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Studies in vivo have demonstrated that the accumulation of D-amino acids (D-AAs) is associated with age-related diseases and increased immune activation. However, the underlying mechanism(s) of these observations are not well defined. The metabolism of D-AAs by D-amino oxidase (DAO) produces hydrogen peroxide (H 2 O 2 ), a reactive oxygen species involved in several physiological processes including immune response, cell differentiation, and proliferation. Excessive levels of H 2 O 2 contribute to oxidative stress and eventual cell death, a characteristic of age-related pathology. Here, we explored the molecular mechanisms of D-serine (D-Ser) and D-alanine (D-Ala) in human liver cancer cells, HepG2, with a focus on the production of H 2 O 2 the downstream secretion of pro-inflammatory cytokine and chemokine, and subsequent cell death. In HepG2 cells, we demonstrated that D-Ser decreased H 2 O 2 production and induced concentration-dependent depolarization of mitochondrial membrane potential (MMP). This was associated with the upregulation of activated NF-кB, pro-inflammatory cytokine, TNF-α, and chemokine, IL-8 secretion, and subsequent apoptosis. Conversely, D-Ala-treated cells induced H 2 O 2 production, and were also accompanied by the upregulation of activated NF-кB, TNF-α, and IL-8, but did not cause significant apoptosis. The present study confirms the role of both D-Ser and D-Ala in inducing inflammatory responses, but each via unique activation pathways. This response was associated with apoptotic cell death only with D-Ser. Further research is required to gain a better understanding of the mechanisms underlying D-AA-induced inflammation and its downstream consequences, especially in the context of aging given the wide detection of these entities in systemic circulation., (© 2024. The Author(s).)

It was long believed that D-amino acids were either unnatural isomers or laboratory artifacts, and that the important functions of amino acids were exerted only by L-amino acids. However, recent investigations have revealed a variety of D-amino acids in mammals that play important roles in physiological functions, including free D-serine and D-aspartate that are crucial in the central nervous system. The functions of several D-amino acids in the periphery and endocrine glands are also receiving increasing attention. Here, we present an overview of recent advances in elucidating the physiological roles of D-amino acids, especially in the periphery and endocrine glands.

The D-amino acids of D-alanine, D-glutamic acid, and D-aspartic acid increase tasting evaluation scores of Sake, a Japanese traditional alcohol beverage. Sake is brewed using seed mash for growth of brewing yeast without growth of contaminating microorganisms. Kimoto is brewed using lactic acid bacteria growth to decrease pH. Sake brewed using the Kimoto method also has a rich taste and a higher tasting evaluation score than Sake brewed using the Sokujyo Syubo (Moto) method, which adds lactic acid instead of using lactic acid bacteria growth. D-alanine, D-glutamic acid, and D-aspartic acid in Sake have the function of increasing tasting evaluation scores. They are converted by enzymes in lactic acid bacteria respectively as alanine racemase (EC 5.1.1.1), glutamate racemase (EC 5.1.1.3), and aspartate racemase (EC 5.1.1.13) in Kimoto Mash. Herein, simultaneous assay methods for D-alanine, D-glutamic acid, and D-aspartic acid are explained. Sample solutions adjusted to alkalinity are derivatized by an L-FDLA solution only for L-amino acid. Results demonstrate that, for D-alanine, D-glutamic acid, and D-aspartic acid, this method can assay them easily using no expensive or specialized equipment., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Some aquatic invertebrates contain free d-alanine. We previously showed copious amounts of free d-glutamate, a novel d-amino acid, in the tissue of the male reproductive organs of Marsupenaeus japonicus. Herein, we clarified the distribution and potential role of d-glutamate and d-alanine in male reproductive tissues, namely the testis, vas deferens and seminal receptacle at different growth stages of M. japonicus. The percentage of d-glutamate to total glutamate was over 50% in these tissues. In particular, the content of d-glutamate was the most abundant in the vas deferens, the ratio of d-glutamate to total glutamate was approximately 80%. In contrast, d-alanine content was the lowest in the vas deferens among these tissues. d-Glutamate content was the highest when the prawn weighed 12 g, indicating that d-glutamate is actively synthesized in the younger stage. Our findings suggest that d-glutamate plays an important role in the reproductive functions of M. japonicus., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

D-Aspartate, D-serine and D-alanine are a regular occurrence in mammalian endocrine tissues, though in amounts varying with the type of gland. The pituitary gland, pineal gland, thyroid, adrenal glands and testis contain relatively large amounts of D-aspartate in all species examined. D-alanine is relatively abundant in the pituitary gland and pancreas. High levels of D-serine characterize the hypothalamus. D-leucine, D-proline and D-glutamate are generally low. The current knowledge of physiological roles of D-amino acids in endocrine tissues is far from exhaustive, yet the topic is attracting increasing interest because of its potential in pharmacological application. D-aspartate is known to act at all levels of the hypothalamus-pituitary-testis axis, playing a key role in reproductive biology in several vertebrate classes. An involvement of D-amino acids in the endocrine function of the pancreas is emerging. D-Aspartate has been immunolocalized in insulin-containing secretory granules in INS-1 E clonal β cells and is co-secreted with insulin by exocytosis. Specific immunolocalization of D-alanine in pituitary ACTH-secreting cells and pancreatic β-cells suggests that this amino acid participates in blood glucose regulation in mammals. By modulating insulin secretion, D-serine probably participates in the control of systemic glucose metabolism by modulating insulin secretion. We anticipate that future investigation will significantly increase the functional repertoire of D-amino acids in homeostatic control.

Fermented foods produced using edible microorganisms have been consumed worldwide since ancient times. Microorganisms produce some healthy functional substances. Recently, many people have been attracted to the notion that D-body amino acids have healthy functional effects. D-body amino acids convert L-body amino acid proteolysis from a substrate such as foods during fermentation. This chapter presents a description of methods used for D-alanine assays in the solution as medium and AARase assays in lactic acid bacteria (LAB) cells for isolation of high AARase-containing strains using D-amino acid oxidase and lactic acid dehydrogenase via a NADH oxidoreduction system.

Some aquatic invertebrates contain free d -alanine. We previously showed copious amounts of free d -glutamate, a novel d -amino acid, in the tissue of the male reproductive organs of Marsupenaeus japonicus. Herein, we clarified the distribution and potential role of d -glutamate and d -alanine in male reproductive tissues, namely the testis, vas deferens and seminal receptacle at different growth stages of M. japonicus. The percentage of d -glutamate to total glutamate was over 50% in these tissues. In particular, the content of d -glutamate was the most abundant in the vas deferens, the ratio of d -glutamate to total glutamate was approximately 80%. In contrast, d -alanine content was the lowest in the vas deferens among these tissues. d -Glutamate content was the highest when the prawn weighed 12 g, indicating that d -glutamate is actively synthesized in the younger stage. Our findings suggest that d -glutamate plays an important role in the reproductive functions of M. japonicus. [ABSTRACT FROM AUTHOR]

The effects of the N-methyl-D-aspartate receptor activators D-serine, D-alanine, and sarcosine against schizophrenia and depression are promising. Nevertheless, high doses of D-serine and sarcosine are associated with undesirable nephrotoxicity or worsened prostatic cancer. Thus, alternatives are needed. DAAO inhibition can increase D-serine as well as D-alanine and protect against D-serine-induced nephrotoxicity. Although several DAAO inhibitors improve the symptoms of schizophrenia and depression, they can increase the plasma levels but not brain levels of D-serine. The mechanism of action of DAAO inhibitors remains unclear. We investigated the effects of the DAAO inhibitor sodium benzoate on the prefrontal cortex and hippocampal level of D-alanine as known another substrate with antipsychotic and antidepressant properties and other NMDAR-related amino acids, such as, L-alanine, D-serine, L-serine, D-glutamate, L-glutamate, and glycine levels. Our results indicate that sodium benzoate exerts antipsychotic and antidepressant-like effects without changing the D-serine levels in the brain prefrontal cortex (PFC) and hippocampus. Moreover, D-alanine levels in the PFC and hippocampus did not change. Despite these negative findings regarding the effects of D-amino acids in the PFC and hippocampus, sodium benzoate exhibited antipsychotic and antidepressant-like effects. Thus, the therapeutic effects of sodium benzoate are independent of D-serine or D-alanine levels. In conclusion, sodium benzoate may be effective among patients with schizophrenia or depression; however, the mechanisms of actions remain to be elucidated. [ABSTRACT FROM AUTHOR]

D-Alanine belongs to nonessential amino acids that have diverse applications in the fields of food and health care. (R)-transaminase [(R)-TA]-catalyzed asymmetric amination of pyruvate is a feasible alternative for the synthesis of D-alanine, but low catalytic efficiency and thermostability limit enzymatic utilization. In this work, several potential (R)-TAs were discovered using NCBI database mining synchronously with enzymatic structure-function analysis, among which Capronia epimyces TA (CeTA) showed the highest activity for amination of pyruvate using (R)-α-methylbenzylamine as the donor. Furthermore, enzymatic residues surrounding a large catalysis pocket were subjected to saturation and combinatorial mutagenesis, and positive mutant F113T showed dramatic improvement in activity and thermostability. Molecular modeling indicated that the substitution of phenylalanine with threonine afforded alleviation of steric hindrance in the pocket and induced formation of additional hydrogen bonds with neighboring residues. Finally, using recombinant cells containing F113T as a biocatalyst, the conversion yield of amination of 100 mM pyruvate to D-alanine achieved up to 95.2%, which seemed to be the highest level in the literature regarding synthesis of D-alanine using TAs. The inherent characteristics rendered CeTA F113T a promising platform for efficient preparation of D-alanine operating with high productivity. [ABSTRACT FROM AUTHOR]

The present study was undertaken to investigate the susceptibility patterns as antifungal and growth of certain D-amino acids inhibitory effects, includes D-alanine(D-ala) and D-proline (D-pro). The obtained results indicated that D- proline acid is potent mostly as antifungal, among tested D-amino acids, then followed by D-ala. The study aim was evaluating the of D-amino acids and nanoparticles against Candida albicans adhered cells and bio-films. Results showed that D-ala MIC value was 25 μg/ml, while in methods of TCP, (D-pro) MIC 50 μg/ml value. Nanoparticles (lithium and silver) and amino acids (D-pro and D-ala) effect in preventing cells adhesion on polystyrene surface and mature bio-film inhibition was studied. Highest inhibition was obtained at D-pro concentration 50 μg /ml, while the lowest at concentration D-ala 25 μg /ml against mature bio-film and cell adhesion. [ABSTRACT FROM AUTHOR]

N-Methyl- and N-benzyl-4H-thieno[3,2-b]pyrrole-5-carboxylic amides containing cytisine, (R)-(+)-α-methylbenzylamine, aniline, and some amino acid fragments have been synthesized. [ABSTRACT FROM AUTHOR]

Purpose: The purpose of this study was to investigate the physiological changes of amino acids in the rat retina caused by ocular hypertension. Methods: Adult Wistar rats were used as an experimental model of ocular hypertension. Retinas were hydrolyzed with HCl at 108°C to isolate amino acids. Residual amino acids were measured by reverse-phase high-performance liquid chromatography and the total volume of residual amino acids and the ratio of D- and L-amino acids were analyzed. Free D- and L-alanine levels were also measured using two-dimensional HPLC. Results: The amount of retinal alanine decreased in ocular hypertension compared with the control (p <.05, Student's t-test); the amounts of other amino acids did not differ between the two conditions. The D/L ratio of alanine was higher than that of other amino acids. Ocular hypertension reduced the D/L ratio of retinal alanine, while that of other amino acids was unchanged. Ocular hypertension increased the D/L ratio of free alanine. Conclusions: Ocular hypertension reduced the D/L ratio of retinal alanine, presumably due in large part to alanine peptides. [ABSTRACT FROM AUTHOR]

For intracellular Chlamydiaceae, there is no need to withstand osmotic challenges, and a functional cell wall has not been detected in these pathogens so far. Nevertheless, penicillin inhibits cell division in Chlamydiaceae resulting in enlarged aberrant bodies, a phenomenon known as chlamydial anomaly. D-alanine is a unique and essential component in the biosynthesis of bacterial cell walls. In free-living bacteria like Escherichia coli, penicillin-binding proteins such as monofunctional transpeptidases PBP2 and PBP3, the putative targets of penicillin in Chlamydiaceae, cross-link adjacent peptidoglycan strands via meso-diaminopimelic acid and D-Ala-D-Ala moieties of pentapeptide side chains. In the absence of genes coding for alanine racemase Alr and DadX homologs, the source of D-Ala and thus the presence of substrates for PBP2 and PBP3 activity in Chlamydiaceae has puzzled researchers for years. Interestingly, Chlamydiaceae genomes encode GlyA, a serine hydroxymethyltransferase that has been shown to exhibit slow racemization of D- and L-alanine as a side reaction in E. coli. We show that GlyA from Chlamydia pneumoniae can serve as a source of D-Ala. GlyA partially reversed the D-Ala auxotrophic phenotype of an E. coli racemase double mutant. Moreover, purified chlamydial GlyA had racemase activity on L-Ala in vitro and was inhibited by D-cycloserine, identifying GlyA, besides D-Ala ligase MurC/Ddl, as an additional target of this competitive inhibitor in Chlamydiaceae. Proof of D-Ala biosynthesis in Chlamydiaceae helps to clarify the structure of cell wall precursor lipid II and the role of chlamydial penicillin-binding proteins in the development of non-dividing aberrant chlamydial bodies and persistence in the presence of penicillin. [ABSTRACT FROM AUTHOR]

The gdaA gene encoding S12 family glycine- d-alanine aminopeptidase (GdaA) was found in the industrial fungus Aspergillus oryzae. GdaA shares 43% amino acid sequence identity with the d-aminopeptidase of the Gram-negative bacterium Ochrobactrum anthropi. GdaA purified from an A. oryzae gdaA-overexpressing strain exhibited high d-stereospecificity and efficiently released N-terminal glycine and d-alanine of substrates in a highly specific manner. The optimum pH and temperature were 8 to 9 and 40°C, respectively. This enzyme was stable under alkaline conditions at pH 8 to 11 and relatively resistant to acidic conditions until pH 5.0. The chelating reagent EDTA, serine protease inhibitors such as AEBSF, benzamidine, TPCK, and TLCK, and the thiol enzyme inhibitor PCMB inhibited the enzyme. The aminopeptidase inhibitor bestatin did not affect the activity. GdaA was largely responsible for intracellular glycine and d-alanine aminopeptidase activities in A. oryzae during stationary-phase growth in liquid media. In addition, the activity increased in response to the depletion of nitrogen or carbon sources in the growth media, although the GdaA-independent glycine aminopeptidase activity highly increased simultaneously. Aminopeptidases of A. oryzae attract attention because the enzymatic release of a variety of amino acids and peptides is important for the enhancement of the palatability of fermented foods. GdaA activity was found in extracts of a solid-state rice culture of A. oryzae (rice koji), which is widely used as a starter culture for Japanese traditional fermented foods, and was largely responsible for the glycine and d-alanine aminopeptidase activity detected at a pH range of 6 to 9. [ABSTRACT FROM AUTHOR]

Abstract: The distribution of d-amino acids was examined on several tissues of kuruma prawn Marsupenaeus japonicus. d-Alanine was found in all tissues, and the ratio of d-alanine to total alanine ranged from 18.7 to 43.7% depending on the tissues. Of these tissues, muscle, heart, and gill contained a relatively large amount of d-alanine. Nervous tissue and eye, on the other hand, contained a large amount of d-aspartate. d-Glutamate was specifically detected in testis. The percentage of d-glutamate to total glutamate was over 50% in testis, suggesting the existence of the biosynthetic enzyme in this tissue. The changes of alanine racemase activity were determined in the muscle and hepatopancreas of M. japonicus before and after molting. The activity after molting increased twice in the muscle. On the other hand, it was not changed in the hepatopancreas. These data suggest that d-alanine plays an important role in the muscle during ecdysis. However, the free d-alanine level in the muscle was not changed significantly before and after ecdysis. From these data, several d-amino acids are considered to be utilized in some essential physiological phenomena in the different tissues of the prawn. [Copyright &y& Elsevier]

Abstract: Two-dimensional-HPLC procedures have been established for the sensitive and selective determination of d-serine (d-Ser) and d-alanine (d-Ala), and their amounts in the tissues and physiological fluids of mice with various d-amino-acid oxidase (DAO) activities have been demonstrated. These two d-amino acids are modulators of the N-methyl-d-aspartate receptor mediated neurotransmission, and the alterations in their amounts following the changes in the DAO activity are matters of interest. After pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), the d-amino acids were determined by the 2D-HPLC system with fluorescence detectors. As the first dimension, a microbore-monolithic-ODS column (750mm×0.53mm I.D.) was adopted and a self-packed narrowbore-Pirkle type enantioselective column (Sumichiral OA-2500S, 250mm×1.5mm I.D.) was selected for the second dimension. The lower limits of quantitation of d-Ser and d-Ala were 500amol, and the within-day and day-to-day precisions were less than 6.8%. Using these methods, the amounts of d-Ser and d-Ala in 6 brain tissues, 4 peripheral tissues, serum and urine of mice having various DAO activities were determined; the amounts of these d-amino acids were drastically increased with a lowering of the DAO activity except for the cases of d-Ser in the frontal brain regions. The present micro-2D-HPLC procedures are powerful tools for the determination of small amounts of d-Ser and d-Ala in mammalian samples, and the obtained results would be useful for developing novel drugs that modulate the DAO activity, such as DAO inhibitors, against neuronal diseases. [Copyright &y& Elsevier]

Aquatic crustaceans and some bivalve mollusks are known to contain copious amounts of free d-alanine in their tissues. For the first time in the animal kingdom, we have isolated a cDNA clone encoding alanine racemase from the muscle and hepatopancreas of the kuruma prawn Marsupenaeus japonicus. The recombinant enzyme expressed in Escherichia coli exhibited alanine recemase activity. The deduced amino-acid sequence showed only 23–31% identity to bacterial alanine racemases. However, the active site residues and some residues that interact with pyridoxal 5′-phosphate were also conserved in M. japonicus enzyme. There was higher alanine racemase mRNA expression in hepatopancreas than in muscle. In contrast, the d-alanine content in hepatopancreas was lower than that in muscle, suggesting that the physiological functions of free d-alanine may differ among tissues. These data suggest that the alanine racemase gene has been conserved from bacteria to invertebrates throughout a long evolutionary time scale. [ABSTRACT FROM PUBLISHER]

The understanding ofd-amino acid metabolism in higher plants lags far behind that in mammals, for which the biological functions of these unique amino acids have already been elucidated. In this article, we report on the biochemical behavior ofd-amino acids (particularlyd-Asp) and relevant metabolic enzymes in Arabidopsis thaliana. During germination and growth of the plant, a transient increase ind-Asp levels was observed, suggesting thatd-Asp is synthesized in the plant. Administration ofd-Asp suppressed growth, although the inhibitory mechanism responsible for this remains to be clarified. Exogenousd-Asp was efficiently incorporated and metabolized, and was converted to otherd-amino acids (d-Glu andd-Ala). We then studied the related metabolic enzymes, and consequently cloned and characterized A. thalianad-amino acid aminotransferase, which is presumably involved in the metabolism ofd-Asp in the plant by catalyzing transamination betweend-amino acids. This is the first report of cDNA cloning and functional characterization of ad-amino acid aminotransferase in eukaryotes. The results presented here provide important information for understanding the significance ofd-amino acids in the metabolism of higher plants. [ABSTRACT FROM AUTHOR]

Abstract: d-amino acids are generally considered to be important markers of bacterial contamination of food products. A screen-printed amperometric biosensor for the detection of d-amino acids has been constructed by the immobilization of d-amino acid oxidase on a graphite working electrode of a screen-printed strip modified with Prussian Blue and Nafion layers. Enzyme immobilization was then carried out by cross-linking of a mixture of the enzyme and bovine serum albumin with glutaraldehyde. As a result of the mediator addition and because of the multi-layer construction of the biosensor, including a polymer layer to avoid the interferences, the limit of the detection of the developed biosensor was two orders of magnitude improved in comparison to other screen-printed biosensors, as far as the determination of amino acids is concerned. Additional modification of the graphite electrode with carbon nanotubes led to a significant enhancement of the signal magnitude. A fast linear response of the developed biosensor was subsequently observed in static measurements for d-alanine in the concentration range from 5 to 200 μM. Excellent enantioselectivity towards d-amino acids was discovered. During the experiment, d-amino acids were detected in fruit juices and some milk samples. The complex matrix of natural milk samples had no influence on the response of the biosensor. The results were in good agreement with those obtained by capillary electrophoresis measurements. [Copyright &y& Elsevier]

Abstract: A mouse monoclonal antibody against d-alanine (d-Ala) has been raised and the immunohistochemical localization of this d-amino acids in the rat pancreas is visualized. The obtained anti-d-Ala monoclonal antibody has no significant cross-reactivity to all proteinogenic l-amino acids and their d-enantiomers. Using this antibody, immunohistochemical staining was performed on the pancreas, and specific staining for d-Ala has been observed only in the Langerhans islets. To identify the types of d-Ala-immunopositive cells, double staining was carried out with antibodies against d-Ala and pancreatic hormones. Similar immunostaining patterns have been observed for d-Ala and insulin, while d-Ala is hardly co-localized with other hormones (glucagon, somatostatin, and pancreatic polypeptide). These results indicate for the first time that d-Ala is localized to insulin producing β-cells in mammalian pancreas, suggesting that this d-amino acid would be involved in the regulation of the blood glucose level. [Copyright &y& Elsevier]

Abstract: Development of Grb2–SH2 domain antagonists is considered to be an effective and non-cytotoxic strategy to develop new antiproliferative agents because of their potential to shut down the Ras signaling pathway. We developed a concise route for the efficient synthesis of G1TE analogs on solid phase. Using this route, a series of cyclic peptides that do not rely on phosphotyrosine or its mimics were designed and synthesized based upon the phage library-derived cyclopeptide, G1TE. Considering that Gly7 plays prominent roles for G1TE binding to the Grb2–SH2 domain, we introduced different amino acids in the 7th position. The d-Ala7-containing peptide 3 demonstrates improved binding affinity by adopting favorable conformation for protein binding. This can be rationalized by molecular modeling. The optimization at the Leu2 position was also studied, and the resulting cyclopeptides exhibited remarkably improved binding affinity. Based upon these global modifications, a highly potent peptide ligand 9 was discovered with a K d =17nM, evaluated by Biacore binding assay. This new analog is one of the most potent non-phosphorus-containing Grb2–SH2 antagonists reported to date. This potent peptidomimetic provides a new template for the development of non-pTyr containing Grb2–SH2 domain antagonists and acts as a chemotherapeutic lead for the treatment of erbB2-related cancer. [Copyright &y& Elsevier]

Abstract: 2-Amino-2,3-dideoxy-d-manno-heptonic acid (7) has been synthesized from 2,5,6,7-tetra-O-acetyl-3-deoxy-d-gluco-heptono-1,4-lactone (1), which was readily prepared from d-glycero-d-gulo-heptono-1,4-lactone. O-Deacetylation of 1 followed by treatment with 13:1 (v/v) 2,2-dimethoxypropane/acetone in the presence of p-toluenesulfonic acid gave methyl 3-deoxy-4,5:6,7-di-O-isopropylidene-d-gluco-heptonate (3) as a crystalline product (80% yield). The free hydroxyl group (OH-2) of 3 was mesylated and substituted by azide to give the corresponding azide derivative 5. Hydrogenolysis and further hydrolysis of the ester function of 5 afforded α-amino acid 7 (43% overall yield from 1). Compound 7 is an analog of l-alanine having a polyhydroxy chain attached to C-3. The diastereoisomer of 7 at C-2, 2-amino-2,3-dideoxy-d-gluco-heptonic acid (12) was also prepared from 3, by a route that involved 2,3-dideoxy-2-iodo derivative 8 as a key intermediate. [Copyright &y& Elsevier]

Abstract: d-Amino acid oxidase of carp (Cyprinus carpio) hepatopancreas was overexpressed in Escherichia coli cells and purified to homogeneity for the first time in animal tissues other than pig kidney. The purified preparation had a specific activity of 293 units mg−1 protein toward d-alanine as a substrate. It showed the highest activity toward d-alanine with a low Km of 0.23 mM and a high kcat of 190 s−1 compared to 10 s−1 of the pig kidney enzyme. Nonpolar and polar uncharged d-amino acids were preferable substrates to negatively or positively charged amino acids. The enzyme exhibited better thermal and pH stabilities than several yeast counterparts or the pig kidney enzyme. Secondary structure topology consisted of 11 α-helices and 17 β-strands that differed slightly from pig kidney and Rhodotorula gracilis enzymes. A three-dimensional model of the carp enzyme constructed from a deduced amino acid sequence resembled that of pig kidney d-amino acid oxidase but with a shorter active site loop and a longer C-terminal loop. Judging from these characteristics, carp d-amino acid oxidase is close to the pig kidney enzyme structurally, but analogous to the R. gracilis enzyme enzymatically in turnover rate and pH and temperature stabilities. [Copyright &y& Elsevier]

A full-length cDNA encoding d-amino acid oxidase (DAO, EC 1.4.3.3) was cloned and sequenced from the hepatopancreas of carp fed a diet supplemented with d-alanine. This clone contained an open reading frame encoding 347 amino acid residues. The deduced amino acid sequence exhibited about 60 and 19–29% identity to mammalian and microbial DAOs, respectively. The expression of full-length carp DAO cDNA in Escherichia coli resulted in a significant level of protein with DAO activity. In carp fed the diet with d-alanine for 14 days, DAO mRNA was strongly expressed in intestine followed by hepatopancreas and kidney, but not in muscle. During d-alanine administration, DAO gene was expressed quickly in hepatopancreas with the increase of DAO activity. The inducible nature of carp DAO indicates that it plays an important physiological role in metabolizing exogenous d-alanine that is abundant in their prey invertebrates, crustaceans, and mollusks. [Copyright &y& Elsevier]

Alanine racemase [EC 5.1.1.1], which catalyzes the interconversion between d- and l-alanine, was purified to homogeneity from the muscle of black tiger prawn Penaeus monodon. The isolated enzyme had a molecular mass of 44 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 90 kDa on gel filtration, indicating a dimeric nature of the enzyme. The enzyme was highly specific to d- and l-alanine and did not catalyze the racemization of other amino acids. Km values toward both d- and l-alanine were almost equal and considerably high compared with those of bacterial enzymes. The purified enzyme retained its activity in the absence of pyridoxal 5′-phosphate as a cofactor but carbonyl reagents inhibited the activity, suggesting the tightly binding of the cofactor to the enzyme protein. Several partial amino acid sequences of peptide fragments of the purified enzyme showed positive homologies from 52 to 76% with bacterial counterparts and a catalytic tyrosine residue of the bacterial enzyme was also retained in the prawn one, indicating alanine racemase gene is well conserved from bacteria to invertebrates. [Copyright &y& Elsevier]

Under hyper-salinity stress from freshwater to 17 and 25 ppt seawater, red swamp crayfish Procambarus clarkii largely accumulated d- and l-alanine together with glycine, l-glutamine, and l-proline in both muscle and hepatopancreas. The increases of d- and l-alanine in muscle were the highest in all amino acids and reached 6.8- and 5.4-fold, respectively, from freshwater to 25 ppt seawater. These results indicate that both d- and l-alanine are the most potent osmolytes for intracellular isosmotic regulation in crayfish as well as other crustaceans thus far examined. Under anoxia stress below 0.1 mg/l dissolved oxygen for 12 h and subsequent recovery in normoxia for 12 h in freshwater, 17 and 25 ppt seawater, muscle ATP decreased dramatically in all salinity levels and almost depleted in seawater. Along with the decrease of muscle glycogen level, the significant increase of l-lactate was found in muscle, hepatopancreas, and hemolymph for each salinity level, suggesting the transport of l-lactate from muscle into hepatopancreas via hemolymph. Under anoxia, d- and l-alanine also largely increased in both muscle and hepatopancreas for each salinity level. The increase was much higher in seawater than in freshwater. Thus, both d- and l-alanine are possible to be anaerobic end products during prolonged anaerobiosis of this species. [Copyright &y& Elsevier]

The net uptake of 14C-D-alanine by the adaxial surfaces of the trap lobes of Dionaea muscipula Ellis (Venus's flytrap) is positively correlated with the rate of H+ efflux from the trap lobes. Effectors that stimulate H+ efflux (secretion elicitor, fusicoccin and auxin) enhance D-alanine uptake whereas effectors that inhibit H+ efflux (diethylstilbestrol) retard uptake. It is proposed that amino acid uptake by the digestive glands of the trap lobes proceeds via a mechanism which mediates the co-transport of H+ ions and amino acids. A model is presented in which a positively charged ternary complex of carrier plus amino acid plus H+ is transported down an inwardly directed H+ electro-chemical potential difference generated by the secretion-elicitor-provoked discharge of HCI into the trap cavity. [ABSTRACT FROM AUTHOR]

The relative amounts of free D-amino acids (D-AA) in the urine of seven healthy volunteers (age 27 to 49 years) were determined using chiral phase (Chirasil-L-Val) capillary gas chromatography in conjunction with selected ion monitoring mass spectrometry. The absolute amounts of free D-AA were determined by pre-column derivatization of the amino acids with o-phthaldialdehyde and N-isobutyryl-L-cysteine followed by high-performance liquid chromatographic separation and fluorescence detection of the isoindol derivatives formed. The following most abundant D-AA were found (highest and lowest absolute and relative amounts): D-Ser (379.8 - 30.1 µMol/L; 56.5 - 19.0%), D-Ala (53.8 - 7.6 µMol/L; 19.6 - 5.7%), D-Thr (5.8 - 0.25 µMol/L; 3.4 - 1.0%), D-Val (3.7 - 0 µMol/L; 4.2 - 0%), and D-Phe (3.5 - 0.35 µMol/L; 4.8 - 1.4%). [ABSTRACT FROM AUTHOR]

d -Alanine (d -Ala) is an unusual endogenous amino acid present in invertebrates and vertebrates. Compared to its l -isomer, the characterization of d -Ala is challenging because of the need for chiral resolution and the low amounts of the d -enantiomer present. With recent improvements in measurement capabilities, research on d -Ala, along with other d -amino acids, has been growing, especially as the functional significance of d -Ala in the mammalian nervous and endocrine systems is becoming known. Here we provide an overview of the distribution, origin, function, and disease implications of d -Ala. Unlabelled Image • d -alanine is widely distributed in invertebrates and vertebrates. • d -alanine originates from diet, microbiota, and potentially endogenous synthesis. • Acts as a putative signaling molecule in the nervous and endocrine systems. • Physiological relevance in the gut-microbiota-brain axis and in the immune response. • d -alanine levels track various disease states suggesting a potential therapeutic role. [ABSTRACT FROM AUTHOR]