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The structure of the metabolic network is highly conserved, but we know little about its evolutionary origins. Key for explaining the early evolution of metabolism is solving a chicken–egg dilemma, which describes that enzymes are made from the very same molecules they produce. The recent discovery of several nonenzymatic reaction sequences that topologically resemble central metabolism has provided experimental support for a “metabolism first” theory, in which at least part of the extant metabolic network emerged on the basis of nonenzymatic reactions. But how could evolution kick-start on the basis of a metal catalyzed reaction sequence, and how could the structure of nonenzymatic reaction sequences be imprinted on the metabolic network to remain conserved for billions of years? We performed an in vitro screening where we add the simplest components of metabolic enzymes, proteinogenic amino acids, to a nonenzymatic, iron-driven reaction network that resembles glycolysis and the pentose phosphate pathway (PPP). We observe that the presence of the amino acids enhanced several of the nonenzymatic reactions. Particular attention was triggered by a reaction that resembles a rate-limiting step in the oxidative PPP. A prebiotically available, proteinogenic amino acid cysteine accelerated the formation of RNA nucleoside precursor ribose-5-phosphate from 6-phosphogluconate. We report that iron and cysteine interact and have additive effects on the reaction rate so that ribose-5-phosphate forms at high specificity under mild, metabolism typical temperature and environmental conditions. We speculate that accelerating effects of amino acids on rate-limiting nonenzymatic reactions could have facilitated a stepwise enzymatization of nonenzymatic reaction sequences, imprinting their structure on the evolving metabolic network., The evolutionary origins of metabolism are largely unknown. This study shows that the prebiotically available proteinogenic amino acid cysteine can promote the metabolism-like rate-limiting formation of ribose-5-phosphate, suggesting that early metabolic pathways could have emerged thought the stepwise enzymatization of non-enzymatic reaction sequences.

—The trace element selenium, which was discovered by Berzelius in 1817, remains poorly investigated and exhibits diverse and surprising functions. We consider it unique for several reasons. First, it is present not only in organic or inorganic compounds but also in the amino acids selenocysteine and selenomethionine, as a key component, and in selenoproteins, which are found in all Domains of life. Second, selenocysteine is the 21st proteinogenic amino acid. Its uniqueness lies not only in the fact that it is encoded by one of the three stop codons of translation but in that its biosynthesis possesses unique features and that this process involves unique cis- and trans-active factors that are necessary for the recognition of this triplet as selenocysteine-encoding to avoid premature translation termination and synthesize full-sized selenoproteins. The maintenance of these cis- and trans-active factors is energy consuming, which suggests the crucial importance of selenoproteins for an organism. In addition, the pathways of amino-acid and selenoprotein biosynthesis have some distinctive features in different Domains of life. Third, the processes and effects of selenium compounds of different origins, in which this micronutrient plays key roles, are strikingly diverse, especially in the regulation of vital functions in mammals. This review uses the most recent data to present a comprehensive view of the properties and functions of selenium and to provide insight into the uniqueness of this micronutrient.

The first product of sulfate assimilation in plants, cysteine, is a proteinogenic amino acid and a source of reduced sulfur for plant metabolism. Cysteine synthesis is the convergence point of the three major pathways of primary metabolism: carbon, nitrate, and sulfate assimilation. Despite the importance of metabolic and genetic coordination of these three pathways for nutrient balance in plants, the molecular mechanisms underlying this coordination, and the sensors and signals, are far from being understood. This is even more apparent in C4 plants, where coordination of these pathways for cysteine synthesis includes the additional challenge of differential spatial localization. Here we review the coordination of sulfate, nitrate, and carbon assimilation, and show how they are altered in C4 plants. We then summarize current knowledge of the mechanisms of coordination of these pathways. Finally, we identify urgent questions to be addressed in order to understand the integration of sulfate assimilation with carbon and nitrogen metabolism particularly in C4 plants. We consider answering these questions to be a prerequisite for successful engineering of C4 photosynthesis into C3 crops to increase their efficiency., This review summarizes current knowledge on the coordination of sulfur, nitrogen, and carbon metabolism and discusses sulfate assimilation in the context of evolution of C4 photosynthesis.

Tellurium is a versatile heavy chalcogen with numerous applications in chemical biology, providing valuable probes in mass cytometry, fluorescence imaging and structural biology. L-Tellurienylalanine (TePhe) is an analogue of the proteinogenic amino acid L-phenylalanine (Phe) in which the phenyl side chain has been replaced by a 5-membered tellurophene moiety. High incorporation level of TePhe in expressed proteins at defined sites is expected to facilitate studies in proteomics, protein NMR spectroscopy, and structure elucidation. As a model we chose immunoglobulin-binding Protein G, B1 domain (GB1) to validate TePhe as a suitable structural analogue for Phe. We demonstrate that approximately 1 in 2 of all Phe sites within GB1 can be substituted with TePhe through expression in standard non-Phe-auxotrophic E. coli in Phe-deficient media containing glyphosate, an inhibitor of aromatic amino acid biosynthesis. The TePhe content of the GB1 sample can be further increased to 85 % through HPLC. Using NMR and CD spectroscopy, we confirm that the Phe-to-TePhe substitution has negligible impact on the global structure and stability of GB1.

More than 50% of proteinogenic amino acid sidechains can bind metal ions, enabling proteins and peptides to bear these ions as cofactors. Nevertheless, post-translational modifications and incorporation of noncanonical amino acids bestow peptides and proteins myriads of other coordination capabilities, thanks to an enhanced metal binding. Here we summarize selected examples of natural and artificial systems that contain one or more noncanonical amino acids coordinating a metal ion and subsequently achieve a new or enhanced function. We report on a wide array of systems: from disease-related proteins that undergo sulfurylation or phosphorylation through natural metallophores that selectively capture precious essential ions to synthetic selfassembly strategies, biocatalysts, and chelating agents against toxic metals. Regardless of their (bio)synthetic routes, all possess unique metal-binding properties that could not be effectively achieved by systems composed of canonical residues.

The proteinogenic composition of yeast extract products varies from experience for manufacturing reasons, though also due to the yeast starting product. Therefore, this study is the first to focus solely on the influence of three industrially applicable cell disruption methods (cell mill, ultrasonic sonotrode and autolysis) on the amino acid and protein composition of a yeast extract. A consistent spent yeast (Saccharomyces cerevisiae TUM 68) produced in a standardized industrial pilot top-fermenting process was used as a raw material for the first time. The disruption effectiveness of autolysis (98%) was higher than that of the mechanical methods (80%), as well as the cleavage of amino acids from the cell protein (307, 155 and 115 mg per g yeast extract for autolysis, sonotrode and cell mill). The proteinogenic amino acid release profiles were dependent upon the disruption methods. The greater the released quantity of an amino acid during autolysis, the more the mean value fluctuated in the prepared yeast extract. Protein size fractionation of the extract using electrophoresis showed differences ranging between 1.5 and 95 kDa. All yeast extracts evidenced good nutritional potential according to FAO/WHO standards. The calculated data showed that the manufacturing method has a big impact on the proteinogenic composition of a yeast extract and the spent yeast TUM 68 used in this study can yield a protein-rich yeast extract.

Amino acids constitute the key sources of nitrogen for growth of Candida albicans. In order to survive inside the host in different and rapidly changing environments, this fungus must be able to adapt via its expression of genes for amino acid metabolism. We analysed the ARO8, ARO9, YER152C, and BNA3 genes with regards to their role in the nutritional flexibility of C. albicans. CaAro8p is undoubtedly the most versatile enzyme among the aminotransferases investigated. It is involved in the catabolism of histidine, lysine, and aromatic amino acids as well as in l-Lys, Phe and Tyr biosynthesis. CaAro9p participates in the catabolism of aromatic amino acids and lysine at high concentrations of these compounds, with no biosynthetic role. Conversely, the CaYer152Cp catalytic potential for aromatic amino acid catabolism observed in vitro appears to be of little importance in vivo. Neither biosynthetic nor catabolic roles of CaBan3p were observed for any proteinogenic amino acid. Finally, none of the analysed aminotransferases was solely responsible for the catabolism of a single particular amino acid or its biosynthesis.

Fluorine is an element with unusual properties that has found significant utility in the design of synthetic small molecules, ranging from therapeutics to materials. In contrast, only a few fluorinated compounds made by living organisms have been found to date, most of which derive from the fluoroacetate/fluorothreonine biosynthetic pathway first discovered in Streptomyces cattleya. While fluoroacetate has long been known to act as an inhibitor of the tricarboxylic acid cycle, the fate of the amino acid fluorothreonine is still not well understood. Here, we show that fluorothreonine can be misincorporated into protein in place of the proteinogenic amino acid threonine. We have identified two conserved proteins from the organofluorine biosynthetic locus, FthB and FthC, that are involved in managing fluorothreonine toxicity. Using a combination of biochemical, genetic, physiological, and proteomic studies, we show that FthB is a trans-acting transfer RNA (tRNA) editing protein, which hydrolyzes fluorothreonyl-tRNA 670-fold more efficiently than threonyl-RNA, and assign a role to FthC in fluorothreonine transport. While trans-acting tRNA editing proteins have been found to counteract the misacylation of tRNA with commonly occurring near-cognate amino acids, their role has yet to be described in the context of secondary metabolism. In this regard, the recruitment of tRNA editing proteins to biosynthetic clusters may have enabled the evolution of pathways to produce specialized amino acids, thereby increasing the diversity of natural product structure while also attenuating the risk of mistranslation that would ensue.

Pyrrolysine is the 22nd proteinogenic amino acid encoded into proteins in response to amber (TAG) codons in a small number of archaea and bacteria. The incorporation of pyrrolysine is facilitated by a specialized aminoacyl-tRNA synthetase (PylRS) and its cognate tRNA (tRNAPyl). The secondary structure of tRNAPyl contains several unique features not found in canonical tRNAs. Numerous studies have demonstrated that the PylRS/tRNAPyl pair from archaea is orthogonal in E. coli and eukaryotic hosts, which has led to the widespread use of this pair for the genetic incorporation of non-canonical amino acids. In this brief review we examine the work that has been done to elucidate the structure of tRNAPyl, its interaction with PylRS, and survey recent progress on the use of tRNAPyl as a tool for genetic code expansion.

The proteinogenic amino acid l-serine is a precursor for various essential biomolecules in all organisms. 3-Phosphoglycerate dehydrogenase (PGDH) is the first committed enzyme of the phosphorylated pathway of l-serine biosynthesis, and is regulated by negative feedback from l-serine in bacteria and plants. In the present study, two Arabidopsis PGDH isoforms were inhibited by l-serine but were activated by l-amino acids such as l-homocysteine in vitro. Activation and inhibition by these amino acids was cooperative, suggesting an allosteric mechanism. Moreover, the half maximal effective concentration of l-homocysteine was 2 orders of magnitude lower than that of l-serine, suggesting greater regulatory potency. These are the first data to show that PGDH is activated by various biomolecules and indicate that serine biosynthesis is regulated by multiple pathways.

Selenium deficiency in various degrees affects around 15% of the world’s population, contributing to a variety of health problems. In this study, we examined the accumulation and biotransformation of soil applied Se-supplementation (sodium selenite and sodium selenate forms) at different concentrations, along with growth and yield formation of green pea, in a greenhouse experiment. Biotransformation of inorganic Se was evaluated using HPLC-ICP-MS for Se-species separation in the above ground parts of green pea. Results showed 3 mg kg−1 SeIV increased green pea growth biomarkers and also caused an increase in protein content in leaves by 17%. Selenomethionine represented 65% of the total selenium content in shoots, but was lower in pods and seeds (54 and 38%, respectively). Selenomethionine was the major species in all plant parts and the only organic selenium form in the lower SeIV concentration range. Elevating the dose of SeIV (≥30 mg kg−1) triggered detrimental effects on growth and protein content and caused higher accumulation of inorganic Se in forms of SeVI and SeIV. Selenocysteine, another organic form of proteinogenic amino acid, was determined when SeIV (≥10 mg kg−1) was applied in higher concentrations. Thus, agronomic biofortification using the appropriate chemical form and concentration of Se will have positive effects on green pea growth and its enriched shoots and seeds provide a value-added protein source for livestock and humans with significant increased selenomethionine.

Spent yeast from beer manufacturing is a cost-effective and nutrient-rich starting material for the production of yeast extracts. In this study, it is shown how physiologically important ingredients in a yeast extract are influenced by the composition of the spent yeast from the brewing process. In pilot fermentations, the time of cropping (primary fermentation, lagering) of the spent yeast and the original gravity (12 ˚P, 16 ˚P, 20 ˚P) of the fermentation medium was varied, and four alternative non-Saccharomyces yeast strains were compared with two commercial Saccharomyces yeast strains. In addition, spent yeast was contaminated with the beer spoiler Lactobacillus brevis. The general nutrient composition (total protein, fat, ash) was investigated as well as the proteinogenic amino acid spectrum, the various folate vitamers (5-CH3-H4folate, 5-CHO-H4folate, 10-CHO-PteGlu, H4folate, PteGlu) and the biological activity (reduction, antioxidative potential) of a mechanically (ultrasonic sonotrode) and an autolytically produced yeast extract. All the investigated ingredients from the yeast extract were influenced by the composition of the spent yeast from the brewing process. The biodiversity of the spent yeast from the brewing process therefore directly affects the content of physiologically valuable ingredients of a yeast extract and should be taken into consideration in industrial manufacturing processes.

Selenocysteine is the only proteinogenic amino acid encoded by a recoded in-frame UGA codon that does not operate as the canonical opal stop codon. A specialized translation elongation factor, eEFSec in eukaryotes and SelB in prokaryotes, promotes selenocysteine incorporation into selenoproteins by a still poorly understood mechanism. Our structural and biochemical results reveal that four domains of human eEFSec fold into a chalice-like structure that has similar binding affinities for GDP, GTP and other guanine nucleotides. Surprisingly, unlike in eEF1A and EF-Tu, the guanine nucleotide exchange does not cause a major conformational change in domain 1 of eEFSec, but instead induces a swing of domain 4. We propose that eEFSec employs a non-canonical mechanism involving the distinct C-terminal domain 4 for the release of the selenocysteinyl-tRNA during decoding on the ribosome., Specialized translation elongation factors (eEFSec and SelB) promote selenocysteine incorporation into proteins. Here, the authors report the structure of human eEFSec, examine its interactions with guanine nucleotides, and propose a non-canonical mechanism for decoding selenocysteine.

Selenocysteine is the 21st proteinogenic amino acid in mammals. The human genome contains 25 genes encoding selenoproteins, and their significance for human health is increasingly recognized through the identification of patients with inborn errors in selenoprotein biosynthetic factors or in individual selenoproteins. Mutations in selenoprotein N (SEPN1) lead to a spectrum of disorders collectively called SEPN1-related myopathy, and mutations in glutathione peroxidase 4 (GPX4) cause respiratory failure and bone defects, and mutations in thioredoxin reductase 2 (TXNRD2) are associated with familial glucocorticoid deficiency. Pathogenic mutations in selenocysteine synthase (SEPSECS) cause neurodevelopmental disorders, but also other factors epistatic to selenoprotein biosynthesis, such as SECIS-binding protein 2 (SECISBP2) and tRNA

Hundreds of non-proteinogenic (np) amino acids (AA) are found in plants and can in principle enter human protein synthesis through foods. While aminoacyl-tRNA synthetase (AARS) editing potentially provides a mechanism to reject np AAs, some have pathological associations. Co-crystal structures show that vegetable-sourced azetidine-2-carboxylic acid (Aze), a dual mimic of proline and alanine, is activated by both human prolyl- and alanyl-tRNA synthetases. However, it inserts into proteins as proline, with toxic consequences in vivo. Thus, dual mimicry increases odds for mistranslation through evasion of one but not both tRNA synthetase editing systems.

Lactococcus lactissubsp.cremorisMG1363 is an important starter culture for dairy fermentation. During industrial fermentations,L. lactisis constantly exposed to stresses that affect the growth and performance of the bacterium. Although the response ofL. lactisto several stresses has been described, the adaptation mechanisms at the level ofin vivofluxes have seldom been described. To gain insights into cellular metabolism,13C metabolic flux analysis and gas chromatography mass spectrometry (GC-MS) were used to measure the flux ratios of active pathways in the central metabolism ofL. lactiswhen subjected to three conditions varying in temperature (30°C, 37°C) and agitation (with and without agitation at 150 rpm). Collectively, the concentrations of proteinogenic amino acids (PAAs) and free fatty acids (FAAs) were compared, and Pearson correlation analysis (r) was calculated to measure the pairwise relationship between PAAs. Branched chain and aromatic amino acids, threonine, serine, lysine and histidine were correlated strongly, suggesting changes in flux regulation in glycolysis, the pentose phosphate (PP) pathway, malic enzyme and anaplerotic reaction catalysed by pyruvate carboxylase (pycA). Flux ratio analysis revealed that glucose was mainly converted by glycolysis, highlighting the stability ofL. lactis’central carbon metabolism despite different conditions. Higher flux ratios through oxaloacetate (OAA) from pyruvate (PYR) reaction in all conditions suggested the activation of pyruvate carboxylate (pycA) inL. lactis, in response to acid stress during exponential phase. Subsequently, more significant flux ratio differences were seen through the oxidative and non-oxidative pentose phosphate (PP) pathways, malic enzyme, and serine and C1 metabolism, suggesting NADPH requirements in response to environmental stimuli. These reactions could play an important role in optimization strategies for metabolic engineering inL. lactis. Overall, the integration of systematic analysis of amino acids and flux ratio analysis provides a systems-level understanding of howL. lactisregulates central metabolism under various conditions.

Streptomyces sp. GSL-6B was isolated from sediment collected from the Great Salt Lake and investigation of its organic extract led to the isolation of three new linear heptapeptides, bonnevillamides A (1), B (2), and C (3). The bonnevillamides represent a new class of linear peptides featuring unprecedented non-proteinogenic amino acids. All three peptides contain the newly characterized bonnevillic acid moiety (3-(3,5-dichloro-4-methoxyphenyl)-2-hydroxyacrylic acid), as well as a heavily modified proline residue. Moreover, in bonnevillamide A, the terminal proline residue found in bonnevillamides B and C is replaced with 4-methyl-azetidine-2-carboxylic acid methyl ester. The structures of the three heptapeptides were elucidated by NMR, high-resolution electrospray ionization mass spectroscopy (HRESIMS), and LC-MS/MS, and the absolute configuration of all proteinogenic amino acid residues were determined by advanced Marfey’s method. Bonnevillamides A, B and C were evaluated for their effects on zebrafish embryo development. All three heptapeptides were shown to modulate heart growth and cardiac function, with bonnevillamide B having the most pronounced effect.

The genetic incorporation of the 22nd proteinogenic amino acid, pyrrolysine (Pyl) at amber codon is achieved by the action of pyrrolysyl-tRNA synthetase (PylRS) together with its cognate tRNA(Pyl). Unlike most aminoacyl-tRNA synthetases, PylRS displays high substrate side chain promiscuity, low selectivity toward its substrate α-amine, and low selectivity toward the anticodon of tRNA(Pyl). These unique but ordinary features of PylRS as an aminoacyl-tRNA synthetase allow the Pyl incorporation machinery to be easily engineered for the genetic incorporation of more than 100 non-canonical amino acids (NCAAs) or α-hydroxy acids into proteins at amber codon and the reassignment of other codons such as ochre UAA, opal UGA, and four-base AGGA codons to code NCAAs.

Secondary metabolites of photoautotrophic organisms have attracted considerable interest in recent years. In particular, molecules of non-proteinogenic amino acids participating in various physiological processes and capable of producing adverse ecological effects have been actively investigated. For example, the non-proteinogenic amino acid β-N-methylamino-L-alanine (BMAA) is neurotoxic to animals including humans. It is known that BMAA accumulation via the food chain can lead to development of neurodegenerative diseases in humans such as Alzheimer's and Parkinson's diseases as well as amyotrophic lateral sclerosis. Moreover, BMAA can be mistakenly incorporated into a protein molecule instead of serine. Natural sources of BMAA and methods for its detection are discussed in this review, as well as the role of BMAA in metabolism of its producers and possible mechanisms of toxicity of this amino acid in different living organisms.

Treatment of α- t -Butyl β-methyl N -Z-(S)-aspartate (2) with lithium amide bases generates the corresponding β-ester enolate, which can be alkylated with suitable electrophiles. The application of this strategy for synthesis of optically active amino acids has been investigated.

The repeated removal of flower, fruit, or vegetative buds is a common treatment to simulate sink limitation. These experiments usually lead to the accumulation of specific proteins, which are degraded during later stages of seed development, and have thus been designated as vegetative storage proteins. We used oligonucleotide microarrays to assess global effects of sink removal on gene expression patterns in soybean leaves and found an induction of the transcript levels of hundreds of genes with putative roles in the responses to biotic and abiotic stresses. In addition, these data sets indicated potential changes in amino acid and phenylpropanoid metabolism. As a response to sink removal we detected an induced accumulation of γ-aminobutyric acid, while proteinogenic amino acid levels decreased. We also observed a shift in phenylpropanoid metabolism with an increase in isoflavone levels, concomitant with a decrease in flavones and flavonols. Taken together, we provide evidence that sink removal leads to an up-regulation of stress responses in distant leaves, which needs to be considered as an unintended consequence of this experimental treatment.

Metabolic flux analysis using (13)C enrichment data of intracellular free amino acids (FAAs) can improve the time resolution of flux estimation compared to analysis of proteinogenic amino acid data owing to the faster turnover times of FAAs. The nature of the (13)C enrichment dynamics of FAAs remains obscure, however, especially with regard to its dependence on culture conditions, even though an understanding of dynamic behavior is important for precise metabolic flux estimation. In this study, we analyzed the (13)C enrichment dynamics of free and proteinogenic amino acids in a series of continuous culture experiments with Escherichia coli. The results indicated that the effect of protein degradation on the (13)C enrichment of FAAs was negligible under cellular growth conditions. Furthermore, they showed that the time scale necessary for (13)C enrichment dynamics of FAAs to reach a steady state depends on culture conditions such as oxygen uptake rate, which was likely due to different pool sizes of intracellular metabolites. The results demonstrate the importance of analyzing (13)C enrichment dynamics for the precise estimation of metabolic fluxes using FAA data.

Focusing on drug discovery non-proteinogenic amino acids have often been used as important building blocks for construction of compound libraries in the filed of combinatorial chemistry and chemical biology. Highly homogeneous L: -mimosine, α-amino-β-(3-hydoxy-4-oxo-1,4-dihydropyridin-1-yl)-propanoic acid, a non-proteinogenic amino acid, has been successfully isolated and purified on an industrial scale from wild leaves of Leucaena (Leucaena leucocephala de Wit) which is a widely distributed legume in Okinawa, a sub-tropical island in Japan. Optical purity determinations used for quality control have been established through diastereomer formation. Physico-chemical properties and biological properties of purified mimosine have been clarified. Mimosine is sparingly soluble in water and organic solvents but can be dissolved in aqueous alkaline solution. The tyrosinase pathway is of particular interest in the cosmetic field, since mimosine is an analog of tyrosine. Thus the present purified mimosine have been tested in tyrosinase inhibitory assays. The IC50 for tyrosinase inhibitory activity of purified Mim was compared with kojic acid. Mimosine shows significant inhibition of melanin production in murine melanoma cells. The derivatization of mimosine has been investigated with a focus on its use in conventional peptide syntheses to generate mimosyl peptides. N-(9-Fluorenylmethoxycarbonyloxy)-mimosine and resin-bound mimosine for solid-phase syntheses have also been performed. Highly homogeneous Mim is a useful material for the development of functional cosmetics or active pharmaceutical ingredients.

The expression of selenoproteins is controlled on each one of the textbook steps of protein biosynthesis, i.e., during gene transcription, RNA processing, translation and posttranslational events as well as via control of the stability of the involved intermediates and final products. Selenoproteins are unique in their dependence on the trace element Se which they incorporate as the 21st proteinogenic amino acid, selenocysteine. Higher mammals have developed unique pathways to enable a fine-tuned expression of all their different selenoproteins according to developmental stage, actual needs, and current availability of the trace element. Tightly controlled and dynamic expression patterns of selenoproteins are present in different tissues. Interestingly, these patterns display some differences in male and female individuals, and can be grossly modified during disease, e.g. in cancer, inflammation or neurodegeneration. Likewise, important health issues related to the selenium status show unexpected sexual dimorphisms. Some detailed molecular insights have recently been gained on how the hierarchical Se distribution among the different tissues is achieved, how the selenoprotein biosynthesis machinery discriminates among the individual selenoprotein transcripts and how impaired selenoprotein biosynthesis machinery becomes phenotypically evident in humans. This review tries to summarize these fascinating findings and highlights some interesting and surprising sex-specific differences.

L-canavanine occurs as a toxic non-protein amino acid in more than 1500 leguminous plants. One mechanism of its toxicity is its incorporation into proteins, replacing L-arginine and giving rise to functionally aberrant polypeptides. A comparison between the recombinant arginyl-tRNA synthetases from a canavanine producer (jack bean) and from a related non-producer (soybean) provided an opportunity to study the mechanism that has evolved to discriminate successfully between the proteinogenic amino acid and its non-protein analogue. In contrast to the enzyme from jack bean, the soybean enzyme effectively produced canavanyl-tRNA(Arg) when using RNA transcribed from the jack bean tRNA(ACG) gene. The corresponding k(cat)/K(M) values gave a discrimination factor of 485 for the jack bean enzyme. The arginyl-tRNA synthetase does not possess hydrolytic post-transfer editing activity. In a heterologous system containing either native Escherichia coli tRNA(Arg) or the modification-lacking E. coli transcript RNA, efficient discrimination between L-arginine and L-canavanine by both plant enzymes (but not by the E. coli arginyl-tRNA synthetase) occurred. Thus, interaction of structural features of the tRNA with the enzyme plays a significant role in determining the accuracy of tRNA arginylation. Of the potential amino acid substrates tested, apart from L-canavanine, only L-thioarginine was active in aminoacylation. As it is an equally good substrate for the arginyl-tRNA synthetase from both plants, it is concluded that the higher discriminatory power of the jack bean enzyme towards L-canavanine does not necessarily provide increased protection against analogues in general, but appears to have evolved specifically to avoid auto-toxicity.

We investigated the effects of nitrogen (N) availability during the vegetative phase on (a) post-anthesis N uptake and (b) its translocation into ears in barley plants grown in a greenhouse at two levels of N: low (50 mg N kg -1 sand) and optimal N supply (150 mg N kg -1 sand). Plants in the two N treatments were fertilised with the same amount of labelled 15 N [50 mg 15 N kg -1 sand at 10% 15 N exc (N excess , i.e. N exc , is defined as the abundance of enriched stable isotope minus the natural abundance of the isotope) applied as 15 NH 4 15 NO 3 ] 10 days after anthesis (daa). In a separate experiment, the uptake and transport into ears of proteinogenic and non-proteinogenic amino acids were studied to determine whether a relationship exists between amino acid transport into ears and their proteinogenic nature. Plants were fed with either 15 N-α-alanine, a proteinogenic amino acid, or 15 N-α-aminoisobutyric acid, a non-proteinogenic amino acid. Both these amino acids were labelled at 95.6% 15 N exc . Results showed that N accumulations in stems, leaves and especially in ears were correlated with their dry matter (dm) weights. The application of 150 mg N kg -1 sand significantly increased plant dm weight and total N accumulation in plants. During their filling period, ears absorbed N from both external (growth substrate) and internal (stored N in plants) sources. Nitrogen concentration in ears was higher in optimal N-fed plants than in low N-fed plants until 10 daa, but from 21 to 35 daa, differences were not detected. Conversely, 15 N exc in ears, leaves and stems was higher in low N-fed plants than in optimal N-fed plants. Ears acted as strong sink organ for the post-anthesis N taken up from the soil independently of pre-anthesis N nutrition: on average, 87% of the N taken up from the soil after anthesis was translocated and accumulated in ears. Low N-fed plants continued to take up N from the post-anthesis N fertiliser during the later grain-filling period. The increase of pre-anthesis N supply rate led to a decrease in the contribution of nitrogen derived from post-anthesis 15 N-labelled fertiliser (N dff ) to total N in all aboveground organs, especially in ears where 44% and 22% of total N originated from post-anthesis N uptake in low N-fed and optimal N-fed plants, respectively. The experiment with labelled amino acids showed that there was greater transport of proteinogenic amino acid into the ear (50% of total 15 N) than non-proteinogenic amino acid (39%). However, this transport of the non-proteinogenic amino acids into ear suggested that the transport of N compounds from source (leaves) to sink organs (ear) might not be intrinsically regulated by their ability to be incorporated into storage protein of ears.

The neurotoxic amino acid β-N-methylamino-L-alanine (BMAA) is produced by cyanobacteria under nitrogen starvation conditions and its metabolism is closely associated with cellular nitrogen control. Very little is known regarding the metabolism or biosynthesis of this amino acid in the producing organisms and current knowledge is limited to the spontaneous formation of carbamate adducts in the presence of aqueous carbon dioxide, the rapid removal of free cellular BMAA upon the addition of ammonia to nitrogen-starved cyanobacterial cultures, and the link between cellular nitrogen status and BMAA synthesis. Data presented here show that exogenous BMAA is readily metabolised by cyanobacteria during which, the primary amino group is rapidly transferred to other cellular amino acids. Furthermore, data suggest that BMAA is metabolised in cyanobacteria via a reversible transamination reaction. This study presents novel data on BMAA metabolism in cyanobacteria and provides the first proposed biosynthetic precursor to BMAA biosynthesis in cyanobacteria.

The proteinogenic amino acid proline has multiple functions in living cells. Depending on whether it is used as a building block in proteins, as a nutrient, or as an osmostress protectant, the cytoplasmic proline con-centration required for these tasks varies dramatically. The soil bacterium Bacillus subtilis solves this problem by precisely regulating and highly integrating catabolic, anabolic, and stress protective processes.

Corynebacterium glutamicum is intensively used for the industrial large-scale (fed-) batch production of amino acids, especially glutamate and lysine. However, metabolic flux analyses based on 13 C-labeling experiments of this organism have hitherto been restricted to small-scale batch conditions and carbon-limited chemostat cultures, and are therefore of questionable relevance for industrial fermentations. To lever flux analysis to the industrial level, a novel Sensor Reactor approach was developed (El Massaoudi et al., Metab. Eng., submitted), in which a 300-L production reactor and a 1-L Sensor Reactor are run in parallel master/slave modus, thus enabling 13 C-based metabolic flux analysis to generate a series of flux maps that document large-scale fermentation courses in detail. We describe the successful combination of this technology with nuclear magnetic resonance (NMR) analysis, metabolite balancing methods and a mathematical description of 13 C-isotope labelings resulting in a powerful tool for quantitative pathway analysis during a batch fermentation. As a first application, 13 C-based metabolic flux analysis was performed on exponentially growing, lysine-producing C. glutamicum MH20-22B during three phases of a pilot-scale batch fermentation. By studying the growth, (co-) substrate consumption and (by-) product formation, the similarity of the fermentations in production and Sensor Reactor was verified. Applying a generally applicable mathematical model, which included metabolite and carbon labeling balances for the analysis of proteinogenic amino acid 13 C-isotopomer labeling data, the in vivo metabolic flux distribution was investigated during subsequent phases of exponential growth. It was shown for the first time that the in vivo reverse C 4 -decarboxylation flux at the anaplerotic node in C. glutamicum significantly decreased (70%) in parallel with threefold increased lysine formation during the investigated subsequent phases of exponential growth.

Methionine is essential in all organisms, as it is both a proteinogenic amino acid and a component of the cofactor,S-adenosyl methionine. The metabolic pathway for its biosynthesis has been extensively characterized inEscherichia coli; however, it is becoming apparent that most bacterial species do not use theE. colipathway. Instead, studies on other organisms and genome sequencing data are uncovering significant diversity in the enzymes and metabolic intermediates that are used for methionine biosynthesis. This review summarizes the different biochemical strategies that are employed in the three key steps for methionine biosynthesis from homoserine (i.e. acylation, sulfurylation and methylation). A survey is presented of the presence and absence of the various biosynthetic enzymes in 1593 representative bacterial species, shedding light on the non-canonical nature of theE. colipathway. This review also highlights ways in which knowledge of methionine biosynthesis can be utilized for biotechnological applications. Finally, gaps in the current understanding of bacterial methionine biosynthesis are noted. For example, the paper discusses the presence of one gene (metC) in a large number of species that appear to lack the gene encoding the enzyme for the preceding step in the pathway (metB), as it is understood inE. coli. Therefore, this review aims to move the focus away fromE. coli, to better reflect the true diversity of bacterial pathways for methionine biosynthesis.

The aim of this work was to study the metabolic changes during germination of Trichoderma atroviride conidia along with selected marker enzyme activities. The increase in proteinogenic amino acid concentrations together with the increase in glutamate dehydrogenase activity suggests a requirement for nitrogen metabolism. Even though the activities of tricarboxylic acid cycle enzymes also increased, detected organic acid pools did not change, which predisposes this pathway to energy production and supply of intermediates for further metabolism. The concentrations of many metabolites, including the main osmolytes mannitol and betaine, also increased during the formation of germ tubes. The activities of H(+)-ATPase and GDPase, the only marker enzymes that did not have detectable activity in non-germinated conidia, were shown with germ tubes.

Balhimycin, a vancomycin-type antibiotic fromAmycolatopsis mediterranei, contains the unusual amino acid (S)-3,5-dihydroxyphenylglycine (Dpg), with an acetate-derived carbon backbone. After sequence analysis of the biosynthetic gene cluster, one gene, dpgA, for a predicted polyketide synthase (PKS) was identified, sharing 20–30% identity with plant chalcone synthases. Inactivation of dpgAresulted in loss of balhimycin production, and restoration was achieved by supplementation with 3,5-dihydroxyphenylacetic acid, which is both a possible product of a PKS reaction and a likely precursor of Dpg. Enzyme assays with the protein expressed in Streptomyces lividans showed that this PKS uses only malonyl-CoA as substrate to synthesize 3,5-dihydroxyphenylacetic acid. The PKS gene is organized in an operon-like structure with three downstream genes that are similar to enoyl-CoA-hydratase genes and a dehydrogenase gene. The heterologous co-expression of all four genes led to accumulation of 3,5-dihydroxyphenylglyoxylic acid. Therefore, we now propose a reaction sequence. The final step in the pathway to Dpg is a transamination. A predicted transaminase gene was inactivated, resulting in abolished antibiotic production and accumulation of 3,5-dihydroxyphenylglyoxylic acid. Interestingly, restoration was only possible by simultaneous supplementation with (S)-3,5-dihydroxyphenylglycine and (S)-4-hydroxyphenylglycine, indicating that the transaminase is essential for the formation of both amino acids.

The ability of a protein to fold into unique functional state or to stay intrinsically disordered is encoded in its amino acid sequence. Both ordered and intrinsically disordered proteins (IDPs) are natural polypeptides that use the same arsenal of 20 proteinogenic amino acid residues as their major building blocks. The exceptional structural plasticity of IDPs, their capability to exist as heterogeneous structural ensembles and their wide array of important disorder-based biological functions that complements functional repertoire of ordered proteins are all rooted within the peculiar differential usage of these building blocks by ordered proteins and IDPs. In fact, some residues (so-called disorder-promoting residues) are noticeably more common in IDPs than in sequences of ordered proteins, which, in their turn, are enriched in several order-promoting residues. Furthermore, residues can be arranged according to their “disorder promoting potencies,” which are evaluated based on the relative abundances of various amino acids in ordered and disordered proteins. This review continues a series of publications on the roles of different amino acids in defining the phenomenon of protein intrinsic disorder and concerns glutamic acid, which is the second most disorder-promoting residue.

Salt stress is primarily osmotic stress, and halophilic/halotolerant microorganisms have evolved two basic mechanisms of osmoadaplation: the KCI-type and the compatible-solute type, the latter representing a very flexible mode of adaptation making use of distinct stabilizing properties of compatible solutes. A comprehensive survey, using HPLC and NMR methods, has revealed the full diversity of euhacterial compatible solutes found in nature. With the exception of proline (a proteinogenic amino acid) they are characterized as amino acid derivatives of the following types: betaines, ectoines, N-acetylated diamino acids and N-derivatized carboxamides of glutamine. From our present knowledge of hiosynthetic pathways it appears that, apart from glycine betaine, all nitrogen-containing compatible solutes originate from two major pathways (the aspartate branch and the glutamate branch). Uptake of compatible solutes from the growth medium (environment) seems to have preference over de novo synthesis. Therefore in the natural ecosystem the solutes of primary producers (mainly glycine betaine), which are readily excreted upon dilution stress, certainly play an important role as a ‘preferred’ solute source for heterolrophic organisms, and as a ‘vital’ source for organisms unable to synthesize their own compatible solutes.

Selenoproteins are defined as proteins containing the 21st proteinogenic amino acid, selenocysteine (Sec). Sec is encoded by UGA (STOP) codons which are re-coded to Sec by the presence of a selenocysteine insertion sequence (SECIS) element in the 3’-untranslated region of selenoprotein mRNAs. The SECIS element is bound by several proteins, including SECIS-binding protein 2 (SBP2). Translation of selenoproteins critically depends on the integrity of the SECIS element – SBP2 interaction. Mutations in a SECIS element can abrogate expression of the respective selenoprotein. Mutations in SBP2 impinge on biosynthesis of a subset of selenoproteins and lead to a syndrome including hormonal, neurological, immunological symptoms as well as myopathy. Several other RNA-binding proteins are involved in selenoprotein translation and mediate the hierarchical response of selenoproteins to selenium deficiency. Global inhibition of selenoprotein translation is lethal in the mouse and hypomorphic mutations in selenocysteine synthase in humans leads to Progressive Cerebello Cerebral Atrophy, a neurodevelopmental and neurodegenerative disease in pediatric patients.

Compatible solutes are best described as organic osmolytes responsible for osmotic balance and at the same time compatible with the cells' metabolism. A comprehensive survey (using HPLC and NMR methods) on halophilic/halotolerant eubacteria has revealed the full diversity of compatible solutes employed in nature. Molecular principles derived from the spectrum of compounds found in the bacterial world may be summarized as follows. Compatible solutes are polar, highly soluble molecules and uncharged at physiological pH. With the exception of proline (a proteinogenic amino acid) they are characterized as amino acid derivatives of the following types: betaines, ectoines, N-acetylated diamino acids and N-derivatized carboxamides of glutamine. Using nearinfrared spectroscopy we have also been able to demonstrate that compatible solutes are strong water-structure formers and as such probably excluded from the hydration shell of proteins. This “preferential exclusion” probably explains their function as effective stabilizers of the hydration shell of native proteins (protection against heating freezing and drying). Hence these typical products of halophilic eubacteria have a considerable potential as stabilizing/protecting agents on both molecular and whole-cell level. Thorough understanding of common structural principles and fundamental water-solute interactions will ultimately enable us to design novel highly efficient stress protectants and stabilizers of biomolecules.

Metabolic flux analysis (MFA) is a powerful tool for rational metabolic and process engineering as it provides a unique insight into the cell. In consideration of the relevant biochemical pathways we developed a metabolic network model for BHK cells.The model included bidirectionality and compartmentation of the fluxes. Mass balancing and isotopomer analysis after feeding with 13C-glucose enabled the quantification of intracellular fluxes. We compared the metabolic fluxes of a BHK cell fine expressing the recombinant yeast pyruvate carboxylase (BHK21-PYC2) with cells carrying the respective plasmid without the PYC2 gene (BHK21-pAG) especially focussing on the anaplerotic pathways.Mass balancing in batch and chemostat cultures advises lower glucose uptake and lactat formation rates and higher growth rates as a result of the genetic modification with recominant pyruvate carboxylase.

In seeds of Hydnocarpus anthelminthica of Flacourtiaceae, cyclopentenylglycine and cyclopentenyl fatty acids are found naturally. The non-proteinogenic amino acid may serve as precursor of cyclopentenyl fatty acids via aleprolic acid, the starter molecule for these long-chain compounds. After administration of cyclopentenyl[2-14C]glycine to maturing seeds of H. anthelminthica, labelled cyclopentenyl fatty acids were synthesized. Comparative activities were observed, when [1-14C]aleprolic acid was supplied to the seeds. Incorporation studies with [1-14C]acetate revealed that the chain-lengthening systems for straight-chain and cyclic fatty acids were still functioning in mature seeds. Endosperm and embryo of H. Anthelminthica seeds synthesized cyclopentenyl fatty acids from cyclopentenyl[2-14C]glycine, [1-14C]aleprolic acid and [1-14C]acetate. In embryonic tissue, a dilution experiment proved the following path for cyclopentenyl fatty acid biosynthesis: cyclopentenylglycine leads to aleprolic acid leads to cyclopentenyl fatty acids. The conversion of cyclopentenylglycine to aleprolic acid may occur via transamination and oxidative decarboxylation; activated aleprolic acid is then lengthened by C2-units to cyclopentenyl fatty acids.

Summary To get insight in the metabolism of the latex-specific amino acid m-tyrosine of Euphorbia myrsinites L., m-tyrosine-[2-14C] was fed to shoots of this plant. The compound damages the plants and is selectively excreted into the latex. In the tissue a partial oxidation occurs to polyphenols and melaninlike products. Another part of the m-tyrosine is transformed in the tissue and the latex to 1-methyl-6-hydroxy-1,2,3,4-tetrahydroisochinoline-3-carboxylic acid by Mannich-condensation with acetaldehyde or its biochemical equivalent. The same reaction occurs spontaneously in the isolated latex. m-Tyrosine-[2-14C] and phenylalanine-[2-14C] fed to sprouts of Euphorbia helioscopia L. are converted to m-hydroxyphenylglycine, occurring in the latex of this species. The unphysiological amino acid is taken up by the plant only in a small degree compared with the proteinogenic amino acid phenylalanine and is preferentially eliminated into the latex. Phenylalanine on its way into the latex is markedly degraded. Incubation studies with isolated latex from Euphorbia helioscopia L. and Euphorbia lathyris L. showed that m-tyrosine is not transformed and not incorporated into m-hydroxyphenylglycine and 3,4-dihydroxyphenylalanine respectively, amino acids occurring in the latex of these species. Shikimic acid[U-14C] is incorporated in a very small amount into m-tyrosine in the isolated latex of Euphorbia myrsinites L., especially after the increase of the pH value about the neutral point. These results are discussed in relation to the physiological significance of the amino acids from latex and of the latex itself and its particular properties.

Background: The non-proteinogenic amino acid p -hydroxyphenylglycine is a crucial component of certain peptidic natural products synthesized by a non-ribosomal peptide synthetase mechanism. In particular, for the vancomycin group of antibiotics p -hydroxyphenylglycine plays a structural role in formation of the rigid conformation of the central heptapeptide aglycone in addition to being the site of glycosylation. Initial labeling studies suggested tyrosine was a precursor of p -hydroxyphenylglycine but the specific steps in p -hydroxyphenylglycine biosynthesis remained unknown. Recently, the sequencing of the chloroeremomycin gene cluster from Amycolatopsis orientalis gave new insights into the biosynthetic pathway and allowed for the prediction of a four enzyme pathway leading to L- p -hydroxyphenylglycine from the common metabolite prephenate. Results: We have characterized three of the four proposed enzymes of the L- p -hydroxyphenylglycine biosynthetic pathway. The three enzymes are encoded by open reading frames (ORFs) 21, 22 and 17 (ORF21: [PCZA361.1, O52791, CAA11761]; ORF22: [PCZA361.2, O52792, CAA11762]; ORF17: [PCZA361.25, O52815, CAA11790]), of the chloroeremomycin biosynthetic gene cluster and we show they have p -hydroxymandelate synthase, p -hydroxymandelate oxidase and L- p -hydroxyphenylglycine transaminase activities, respectively. Conclusions: The L- p -hydroxyphenylglycine biosynthetic pathway shown here is proposed to be the paradigm for how this non-proteinogenic amino acid is synthesized by microorganisms incorporating it into peptidic natural products. This conclusion is supported by the finding of homologs for the four L- p -hydroxyphenylpyruvate biosynthetic enzymes in four organisms known to synthesize peptidic natural products that contain p -hydroxyphenylglycine. Three of the enzymes are proposed to function in a cyclic manner in vivo with L-tyrosine being both the amino donor for L- p -hydroxyphenylglycine and a source of p -hydroxyphenylpyruvate, an intermediate in the biosynthetic pathway.

Delivery mode and perinatal antibiotics influence gut microbiome composition in children. Most microbiome studies have used the sequencing of the bacterial 16S marker gene but have not reported the metabolic function of the gut microbiome, which may mediate biological effects on the host. Here, we used the PICRUSt2 bioinformatics tool to predict the functional profiles of the gut microbiome based on 16S sequencing in two child cohorts. Both Caesarean section and perinatal antibiotics markedly influenced the functional profiles of the gut microbiome at the age of 1 year. In machine learning analysis, bacterial fatty acid, phospholipid, and biotin biosynthesis were the most important pathways that differed according to delivery mode. Proteinogenic amino acid biosynthesis, carbohydrate degradation, pyrimidine deoxyribonucleotide and biotin biosynthesis were the most important pathways differing according to antibiotic exposure. Our study shows that both Caesarean section and perinatal antibiotics markedly influence the predicted metabolic profiles of the gut microbiome at the age of 1 year.

Efficient and inexpensive methods are required for the high-throughput quantification of amino acids in physiological fluids or microbial cell cultures. Here we develop an array of Escherichia coli biosensors to sensitively quantify eleven different amino acids. By using online databases, genes involved in amino acid biosynthesis were identified that – upon deletion – should render the corresponding mutant auxotrophic for one particular amino acid. This rational design strategy suggested genes involved in the biosynthesis of arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, and tyrosine as potential genetic targets. A detailed phenotypic characterization of the corresponding single-gene deletion mutants indeed confirmed that these strains could neither grow on a minimal medium lacking amino acids nor transform any other proteinogenic amino acid into the focal one. Site-specific integration of the egfp gene into the chromosome of each biosensor decreased the detection limit of the GFP-labeled cells by 30% relative to turbidometric measurements. Finally, using the biosensors to determine the amino acid concentration in the supernatants of two amino acid overproducing E. coli strains (i.e. ΔhisL and ΔtdcC) both turbidometrically and via GFP fluorescence emission and comparing the results to conventional HPLC measurements confirmed the utility of the developed biosensor system. Taken together, our study provides not only a genotypically and phenotypically well-characterized set of publicly available amino acid biosensors, but also demonstrates the feasibility of the rational design strategy used.

Background Pseudomonas fluorescens SBW25 has been extensively studied because of its plant growth promoting properties and potential as a biocontrol agent. The genome of SBW25 has been sequenced, and among sequenced strains of pseudomonads, SBW25 appears to be most closely related to P. fluorescens WH6. In the authors’ laboratories, WH6 was previously shown to produce and secrete 4-formylaminooxyvinylglycine (FVG), a non-proteinogenic amino acid with selective herbicidal and antimicrobial activity. Although SBW25 does not have the genetic capacity to produce FVG, we were interested in determining whether this pseudomonad might produce some other type of non-proteinogenic amino acid. Results P. fluorescens SBW25 was found to produce and secrete a ninhydrin-reactive compound with selective antimicrobial properties. This compound was purified from SBW25 culture filtrate and identified as the non-proteinogenic amino acid L-furanomycin [2S,2′R,5′S)-2-amino-2-(5′methyl-2′,5′-dihydrofuran-2′-yl)acetic acid]. Conclusions The identification of furanomycin as a secondary metabolite of SBW25 is the first report of the production of furanomycin by a pseudomonad. This compound was known previously only as a natural product produced by a strain of Streptomyces. This report adds furanomycin to the small list of non-proteinogenic amino acids that have been identified as secondary products of pseudomonads. This study also extends the list of bacteria that are inhibited by furanomycin to include several plant pathogenic bacteria.