn) and glutamine (Gln) amide rotamers in protein crystal structures is in the order of 20% and as a consequence the current Protein Database (PDB) contains approximately half a million incorrect Asn and Gln side-chain rotamers. Here we present NQ-Flipper, a web service based on knowledge-based potentials of mean force to automatically detect and correct erroneous rotamers. We achieve excellent agreement with expert curated data.     

Phloem Loading and Metabolism of Xylem-Borne Amino Compounds in Fruiting Shoots of a Legume

Cut, fruiting shoots of Lupinus albus L. supplied with ¹⁴C-and ¹⁵N-labelled L-asparagine, L-glutamine, L-aspartic acid,or L-glutamic acid through the transpiration stream readilytransferred the labelled carbon and nitrogen of each compoundto pods and seeds of fruits. A time course of labelling of phloemsap collected from petioles and fruit tips following feedingof labelled asparagine indicated that xylem to phloem exchangein leaflets was an immediate and effective route of transferof the amide to fruits and that this and the loading onto phloemof additional asparagine from unlabelled pools of the amidein stems furnished a major source of the nitrogen for fruitfilling. Xylem to phloem exchange of nitrogen was accomplishedin different ways for each amino acid. The amide nitrogen ofasparagine was transferred mainly in the unmetabolized compound,the nitrogen of aspartate and glutamate largely in a wide rangeof amino acids synthesized in the leaf, and the amide nitrogenof glutamine was transferred in a manner intermediate betweenthese extremes. Glutamine and asparagine were the principalphloem solutes labelled with nitrogen from any of the suppliedcompounds, but the photosynthetically produced amino acids,glutamate, aspartate, serine, alanine, and valine also became¹⁵N-labelled in phloem. The main pathway for glutamine synthesisin vegetative parts of the shoot appeared to be by amidationof glutamate, but asparagine was not considered to be derivedsimilarly from aspartate. Leaflets metabolized glutamine morereadily than asparagine, but in each case the amide nitrogenwas used for synthesis of a variety of amino acids and the carbonwas recovered largely in non-amino compounds.