Occurrence Of D-amino Acids Research Articles

D-amino acids metabolism reflects the evolutionary origin of higher plants and their adaptation to the environment.

d-amino acids are the d stereoisomers of the common l-amino acids found in proteins. Over the past two decades, the occurrence of d-amino acids in plants has been reported and circumstantial evidence for a role in various processes, including interaction with soil microorganisms or interference with cellular signalling, has been provided. However, examples are not numerous and d-amino acids can also be detrimental, some of them inhibiting growth and development. Thus, the persistence of d-amino acid metabolism in plants is rather surprising, and the evolutionary origins of d-amino acid metabolism are currently unclear. Systemic analysis of sequences associated with d-amino acid metabolism enzymes shows that they are not simply inherited from cyanobacterial metabolism. In fact, the history of plant d-amino acid metabolism enzymes likely involves multiple steps, cellular compartments, gene transfersand losses. Regardless of evolutionary steps, enzymes of d-amino acid metabolism, such as d-amino acid transferases or racemases, have been retained by higher plants and have not simply been eliminated, so it is likely that they fulfil important metabolic roles such as serine, folateor plastid peptidoglycan metabolism. We suggest that d-amino acid metabolism may have been critical to support metabolic functions required during the evolution of land plants.

Enantiomeric Ratio of Amino Acids as a Tool for Determination of Aging and Disease Diagnostics by Chromatographic Measurement

Occurrence of d-amino acids in living organisms is a useful indicator of various changes, diseases, or disorders. Determination of amino acid enantiomers, namely the enantiomeric ratio of amino acids or excess of certain d-amino acids, represents a useful tool in the studies of aging processes or biomarkers in disease/disorder diagnosis in humans. The amount of d-amino acids is usually very low. Therefore, suitable sample pretreatment, often derivatization, and highly selective and sensitive separation methods are essential for d-amino acid analysis in this field. Chromatographic techniques offer appropriate choices for solving these tasks. This review covers the advances in methodology and development of improved instrumental chromatographic methods focused on d,l-amino acid separation and determination. New findings in the area of possible d-amino acid biomarkers are also included.

The stereoselective separation of serine containing peptides by zwitterionic ion exchanger type chiral stationary phases and the study of serine racemization mechanisms by isotope exchange and tandem mass spectrometry

The occurrence of d-amino acids (d-AAs) in higher-developed organisms in their free form, and within peptides and proteins, has been investigated with an increasing number of studies. Often the inversion of the stereochemical configuration of an individual amino acid drastically changes its biological activity. Alongside Asn and Asp, Ser is most prone to racemization within peptides. Specific enzymes catalyzing d-Ser generation and breakdown have been described. Hence, the applicability of enantioselective ZWIX(+)® and ZWIX(−)® chiral stationary phases (CSPs) to peptide separations was assessed and a set of 14 pairs of diastereomeric and enantiomeric Ser and Thr containing di-, tri- and tetra-peptides was chromatographically separated without prior hydrolysis to the individual amino acids. To a certain extent, RP chromatography also enabled the separation of peptide diastereomers. The ZWIX CSPs delivered chromatographic selectivities between 1.04 and 7.23, allowing a change of elution order by switching between the ZWIX(+) and the ZWIX(−) CSP. Coupling these highly selective chromatographic columns with an LTQ-Orbitrap XL™ mass spectrometer and performing high resolution MS2 measurements enabled us to investigate mechanistic aspects of chemically induced racemization of Ser embedded in short peptides. As reaction medium an alkaline aqueous solution (pH 12.3) was selected. Proton/deuterium exchange experiments provided evidence of a fast Cα proton exchange with simultaneous racemization. Additionally, 18O/16O exchange allowed the identification of an alternative, and somewhat retarded racemization via a reversible β-elimination and reintroduction of water at the hydroxymethyl side chain of Ser. This involved the intermediate generation of the prochiral didehydro alanine unit.

D- and L-amino acids in Antarctic lakes: assessment of a very sensitive HPLC-MS method.

Amino acids represent a fraction of organic matter in marine and freshwater ecosystems, and a source of carbon, nitrogen and energy. L-Amino acids are the most common enantiomers in nature because these chiral forms are used during the biosynthesis of proteins and peptide. To the contrary, the occurrence of D-amino acids is usually linked to the presence of bacteria. We investigated the distribution of L- and D-amino acids in the lacustrine environment of Terra Nova Bay, Antarctica, in order to define their natural composition in this area and to individuate a possible relationship with primary production. A simultaneous chromatographic separation of 40 L- and D-amino acids was performed using a chiral stationary phase based on teicoplainin aglycone (CHIROBIOTIC TAG). The chromatographic separation was coupled to two different mass spectrometers--an LTQ-Orbitrap XL (Thermo Fisher Scientific) and an API 4000 (ABSciex)--in order to investigate their quantitative performance. High-performance liquid chromatography coupled with mass spectrometry methods were evaluated through the estimation of their linear ranges, repeatability, accuracy and detection and quantification limits. The high-resolution mass spectrometer LTQ-Orbitrap XL presented detection limits between 0.4 and 7 μg L (-1), while the triple quadrupole mass spectrometer API 4000 achieved the best detection limits reported in the literature for the quantification of amino acids (between 4 and 200 ng L (-1)). The most sensitive method, HPLC-API 4000, was applied to lake water samples.

D-amino Acids: Prospects for New Therapeutic Agents

 Abstract—D-amino acids are predominantly produced and utilized by bacteria. They are involved in the synthesis and cross-linking of peptidoglycan. Furthermore, oxidative catabolism of D-amino acids, via the D-amino acid dehydrogensae pathway, sustains energy production for cellular functions. Only a few decades ago, it was largely believed that free D-amino acids were restricted to bacteria. Often, D-amino acids were considered as the by-products of bacterial metabolism. Nevertheless, the occurrence of Damino acids in mammals was recently confirmed by means of sensitive and advanced analytical methods. The physiological functions of D-amino acids in humans are still under investigation. The presence of astonishing amounts of certain D-amino acids in the human Central Nervous System suggests a vital role of D-amino acids in neuromodulation. Apparently, both prokaryotes and eukaryotes maintain tight regulation on the occurrence of D-amino acids in their systems through specific metabolic pathways. Previously, it had been reported that the accumulation of certain D-amino acids results in cellular toxicity. In this study, we investigate the potential of Damino acids as prospective new therapeutic agents. Antibacterial, antifungal, and cytotoxic activities were evaluated against representative cellular models. Our findings indicate that, although D-amino acids are toxic on their own, their efficacy can be significantly improved by synergism with other therapeutic agents. The ability to use lower doses of both, the drug and D-amino acids, may be beneficial for the development of combinational remedies against resistant pathogens or cancerous cells. Index Terms—D-amino acids, antibacterial, antifungal, cytotoxicity

Correlation between amino acid racemization and processing conditions for various wheat products, oil seed press cakes and lignin samples

The occurrence of d-amino acids in native and processed plant products is brought into context with the harshness of their treatment condition. It was found that already a small increase in processing harshness such as the milling efficiency of wheat straw or the increased pressure and duration of pumpkin seed oil extraction leads to traceable changes in the overall amino acid content as well as the racemization rate of free and protein bound amino acids. In the top position of our process harshness scale lies the strong alkali and heat-induced extraction of polyphenolic compound lignin from wood and other cellulose-rich plant fibers, e.g. wheat straw, during paper production. Since lignin accumulates in large quantities and its transformation into value-added “bio” products necessitates the recovery of native, functionally preserved lignin, milder pulping methods gain increasing interest. An unexpected drawback of such mild extraction conditions is however the high nitrogen content of such lignin products, which further increase with increasing mildness of the pulping procedure. The presence of amide bands in the FT-IR spectra of such mildly processed lignin has revealed that this nitrogen originated from co-extracted proteins and peptides. The total amino acid content as well as the free amino acid content and the degree of racemization was determined by gas chromatography–ion trap mass spectrometry (GC–Iontrap MS) employing enantiomer labeling for quantification. Cross-determination of the average amino acid content was calculated from nitrogen values obtained by elemental analysis and correlation factors were calculated from measured and calculated data. Since those amino acids with two chiral centers, such as isoleucine, threonine and hydroxyproline exhibit the highest possible diversity in terms of their relative amount to one another as well as their internal enantiomer distribution, this set of two-centered amino acids provided very distinct amino acid pattern, which proofed to be characteristic for the type of plant material as well as the harshness of the different processing conditions.

Alterations in d-amino acid concentrations and microbial community structures during the fermentation of red and white wines

Alterations in D-amino acid concentrations and microbial community structures during the fermentation of red and white wines were analyzed to clarify the relationship between the occurrence of d-amino acids and the actions of fermentative microorganisms. Relatives of Saccharomyces cerevisiae and Oenococcus oeni were detected in red wine samples, and relatives of S. cerevisiae, O. oeni, and Gluconacetobacter saccharivorans were detected in white wine samples. The S. cerevisiae relatives were detected throughout the fermentation process, whereas the O. oeni relatives were detected at the late stage of fermentation in both the red and white wine samples. The G. saccharivorans relative was detected in the early stage of fermentation. The amino acid analysis showed that D-alanine, D-glutamic acid, and D-lysine were present in both the red and white wine samples. The concentrations of these D-amino acids increased as the fermentation continued, especially from the malolactic fermentation stage to the end of the fermentation processes. These increases seem to be linked to the presence of O. oeni relatives.

Generation of Enantiomeric Amino Acids during Acid Hydrolysis of Peptides Detected by the Liquid Chromatography/Tandem Mass Spectroscopy

The number of reports indicating the occurrence of D-amino acids in various proteins and natural peptides is increasing. For a usual detection of peptidyl D-amino acids, proteins or peptides are subjected to acid hydrolysis, and the products obtained are analyzed after cancellation of the effect of amino acid racemization during the hydrolysis. However, this method does not seem reliable enough to determine the absence or presence of a small amount of innate D-amino acids. We introduce a modification of an alternative way to distinguish true innate D-amino acids from those artificially generated during hydrolysis incubation. When model peptides (L-Ala)(3), D-Ala-(L-Ala)(2) are hydrolyzed in deuterated hydrochloric acid (DCl), only newly generated D-amino acids are deuterated at the alpha-H-atom. Both innate D-amino acids and artificially generated ones are identified by the combination of high-performance liquid chromatography and liquid chromatography/tandem mass spectrometry equipped with a chiral column. When a peptide containing D-Phe residues was analyzed by this method, the hydrolysis-induced conversion to L-Phe was similarly identified.

D-Amino Acid Formation in Sterilized Alkali-Treated Olives

The occurrence of d-amino acids in commercial ripe olives, a well-known sterilized alkali-treated product, was investigated by high-performance liquid chromatography (HPLC) with precolumn automatic derivatization. Absolute amounts of D-amino acids were in total 18.6-38.2 mg/100 g edible portion. The major D-amino acids were D-aspartic acid, D-glutamic acid, D-serine, and D-leucine. Furthermore, to evaluate the effects of sterilization time and olive pH on amino acid racemization, a simulated processing of green ripe olives was carried out. Serine (both free and bound form) was the most-racemized amino acid after heat treatment. Sterilization (15-35 min at 121 degrees C) increased the racemization values of both free and protein-bound amino acids, although in case of protein-bound phenylalanine the increase was not statistically significant. With an increase of pH from 8 to 10 units, the racemization values of all amino acids increased significantly, except for free forms of aspartic and glutamic acids. In general, the effects of the sterilization time and olive pH on total concentration (L + D enantiomers) of each amino acid were also significant.

Neurobiology through the looking-glass: d-serine as a new glial-derived transmitter

d-Amino acids have been known to be present in bacteria for more than 50 years, but only recently they were identified in mammals. The occurrence of d-amino acids in mammals challenge classic concepts in biology in which only l-amino acids would be present or thought to play important roles. Recent discoveries uncovered a role of endogenous d-serine as a putative glial-derived transmitter that regulates glutamatergic neurotransmission in mammalian brain. Free d-serine levels in the brain are about one third of l-serine values and its extracellular concentration is higher than many common l-amino acids. d-Serine occurs in protoplasmic astrocytes, a class of glial cells that ensheath the synapses and modulate neuronal activity. Biochemical and electrophysiological studies suggest that endogenous d-serine is a physiological modulator at the co-agonist site of NMDA-type of glutamate receptors. We previously showed that d-serine is synthesized by a glial serine racemase, a novel enzyme converting l- to d-serine in mammalian brain. The enzyme requires pyridoxal 5′-phosphate and it was the first racemase to be cloned from eucaryotes. Inhibitors of serine racemase have therapeutic implications for pathological processes in which over-stimulation of NMDA receptors takes place, such as stroke and neurodegenerative diseases. Here, we review the role of endogenous d-serine in modulating NMDA neurotransmission, its biosynthetic apparatus and the potential usefulness of serine racemase inhibitors as a novel neuroprotective strategy to decrease glutamate/NMDA excitotoxicity.

Occurrence of d-amino acids in a few archaea and dehydrogenase activities in hyperthermophile Pyrobaculum islandicum

The contents of d-enantiomers of serine, alanine, proline, glutamate (glutamine) and aspartate (asparagine) were examined in the membrane fractions, soluble proteins and free amino acids from some species of archaea, Pyrobaculum islandicum, Methanosarcina barkeri and Halobacterium salinarium. Around 2% ( d/ d+ l) of d-aspartate was found in the membrane fractions. In the soluble proteins, the d-amino acid content was higher in P. islandicum than that in the other archaeal cells: the concentrations in P. islandicum were 3 and 4% for d-serine and d-aspartate, respectively. High concentrations of free d-amino acids were found in P. islandicum and H. salinarium; the concentrations of d-serine (12–13%), d-aspartate (4–7%) and d-proline (3–4%) were higher than those of d-alanine and d-glutamate. This result showed a resemblance between these archaea and not bacterial, but eukaryotic cells. The presence of d-amino acids was confirmed by their digestion with d-amino acid oxidase and d-aspartate oxidase. The occurrence of d-amino acids was also confirmed by the presence of activities catalyzing catabolism of d-amino acids in the P. islandicum homogenate, as measured by 2-oxo acid formation. The catalytic activities oxidizing d-alanine, d-aspartate and d-serine at 90°C were considerably high. Under anaerobic conditions, dehydrogenase activities of the homogenate were 69, 84 and 30% of the above oxidase activities toward d-alanine, d-aspartate and d-serine, respectively. Comparable or higher dehydrogenase activities were also detected with these d-amino acids as substrate by the reduction of 2,6-dichlorophenolindophenol. No d-amino acid oxidase activity was detected in the homogenates of M. barkeri and H. salinarium.

Occurrence of D-amino acids in a typical semi-hard cheese

D-alanine (D-Ala), D-aspartic acid (D-Asp) and D-glutamic acid (D-Glu) are important constituents of the cell walls of bacteria responsible for fermentation processes, and have been detected in several fermented foods, such as cheeses, yogurt and vinegar (Palla et al. 1989; Brückner & Hausch, 1990; Dossena et al. 1991; Brückner et al. 1992; Gandolfi et al. 1992). In particular, D-amino acids have been found, free and abundant, in cheeses requiring long ripening periods (1–2 years; Santaguida et al. 1995). Their presence in cheeses could be related mainly to lysis of the bacterial wall, to which short-chain D-amino acids are bound, but also to the activity of bacterial racemases (Adams, 1972), which convert free L-amino acids generated during proteolysis. The content of these D-amino acids and the D:L ratio have been recently proposed as indicators of ripening and for quality assessment of Parmigiano Reggiano and Grana Padano cheeses (Marchelli et al. 1997).The aim of the present study was to determine D-amino acids in cheeses with shorter ripening periods to evaluate whether they could be useful as indicators of ripening and for quality assessment. The work was carried out with Montasio, a typical cheese produced in north-east Italy using traditional methods. Montasio is a semi-hard cheese produced from unpasteurized milk, cooked at 44–46 °C and consumed after at least 2 months ripening. This study represents an extension of previous work on free amino acid contents and ripening in semi-hard cheeses (Innocente, 1997).

Occurrence of D-Amino Acids in Fish Sauces and Other Fermented Fish Products

Occurrence of D-Amino Acids in Fish Sauces and Other Fermented Fish Products

Biological role of D-amino acid oxidase and D-aspartate oxidase. Effects of D-amino acids.

D-Amino acids administered to animals are absorbed by the intestine and transported through the blood-stream to solid tissues where they are oxidized in vivo by D-amino acid oxidase and D-aspartate oxidase to produce the same compounds they do in vitro; i.e. NH3, H2O2, and the keto acid corresponding to the amino acid ingested. In the liver and kidneys of the animals, an inverse relationship exists between the occurrence of D-amino acids and these oxidative enzymes. For example, younger animals have lower amounts of these oxidases and consequently higher concentrations of free D-amino acids compared to adult animals. If the ingested D-amino acids are not metabolized by these enzymes, they will accumulate in the tissues and may provoke serious damage, e.g. suppression of the synthesis of other essential enzymes and inhibition of the growth rate of the animals. A specific enzyme induction for these D-amino acid oxidases exists in young rats following ingestion of free D-amino acids by the mother. Specifically, when a mother rat ingests D-Ala or D-Asp during pregnancy and suckling, an increase in D-amino acid oxidase or D-aspartate oxidase is observed in the liver and kidneys of the baby rats. These results suggest that the in vivo biological role of these oxidases in animals is to act as detoxifying agents to metabolize D-amino acids which may have accumulated during aging.

Occurrence of D-amino acids in food : Detection by capillary gas chromatography and by reversed-phase high-performance liquid chromatography with L-phenylalaninamides as chiral selectors

The presence of D-amino acids was investigated in dairy products, in raw ham and in roasted coffee. The analysis was carried on by capillary gas chromatography, using a very stable tetraamidic selector derived from L-phenylalanine, which allowed the detection of D-alanine, D-aspartic acid, D-glutamic acid and other D-amino acids. Moreover, D-glutamic acid and D-alanine were detected also by reversed-phase high-performance liquid chromatography, using L-phenylalaninamide and copper(II) acetate as chiral additives to the eluent. The presence of D-amino acids may be ascribed to thermal racemization or to microbial activity.

Occurrence of d-amino acids in higher organisms: A survey of the distribution of d-amino acids in marine invertebrates

1. 1. d-amino acids assayed using a d-amino acid oxidase (DAO) technique have been found in 18 species of marine invertebrates in six phyla out of a total of 43 species in eight phyla that were analyzed (42%). 2. 2. The apparent tissue concentrations ranged from 44 mM in the polychaete, Glycera dibranchiata to 0.04 mM in the nemertine, Parborlasia corrugatus. 3. 3. Endogenous inhibitors (mol. wt 1000–2000 as determined by gel filtration) were discovered which drastically decrease the sensitivity of the DAO assay technique. It is therefore possible that the reported concentrations are underestimates of the true tissue concentrations. 4. 4. Their wide distribution in marine invertebrates suggests there may be undiscovered physiological and nutritional roles for d-amino acids.

D-amino acid in irradiated and aged mouse.

A single high dose of X-rays (900R to the whole body or 1500R to the head alone) or gamma-rays (500R to the whole body) was applied to the mouse to promote the aging process, and the correlation of occurrence of D-amino acids to aging or cataract formation was examined in the skin and lenses. No D-amino acid was found in the skin even after irradiation. On the other hand, D-aspartic acid was detected in the insoluble fraction of lens protein from aged mice after irradiation. Irradiation simultaneously induced cataracts depending on the radiation dose. Thus, the irradiation promoted both the formation of cataract and D-aspartic acid in the lens protein, suggesting that the presence of D-aspartic acid in the lens might be considered as a molecular indicator of aging.

The Occurrence of D-Amino Acids in Soil Organic Matter and Its Significance for Soil Nitrogen Metabolism : I.Methods for the Optical Resolution of Amino Acids

The Occurrence of D-Amino Acids in Soil Organic Matter and Its Significance for Soil Nitrogen Metabolism : I.Methods for the Optical Resolution of Amino Acids

OCCURRENCE OF d-AMINO ACIDS IN SOME NATURAL MATERIALS

OCCURRENCE OF d-AMINO ACIDS IN SOME NATURAL MATERIALS