Production of an aromatic amino acid auxotroph of a trimethoprim-resistant Escherichia coli and deuteration of the aromatic amino acids in its dihydrofolate

Interpretation of the ¹H-NMR spectra of Escherichia coli dihydrofolate reductase is complicated by the large number of overlapping resonances due to protonated aromatic amino acids. Deuteration of the aromatic protons of aromatic amino acid residues is one technique useful for simplifying the ¹H-NMR spectra. Previous attempts to label the dihydrofolate reductase from over-producing strains of Escherichia coli were not completely successful. This labeling problem was solved by transducing via P1 phage a genetic block into the de novo biosynthetic pathway of aromatic amino acids in a trimethoprim resistant strain of E. coli, MB 3746. A new strain, MB 4065, is a very high level producer of dihydrofolate reductase and requires exogenous aromatic amino acids for growth, therefore allowing efficient labeling of its dihydrofolate reductase with exogenous deuterated aromatic amino acid.     

Sites of synthesis of plasma proteins in the foetal rat

1. The foetal rat of 16 or more days incorporates ¹⁴C-labelled amino acids into all the demonstrable plasma protein fractions in vivo. 2. Slices of foetal rat liver incubated in vitro incorporate ¹⁴C-labelled amino acids into the main plasma protein fractions, including the foetal-specific `post-albumin'. 3. Slices of placenta are unable to incorporate ¹⁴C-labelled amino acids into plasma proteins in vitro. 4. Liver slices from maternal rats incubated in vitro incorporate ¹⁴C-labelled amino acids into plasma proteins. The presence of post-albumin cannot be demonstrated after incubation. 5. Liver slices from foetal rats, but not from adult rats, contain demonstrable amounts of haemoglobin into which ¹⁴C-labelled amino acids are incorporated.     

Amino Acid Transport across the Tonoplast of Vacuoles Isolated from Barley Mesophyll Protoplasts : Uptake of Alanine, Leucine, and Glutamine

Mesophyll protoplasts from leaves of well-fertilized barley (Hordeum vulgare L.) plants contained amino acids at concentrations as high as 120 millimoles per liter. With the exception of glutamic acid, which is predominantly localized in the cytoplasm, a major part of all other amino acids was contained inside the large central vacuole. Alanine, leucine, and glutamine are the dominant vacuolar amino acids in barley. Their transport into isolated vacuoles was studied using ¹⁴C-labeled amino acids. Uptake was slow in the absence of ATP. A three- to sixfold stimulation of uptake was observed after addition of ATP or adenylyl imidodiphosphate an ATP analogue not being hydrolyzed by ATPases. Other nucleotides were ineffective in increasing the rate of uptake. ATP-Stimulated amino acid transport was not dependent on the transtonoplast pH or membrane potential. p-Chloromercuriphenylsulfonic acid and n-ethyl maleimide increased transport independently of ATP. Neutral amino acids such as valine or leucine effectively decreased the rate of alanine transport. Glutamine and glycine were less effective or not effective as competitive inhibitors of alanine transport. The results indicate the existence of a uniport translocator specific for neutral or basic amino acids that is under control of metabolic effectors.     

Amino Acid Uptake and Metabolism of Legionella pneumophila Hosted by Acanthamoeba castellanii

Legionella pneumophila survives and replicates within a Legionella-containing vacuole (LCV) of amoebae and macrophages. Less is known about the carbon metabolism of the bacteria within the LCV. We have now analyzed the transfer and usage of amino acids from the natural host organism Acanthamoeba castellanii to Legionella pneumophila under in vivo (LCV) conditions. For this purpose, A. castellanii was ¹³C-labeled by incubation in buffer containing [U-¹³C₆]glucose. Subsequently, these ¹³C-prelabeled amoebae were infected with L. pneumophila wild type or some mutants defective in putative key enzymes or regulators of carbon metabolism. ¹³C-Isotopologue compositions of amino acids from bacterial and amoebal proteins were then determined by mass spectrometry. In a comparative approach, the profiles documented the efficient uptake of Acanthamoeba amino acids into the LCV and further into L. pneumophila where they served as precursors for bacterial protein biosynthesis. More specifically, A. castellanii synthesized from exogenous [U-¹³C₆]glucose unique isotopologue mixtures of several amino acids including Phe and Tyr, which were also observed in the same amino acids from LCV-grown L. pneumophila. Minor but significant differences were only detected in the isotopologue profiles of Ala, Asp, and Glu from the amoebal or bacterial protein fractions, respectively, indicating partial de novo synthesis of these amino acids by L. pneumophila. The similar isotopologue patterns in amino acids from L. pneumophila wild type and the mutants under study reflected the robustness of amino acid usage in the LCV of A. castellannii.     

Amino Acid Synthesis in Photosynthesizing Spinach Cells : EFFECTS OF AMMONIA ON POOL SIZES AND RATES OF LABELING FROM CO(2)

Isolated cells from leaves of Spinacia oleracea have been maintained in a state capable of high rates of photosynthetic CO₂ fixation for more than 60 hours. The incorporation of ¹⁴CO₂ under saturating CO₂ conditions into carbohydrates, carboxylic acids, and amino acids, and the effect of ammonia on this incorporation have been studied. Total incorporation, specific radioactivity, and pool size have been determined as a function of time for most of the protein amino acids and for γ-aminobutyric acid. The measurements of specific radio-activities and of the approaches to ¹⁴C “saturation” of some amino acids indicate the presence and relative sizes of metabolically active and passive pools of these amino acids.     

Role of Amino Acids and Vitamins in Nutrition of Mesophilic Methanococcus spp

In this study we found that autotrophic methanococci similar to Methanococcus maripaludis obtained up to 57% of their cellular carbon from exogenous amino acids. About 85% of the incorporation was into protein. Primarily nonpolar and basic amino acids and glycine were incorporated; only small amounts of acidic and some polar amino acids were taken up. An additional 10% of the incorporation was into the nucleic acid fraction. Because little ¹⁴CO₂ was formed from the ¹⁴C-amino acids, little metabolism of the amino acids occurred. Therefore the growth stimulation by amino acids was probably due to the sparing of anabolic energy requirements. Of the amino acids incorporated, only alanine was also a sole nitrogen source for these methanococci. In contrast, Methanococcus vannielii and “Methanococcus aeolicus” are autotrophic methanococci which did not incorporate amino acids and did not utilize alanine as a sole nitrogen source. Although glutamine served as a sole nitrogen source for the autotrophic methanococci and Methanococcus voltae, a heterotrophic methanococcus, growth was due to chemical deamination in the medium. M. voltae requires leucine and isoleucine for growth. However, these amino acids were not significant nitrogen sources, and alanine was not a sole nitrogen source for the growth of M. voltae. The branched-chain amino acids were not extensively metabolized by M. voltae. Pantoyl lactone and pantoic acid were readily incorporated by M. voltae. The intact vitamin pantothenate was neither stimulatory to growth nor incorporated. In conclusion, although amino acids and vitamins are nutritionally important to both autotrophic and heterotrophic methanococci, generally they are not subject to extensive catabolism.     

Minimal Requirements for Exponential Growth of Lactococcus lactis

A minimal growth medium containing glucose, acetate, vitamins, and eight amino acids allowed for growth of Lactococcus lactis subsp. lactis, with a specific growth rate in batch culture of μ = 0.3 h^-1. With 19 amino acids added, the growth rate increased to μ = 0.7 h^-1 and the exponential growth phase proceeded until high cell concentrations were reached. We show that morpholinepropanesulfonic acid (MOPS) is a suitable buffer for L. lactis and may be applied in high concentrations.     

De Novo Synthesis of Amino Acids by the Ruminal Bacteria Prevotella bryantii B14, Selenomonas ruminantium HD4, and Streptococcus bovis ES1

The influence of peptides and amino acids on ammonia assimilation and de novo synthesis of amino acids by three predominant noncellulolytic species of ruminal bacteria, Prevotella bryantii B₁4, Selenomonas ruminantium HD4, and Streptococcus bovis ES1, was determined by growing these bacteria in media containing ¹⁵NH₄Cl and various additions of pancreatic hydrolysates of casein (peptides) or amino acids. The proportion of cell N and amino acids formed de novo decreased as the concentration of peptides increased. At high concentrations of peptides (10 and 30 g/liter), the incorporation of ammonia accounted for less than 0.16 of bacterial amino acid N and less than 0.30 of total N. At 1 g/liter, which is more similar to peptide concentrations found in the rumen, 0.68, 0.87, and 0.46 of bacterial amino acid N and 0.83, 0.89, and 0.64 of total N were derived from ammonia by P. bryantii, S. ruminantium, and S. bovis, respectively. Concentration-dependent responses were also obtained with amino acids. No individual amino acid was exhausted in any incubation medium. For cultures of P. bryantii, peptides were incorporated and stimulated growth more effectively than amino acids, while cultures of the other species showed no preference for peptides or amino acids. Apparent growth yields increased by between 8 and 57%, depending on the species, when 1 g of peptides or amino acids per liter was added to the medium. Proline synthesis was greatly decreased when peptides or amino acids were added to the medium, while glutamate and aspartate were enriched to a greater extent than other amino acids under all conditions. Thus, the proportion of bacterial protein formed de novo in noncellulolytic ruminal bacteria varies according to species and the form and identity of the amino acid and in a concentration-dependent manner.     

Fluorometric analysis of N alpha-methylamino acids using fluorescamine

The fluorometric amino acid analyzer based on fluorescamine has been utilized for quantitative determination of N^α-methylamino acids. N-Chlorosuccinimide (1 × 10^?3m in 0.05 m HCl) was continuously introduced into the column eluate to convert N^α-methylamino acids to fluorescamine-sensitive methylamine. As little as 100 pmoles of l-N-methylalanine was detectable with a linear fluorescence response up to 10.0 nmoles. Distinction of primary and secondary amino acids was achieved by carrying out duplicate analyses with and without the introduction of the N-chlorosuccinimide solution.     

Loss of organic acids, amino acids, k, and cl from barley roots treated anaerobically and with metabolic inhibitors

Excised roots of barley (Hordeum vulgare, var. Campana) lost organic acids, amino acids, K⁺, and Cl⁻ within 15 minutes after initiation of anaerobic treatment or treatment with NaCN and 2,4-dinitrophenol. Initial loss of organic acids when roots were placed under N₂ is attributed to a decarboxylation reaction, possibly catalyzed by phosphoenolpyruvate carboxykinase. Organic and amino acids began to leak from the roots to the bathing medium after 1 to 2 hours under N₂, indicating injury to cell membranes. During the first hour of anaerobic treatment, K⁺ loss from low-salt roots was equivalent to organic acid loss. Potassium loss from roots containing high levels of KCl was approximately equal to organic acid plus amino acid loss; and Cl⁻ loss was approximately equal to amino acid loss. It is postulated that, within cells, organic acids may electrostatically bind an equivalent quantity of cations and that amino acids may bind an equivalent quantity of both cations and anions.     

The influence of dietary calcium and phosphorus on intestinal calcium transport in rats given vitamin D metabolites.

A new method for the simple analysis of methylated amino acids based on autoradiography is introduced. With this technique a survey of protein methylation in a prokaryote, Escherichia coli, and a eukaryote, fibroblasts in culture, was carried out in an attempt to identify, quantitate, and determine the subcellular localization of all the methylated amino acids found in the proteins of these organisms.In mammalian cells using an established mouse fibroblast line (3T3), we have found that nuclei-free and mitochondria-free cytoplasm contain readily detectable amounts of four identifiable methylated amino acids: N^?,N^?-dimethyllysine, N^?,N^?,N^?-trimethyllysine, N^G,N^G-dimethylarginine (or N^G-methylarginine), and N^G,N′^G-dimethylarginine. The crude nuclear pellet also contains these methylated amino acids, but in addition contains N^?-methyllysine and a new as yet unidentified methylated compound. Histones purified from these nuclei contain essentially the same array of methylated compounds.The ribosomal subunits of the mammalian cells contained only small amounts of the methylated amino acids; the 40S subunit contained a substantial amount of just one, N^G,N^G-dimethylarginine (or N^G-methylarginine), and smaller amounts of N^G,N′^G-dimethylarginine, and an as yet unidentified methylated compound. The 60S subunit contained even smaller amounts of methylated amino acids, 50% of which was N^?,N^?,N^?-trimethyllysine and smaller amounts of N^?-methyllysine, N^?,N^?-dimethyllysine, and N^G,N^G-dimethylarginine. These subunits also contained an as yet unidentified methylated compoundThese results were in marked contrast to those that we obtained with the prokaryote, Escherichia coli. Only the proteins of the 50S ribosomal subunit of the bacteria contained methylated amino acids. Of those present 50% was N^?,N^?,N^?-trimethyllysine, with the remainder distributed about equally between N^?-methyllysine and three unknowns, one of which is apparently the same as that found in the 60S subunit of the mouse fibroblasts. All of the N^?-methyllysine was apparently in the small acidic proteins, L7 and L12.     

Monoiodo-d-tyrosine,an artificial amino acid radiopharmaceutical for selective measurement of membrane amino acid transport in the pancreas

Using ¹¹C-labeled natural amino acids, the functional diagnosis of tissue metabolism has been actively studied. Our interest has been focused on developing a clinically available ¹²³I-labeled artificial amino acid with a single metabolic function. For this study, [¹²³I]3-iodo-d-tyrosine ([¹²³I]d-MIT) was selected. In vitro and in vivo studies using ¹²⁵I-labeled d-MIT indicated that it showed a high pancreatic accumulation, selective affinity for membrane active transport systems, and was stable against enzymatic deiodination. A canine scintigraphic study using ¹²³I-labeled d-MIT and kinetic analysis showed that it behaved as an “artificial amino acid” radiopharmaceutical with selective membrane amino acid transport affinity in the pancreas.     

Gas Chromatography-Mass Spectrometry of N- Heptafluorobutyryl Isobutyl Esters of Amino Acids in the Analysis of the Kinetics of [N]H(4) Assimilation in Lemna minor L

Rapid, sensitive, and selective methods for the determination of the ¹⁵N abundance of amino acids in isotopic tracer experiments with plant tissues are described and discussed. Methodology has been directly tested in an analysis of the kinetics of [¹⁵N]H₄⁺ assimilation in Lemna minor L. The techniques utilize gas chromatography-mass spectrometry selected ion monitoring of major fragments containing the N moiety of N-heptafluorobutyryl isobutyl esters of amino acids. The ratio of selected ion pairs at the characteristic retention time of each amino acid derivative can be used to calculate ¹⁵N abundance with an accuracy of ±1 atom% excess ¹⁵N using samples containing as little as 30 picomoles of individual amino acids. Up to 11 individual amino acid derivatives can be selectively monitored in a single chromatogram of 30 minutes. It is suggested that these techniques will be useful in situations where the small quantities of N available for analysis have hitherto hindered the use of ¹⁵N-labeled precursors.     

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.     

Identification of endogenous acyl amino acids based on a targeted lipidomics approach

Using a partially purified bovine brain extract, our lab identified three novel endogenous acyl amino acids in mammalian tissues. The presence of numerous amino acids in the body and their ability to form amides with several saturated and unsaturated fatty acids indicated the potential existence of a large number of heretofore unidentified acyl amino acids. Reports of several additional acyl amino acids that activate G-protein coupled receptors (e.g., N-arachidonoyl glycine, N-arachidonoyl serine) and transient receptor potential channels (e.g., N-arachidonoyl dopamine, N-acyl taurines) suggested that some or many novel acyl amino acids could serve as signaling molecules. Here, we used a targeted lipidomics approach including specific enrichment steps, nano-LC/MS/MS, high-throughput screening of the datasets with a potent search algorithm based on fragment ion analysis, and quantification using the multiple reaction monitoring mode in Analyst software to measure the biological levels of acyl amino acids in rat brain. We successfully identified 50 novel endogenous acyl amino acids present at 0.2 to 69 pmol g⁻¹ wet rat brain.     

Modular pathways for editing non-cognate amino acids by human cytoplasmic leucyl-tRNA synthetase

To prevent potential errors in protein synthesis, some aminoacyl-transfer RNA (tRNA) synthetases have evolved editing mechanisms to hydrolyze misactivated amino acids (pre-transfer editing) or misacylated tRNAs (post-transfer editing). Class Ia leucyl-tRNA synthetase (LeuRS) may misactivate various natural and non-protein amino acids and then mischarge tRNA^Leu. It is known that the fidelity of prokaryotic LeuRS depends on multiple editing pathways to clear the incorrect intermediates and products in the every step of aminoacylation reaction. Here, we obtained human cytoplasmic LeuRS (hcLeuRS) and tRNA^Leu (hctRNA^Leu) with high activity from Escherichia coli overproducing strains to study the synthetic and editing properties of the enzyme. We revealed that hcLeuRS could adjust its editing strategy against different non-cognate amino acids. HcLeuRS edits norvaline predominantly by post-transfer editing; however, it uses mainly pre-transfer editing to edit α-amino butyrate, although both amino acids can be charged to tRNA^Leu. Post-transfer editing as a final checkpoint of the reaction was very important to prevent mis-incorporation in vitro. These results provide insight into the modular editing pathways created to prevent genetic code ambiguity by evolution.     

Accumulation of putrescine and related amino acids in potassium deficient Sesamum

Sesamum indicum was grown in complete or potassium deficient nutrient solution and amino acids, amines, nitrogen and potassium were determined weekly in the leaves. The incorporation of l-arginine-[U-¹⁴C] into protein was also followed. The interconversions of the amino acids of the ordithine-urea cycle, and their contribution to the formation of amines, were studied in cell-free extracts and intact leaves using labelled amino acids. As the level of potassium in the leaves decreased, the levels of the amino acids ornithine, citrulline and arginine, and of the amines putrescine, N-carbamylputrescine and agmatine increased. Potassium deficiency also reduced the rate of protein synthesis. Putrescine appears to be formed preferentially from citrulline with N-carbamylputrescine as intermediate.     

An Efficient and Epimerization Free Synthesis of C-Terminal Arylamides Derived from α-Amino Acids and Peptide Acids via T3P Activation

A high yield and rapid synthesis of enantiomerically pure N ^α -protected amino/peptide acid arylamides using n-propylphosphonic anhydride (T3P) in presence of N-methylmorpholine is described. The generality of the reaction has been studied for various N ^α -protected amino acids with diverse range of aromatic amines and coumarin derivatives.     

Uptake of intact amino acids by plants depends on soil amino acid concentrations

Studies in different ecosystems have shown that plants take up intact amino acids directly but little is known about the influence of free amino acid concentrations in the soil on this process. We investigated the effect of three different soil amino acid N concentrations (0.025, 0.13 and 2.5 μg N g^?1 soil) on direct uptake of four dual labelled (¹⁵N, ¹³C) amino acids (glycine, tyrosine, lysine, valine) in a greenhouse experiment using Anthoxantum odoratum as a model plant.Our results revealed that 8–45% of applied ¹⁵N was incorporated into plant root and shoot tissue 48 h after labelling. Additional ¹³C enrichment showed that 2–70% of this incorporated ¹⁵N was taken up as intact amino acid. Total ¹⁵N uptake and ¹⁵N uptake as intact amino acids were significantly affected by soil amino acid N concentrations and significantly differed between the four amino acids tested.We found a positive effect of soil amino acid concentrations on uptake of mineralized ¹⁵N relative to amino acid concentrations for all amino acids which was presumably due to higher diffusion rates of mineralized tracer to the root surface. However, intact amino acid uptake relative to amino acid concentrations as well as the proportion of total ¹⁵N taken up directly decreased with increasing soil amino acid N concentrations for all amino acids, irrespective of their microbial degradability. This effect is most likely controlled by the mineral N concentration in soil and perhaps in plants which inhibits direct amino acids uptake.Overall, we conclude that plant internal regulation of amino acid uptake controlled by mineral N is the main mechanism determining direct uptake of amino acids and thus a lower contribution of intact amino acid uptake to the plants N nutrition has to be expected for higher amino acid concentrations accompanied by mineralization in soil.     

Aminoacylation and translational quality control strategy employed by leucyl-tRNA synthetase from a human pathogen with genetic code ambiguity

Aminoacyl-tRNA synthetases should ensure high accuracy in tRNA aminoacylation. However, the absence of significant structural differences between amino acids always poses a direct challenge for some aminoacyl-tRNA synthetases, such as leucyl-tRNA synthetase (LeuRS), which require editing function to remove mis-activated amino acids. In the cytoplasm of the human pathogen Candida albicans, the CUG codon is translated as both Ser and Leu by a uniquely evolved CatRNA^Ser(CAG). Its cytoplasmic LeuRS (CaLeuRS) is a crucial component for CUG codon ambiguity and harbors only one CUG codon at position 919. Comparison of the activity of CaLeuRS-Ser⁹¹⁹ and CaLeuRS-Leu⁹¹⁹ revealed yeast LeuRSs have a relaxed tRNA recognition capacity. We also studied the mis-activation and editing of non-cognate amino acids by CaLeuRS. Interestingly, we found that CaLeuRS is naturally deficient in tRNA-dependent pre-transfer editing for non-cognate norvaline while displaying a weak tRNA-dependent pre-transfer editing capacity for non-cognate α-amino butyric acid. We also demonstrated that post-transfer editing of CaLeuRS is not tRNA^Leu species-specific. In addition, other eukaryotic but not archaeal or bacterial LeuRSs were found to recognize CatRNA^Ser(CAG). Overall, we systematically studied the aminoacylation and editing properties of CaLeuRS and established a characteristic LeuRS model with naturally deficient tRNA-dependent pre-transfer editing, which increases LeuRS types with unique editing patterns.