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Previously, we demonstrated that the animal aspartate racemase (AspR) gene has evolved from the serine racemase (SerR) gene by acquisition of three consecutive serine residues (Ser155-Ser156-Ser157) involved in the strong AspR activity, and this event has occurred independently and frequently during animal evolution. In the present study, we cloned and characterized two mammalian SerR homologous genes from the hemichordate acorn worm (Saccoglossus kowalevskii). The enzymes have been identified as an AspR and an aspartate/glutamate racemase (Asp/GluR) on the basis of their kinetic parameters. The S. kowalevskii Asp/GluR shows comparable substrate affinity and high catalytic efficiency (kcat/Km) for both aspartate and glutamate and is the first reported enzyme from animals that can synthesize d-glutamate. Amino acid sequence alignment analysis and site-directed mutagenesis studies have revealed that the amino acid residue at position 156, which is serine in AspR and alanine in Asp/GluR, is associated with binding and recognition of glutamate and aspartate. Phylogenetic analysis suggests that the S. kowalevskii AspR gene has evolved from the SerR gene after the divergence of hemichordata and vertebrate lineages by acquisition of the three serine residues at position 155 to 157 as in the case of other animal AspR genes. Furthermore, the S. kowalevskii Asp/GluR gene is the result of AspR gene duplication and several amino acid substitutions including that of the 156th serine residue with alanine. The fact that SerR has acquired substrate specificity towards aspartate or glutamate raises the possibility that synthesis of other d-amino acids is carried out by enzymes evolved from SerR.

Recently, we cloned and characterized eleven serine and aspartate racemases (SerR and AspR, respectively) from animals. These SerRs and AspRs are not separated by their racemase functions and form a serine/aspartate racemase family cluster based on phylogenetic analysis. Moreover, we have proposed that the AspR-specific triple serine loop region at amino acid positions 150-152 may be responsible for the large AspR activity. In the present study, to test this hypothesis, we prepared and characterized fourteen mutants in this region of animal SerRs and AspRs. The large AspR activity in Acropora and Crassostrea AspR was reduced to

Recently, substantial levels of acidic D-amino acids, such as D-aspartate and D-glutamate, have been identified in many organisms, from bacteria to mammals, suggesting that acidic D-amino acids have multiple physiological significances. Although acidic D-amino acids found in animals primarily originate from foodstuffs and/or bacteria, the D-aspartate-synthesizing enzyme aspartate racemase is identified in various animals. In eukaryotic organisms, acidic D-amino acids are primarily degraded by the flavoenzyme D-aspartate oxidase (DDO). DDO is found in multiple eukaryotic organisms and may play important roles in acidic D-amino acid utilization, elimination, and intracellular level regulation. Moreover, owing to its perfect enantioselectivity and stereoselectivity, DDO may be a valuable tool in several biotechnological applications, including the identification and quantification of acidic D-amino acids. In this mini-review, previous DDO reports are summarized and the potential bioengineering and biotechnological applications of DDO are discussed. Key Points ・Occurrence and distribution ofd-aspartate oxidase. ・Fundamental properties of d -aspartate oxidase of various eukaryotic organisms. ・Biotechnological applications and potential engineering ofd-aspartate oxidase.

d-Aspartate, aspartate racemase activity, and d-aspartate oxidase activity were detected in tissues from several types of starfish. Aspartate racemase activity in male testes of Patiria pectinifera was significantly elevated in the summer months of the breeding season compared with spring months. We also compared aspartate racemase activity with the gonad index and found that activity in individuals with a gonad index ≥6% was four-fold higher than that of individuals with a gonad index

D-Aspartate (D-Asp) has important physiological functions, and recent studies have shown that substantial amounts of free D-Asp are present in a wide variety of mammalian tissues and cells. Biosynthesis of D-Asp has been observed in several cultured rat cell lines, and a murine gene (glutamate-oxaloacetate transaminase 1-like 1, Got1l1) that encodes Asp racemase, a synthetic enzyme that produces D-Asp from L-Asp, was proposed recently. The product of this gene is homologous to mammalian glutamate-oxaloacetate transaminase (GOT). Here, we tested the hypothesis that rat and human homologs of mouse GOT1L1 are involved in Asp synthesis. The following two approaches were applied, since the numbers of attempts were unsuccessful to prepare soluble GOT1L1 recombinant proteins. First, the relationship between the D-Asp content and the expression levels of the mRNAs encoding GOT1L1 and D-Asp oxidase, a primary degradative enzyme of D-Asp, was examined in several rat and human cell lines. Second, the effect of knockdown of the Got1l1 gene on D-Asp biosynthesis during culture of the cells was determined. The results presented here suggest that the rat and human homologs of mouse GOT1L1 are not involved in D-Asp biosynthesis. Therefore, D-Asp biosynthetic pathway in mammals is still an urgent issue to be resolved.

Aspartate racemase (AspR) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that is responsible for D-aspartate biosynthesis in vivo. To the best of our knowledge, this is the first study to report an X-ray crystal structure of a PLP-dependent AspR, which was resolved at 1.90 Å resolution. The AspR derived from the bivalve mollusc Scapharca broughtonii (SbAspR) is a type II PLP-dependent enzyme that is similar to serine racemase (SR) in that SbAspR catalyzes both racemization and dehydration. Structural comparison of SbAspR and SR shows a similar arrangement of the active-site residues and nucleotide-binding site, but a different orientation of the metal-binding site. Superposition of the structures of SbAspR and of rat SR bound to the inhibitor malonate reveals that Arg140 recognizes the β-carboxyl group of the substrate aspartate in SbAspR. It is hypothesized that the aromatic proline interaction between the domains, which favours the closed form of SbAspR, influences the arrangement of Arg140 at the active site.

d-Aspartate is an endogenous free amino acid in the brain, endocrine tissues, and exocrine tissues in mammals, and it plays several physiological roles. In the testis, d-aspartate is detected in elongate spermatids, Leydig cells, and Sertoli cells, and implicated in the synthesis and release of testosterone. In the hippocampus, d-aspartate strongly enhances N-methyl-d-aspartate receptor-dependent long-term potentiation and is involved in learning and memory. The existence of aspartate racemase, a candidate enzyme for d-aspartate production, has been suggested. Recently, mouse glutamic-oxaloacetic transaminase 1-like 1 (Got1l1) has been reported to synthesize substantially d-aspartate from l-aspartate and to be involved in adult neurogenesis. In this study, we investigated the function of Got1l1 in vivo by generating and analyzing Got1l1 knockout (KO) mice. We also examined the enzymatic activity of recombinant Got1l1 in vitro. We found that Got1l1 mRNA is highly expressed in the testis, but it is not detected in the brain and submandibular gland, where d-aspartate is abundant. The d-aspartate contents of wild-type and Got1l1 KO mice were not significantly different in the testis and hippocampus. The recombinant Got1l1 expressed in mammalian cells showed l-aspartate aminotransferase activity, but lacked aspartate racemase activity. These findings suggest that Got1l1 is not the major aspartate racemase and there might be an as yet unknown d-aspartate-synthesizing enzyme.

It was long believed that D-amino acids were either unnatural isomers or laboratorial artifacts and that the important functions of amino acids were exerted only by l-amino acids. However, recent investigations have shown that a variety of D-amino acids are present in mammals and that they play important roles in physiological functions in the body. Among the free d-amino acids that have been identified in mammals, D-aspartate (D-Asp) has been shown to play a crucial role in the neuroendocrine and endocrine systems as well as in the central nervous system. Here, we present an overview of recent studies of free D-Asp, focusing on the analytical methods in real biological matrices, expression and localization in tissues and cells, biological and physiological activities, biosynthesis, degradation, cellular transport, and possible relevance to disease. In addition to frequently used techniques for the enantiomeric determination of amino acids, including high-performance liquid chromatography and enzymatic methods, the recent development of analytical methods is also described.

d-Aspartate (d-Asp) is found in specific neurons, transported to neuronal terminals and released in a stimulation-dependent manner. Because d-Asp formation is not well understood, determining its function has proved challenging. Significant levels of d-Asp are present in the cerebral ganglion of the F- and C-clusters of the invertebrate Aplysia californica, and d-Asp appears to be involved in cell-cell communication in this system. Here, we describe a novel protein, DAR1, from A. californica that can convert aspartate and serine to their other chiral form in a pyridoxal 5′-phosphate (PLP)-dependent manner. DAR1 has a predicted length of 325 amino acids and is 55% identical to the bivalve aspartate racemase, EC 5.1.1.13, and 41% identical to the mammalian serine racemase, EC 5.1.1.18. However, it is only 14% identical to the recently reported mammalian aspartate racemase, DR, which is closely related to glutamate-oxaloacetate transaminase, EC 2.6.1.1. Using whole-mount immunohistochemistry staining of the A. californica central nervous system, we localized DAR1-like immunoreactivity to the medial region of the cerebral ganglion where the F- and C-clusters are situated. The biochemical and functional similarities between DAR1 and other animal serine and aspartate racemases make it valuable for examining PLP-dependent racemases, promising to increase our knowledge of enzyme regulation and ultimately, d-serine and d-Asp signaling pathways.

D-Aspartic acid is abundant in the developing brain. We have identified and cloned mammalian aspartate racemase (DR), which converts L-aspartate to D-aspartate and colocalizes with D-aspartate in the brain and neuroendocrine tissues. Depletion of DR by retrovirus-mediated expression of short-hairpin RNA in newborn neurons of the adult hippocampus elicits profound defects in the dendritic development and survival of newborn neurons and survival. Because D-aspartate is a potential endogenous ligand for NMDA receptors, the loss of which elicits a phenotype resembling DR depletion, D-aspartate may function as a modulator of adult neurogenesis.

Free d-amino acids have been found in various invertebrate phyla, while amino acid racemase genes have been identified in few species. The purpose of this study is to elucidate the distribution, function, and evolution of amino acid racemases in invertebrate animals. We searched the GenBank databases, and found 11 homologous serine racemase genes from eight species in eight different invertebrate phyla. The cloned genes were identified based on their maximum activity as Acropora millepora (Cnidaria) serine racemase (SerR) and aspartate racemase (AspR), Caenorhabditis elegans (Nematoda) SerR, Capitella teleta (Annelida) SerR, Crassostrea gigas (Mollusca) SerR and AspR, Dugesia japonica (Platyhelminthes) SerR, Milnesium tardigradum (Tardigrada) SerR, Penaeus monodon (Arthropoda) SerR and AspR and Strongylocentrotus purpuratus (Echinodermata) AspR. We found that Acropora, Aplysia, Capitella, Crassostrea and Penaeus had two amino acid racemase paralogous genes and these paralogous genes have evolved independently by gene duplication at their recent ancestral species. The transcriptome analyses using available SRA data and enzyme kinetic data suggested that these paralogous genes are expressed in different tissues and have different functions in vivo. Phylogenetic analyses clearly indicated that animal SerR and AspR are not separated by their particular racemase functions and form a serine/aspartate racemase family cluster. Our results revealed that SerR and AspR are more widely distributed among invertebrates than previously known. Moreover, we propose that the triple serine loop motif at amino acid positions 150–152 may be responsible for the large aspartate racemase activity and the AspR evolution from SerR.

D-Aspartate is present at high concentrations in the tissues of Scapharca broughtonii, and its production depends on aspartate racemase. This enzyme is the first aspartate racemase purified from animal tissues and unique in its pyridoxal 5'-phosphate (PLP)-dependence in contrast to microbial aspartate racemases thus far characterized. The enzyme activity is markedly increased in the presence of AMP and decreased in the presence of ATP. To analyze the structure-function relationship of the enzyme further, we cloned the cDNA of aspartate racemase, and then purified and characterized the recombinant enzyme expressed in Escherichia coli. The cDNA included an open reading frame of 1,017 bp encoding a protein of 338 amino acids, and the deduced amino acid sequence contained a PLP-binding motif. The sequence exhibits the highest identity (43-44%) to mammalian serine racemase, followed mainly by threonine dehydratase. These relationships are fully supported by phylogenetic analyses of the enzymes. The active recombinant aspartate racemase found in the Escherichia. coli extract represented about 10% of total bacterial protein and was purified to display essentially identical physicochemical and catalytic properties with those of the native enzyme. In addition, the enzyme showed a dehydratase activity toward L-threo-3-hydroxyaspartate, similar to the mammalian serine racemase that produces pyruvate from D- and L-serine.

In the lizard Podarcis s. sicula, a substantial amount of d-aspartate (d-Asp) is endogenous to the testis and shows cyclic changes of activity connected with sex hormone profiles during the annual reproductive phases. Testicular d-Asp content shows a direct correlation with testosterone titres and a reverse correlation with 17β-estradiol titres. In vivo experiments, consisting of i.p. injections of 2.0 μmol/g body weight of d-Asp or other amino acids, in lizards collected during the three main phases of the reproductive cycle (pre-reproductive, reproductive and post-reproductive period), revealed that the testis can specifically take up and accumulate d-Asp alone. Moreover, this amino acid influences the synthesis of testosterone and 17β-estradiol in all phases of the cycle. This phenomenon is particularly evident during the pre- and post-reproductive period, when endogenous testosterone levels observed in both testis and plasma were the lowest and 17β-estradiol concentrations were the highest. d-Asp rapidly induces a fall in 17β-estradiol and a rise in testosterone at 3 h post-injection in the testis and at 6 h post-injection in the blood. In vitro experiments show that testicular tissue converted l-Asp into d-Asp through an aspartate racemase. d-Asp synthesis was measured in all phases of the cycle, but was significantly higher during the reproductive period with a peak at pH 6.0. The exogenous d-Asp also induces a significant increase in the mitotic activity of the testis at 3 h (PPd-Asp is recognized by an intense immunoreactivity of the germinal epithelium (spermatogonia and spermatids) for proliferation cell nuclear antigen (PCNA). The effects of d-Asp on the testis appear to be specific since they were not seen in lizards injected with other d- or l-forms of amino acids with known excitatory effects on neurosecretion. Our results suggest a regulatory role for d-Asp in the steroido-genesis and spermatogenesis of the testis of the lizard Podarcis s. sicula.

Blood shell, Scapharca broughtonii, contains large quantities of free D-aspartate comparable to free L-aspartate in its tissues. When the shell was reared in hypoxic seawater, D-aspartate as well as L-aspartate in the foot muscle decreased rapidly, and their total level became about one-fourth within 24 hr. None of the other amino acids examined showed a similar behavior, but many of them rather increased during the same period. The increase in L-alanine was especially remarkable and was almost equal to the sum of the decrease in aspartate enantiomers. When the shell that had been acclimated to hypoxic seawater for 96 hr was transferred to normoxic seawater, all the amino acid levels mostly returned to the control levels within 96 hr. In contrast to these effects of hypoxic stress, hyperosmotic stress of 150% seawater had no effect on the D- and L-aspartate levels in the same tissue. These results suggest that D-aspartate is involved in anaerobic energy metabolism of this bivalve as well as L-aspartate, whose vital role in anoxia-tolerant bivalves is well known.

Probes for the occurrence of endogenous D-aspartic acid (D-Asp) and N-methyl-D-aspartic acid (NMDA) in the neural complex and gonads of a protochordate, the ascidian Ciona intestinalis, have confirmed the presence of these two excitatory amino acids and their involvement in hormonal activity. A hormonal pathway similar to that which occurs in vertebrates has been discovered. In the cerebral ganglion D-Asp is synthesized from L-Asp by an aspartate racemase. Then, D-Asp is transferred through the blood stream into the neural gland where it gives rise to NMDA by means of an NMDA synthase. NMDA, in turn, passes from the neuronal gland into the gonads where it induces the synthesis and release of a gonadotropin-releasing hormone (GnRH). The GnRH in turn modulates the release and synthesis of testosterone and progesterone in the gonads, which are implicated in reproduction. © 2003 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

The activity of d -aspartate racemase purified from Scapharca broughtonii has been found to depend markedly on some nucleotides. Purine nucleoside monophosphates enhanced the enzyme activity, which was, on the contrary, lowered by purine nucleoside triphosphates and not affected by pyrimidine nucleotides. AMP produced the highest increase of seven-fold in the enzyme activity at 6 mM and a half-maximum increase at approximately 3.8 mM. ATP caused a half-maximum decrease in the activity at approximately 1.4 mM and the remaining activity was lower than 7% at saturating ATP concentrations. AMP and ATP both brought about changes in Vmax and not in Km. Analysis of the effect of AMP and ATP suggests that each of them has its own primary binding site, which is different from the substrate-binding site. In view of these effects of the nucleotides, the roles of the racemase and d -aspartate in energy metabolism under anoxic conditions are discussed.

Free D-aspartate (D-Asp) occurs in substantial amounts in glandular tissues. This paper reviews the existing work on D-Asp in vertebrate exocrine and endocrine glands, with emphasis on functional roles. Endogenous D-Asp was detected in salivary glands. High D-Asp levels in the parotid gland during development suggest an involvement of the amino acid in the regulation of early developmental phases and/or differentiation processes. D-Asp has a prominent role in the Harderian gland, where it elicits exocrine secretion through activation of the ERK1/2 pathway. Interestingly, the increase in NOS activity associated with D-Asp administration in the Harderian gland suggests a potential capability of D-Asp to induce vasodilatation. In mammals, an increase in local concentrations of D-Asp facilitates the secretion of anterior pituitary hormones, i.e., PRL, LH and GH, whereas it inhibits the secretion of POMC/α-MSH from the intermediate pituitary and of oxytocin from the posterior pituitary. D-Asp also acts as a negative regulator for melatonin synthesis in the pineal gland. Further, D-Asp can stereo-specifically modulate the production of sex steroids, thus taking part in the endocrine control of reproductive activity. Although D-Asp receptors remain to be characterized, gene expression of NR1 and NR2 subunits of NMDAr responds to D-Asp in the testis. © 2014 Springer-Verlag.

High levels of D-aspartate occur in the brain and endocrine glands, such as pineal, adrenal and pituitary. In the brain, D-aspartate levels are highest in embryonic and early postnatal stages. Notably high levels occur in the early postnatal cortical plate and subventricular zone of the cerebral cortical cultures, implying a role in development. In embryonic neuronal primary culture cells, we detected high levels of endogenous D-aspartate and demonstrated biosynthesis of [14C]D-aspartate using [14C]L-aspartate as precursor. Synthesis of D-aspartate in cell cultures is inhibited by amino-oxyacetic acid, an inhibitor of pyridoxal phosphate-dependent enzymes. In the rat adrenal medulla, D-aspartate is depleted by treatment of the animals with intraperitoneal nicotine injections. In adrenal slices, D-aspartate is released by depolarization with KCl or acetylcholine, implying physiological release by activation of the cholinergic innervation of the adrenal. Our characterization of D-aspartate ontogeny, biosynthesis and depolarization-induced release implies specific physiological roles for this amino acid.

Functional activity of N -methyl- d -aspartate (NMDA) receptors requires both glutamate binding and the binding of an endogenous coagonist that has been presumed to be glycine, although d -serine is a more potent agonist. Localizations of d -serine and it biosynthetic enzyme serine racemase approximate the distribution of NMDA receptors more closely than glycine. We now show that selective degradation of d -serine with d -amino acid oxidase greatly attenuates NMDA receptor-mediated neurotransmission as assessed by using whole-cell patch–clamp recordings or indirectly by using biochemical assays of the sequelae of NMDA receptor-mediated calcium flux. The inhibitory effects of the enzyme are fully reversed by exogenously applied d -serine, which by itself did not potentiate NMDA receptor-mediated synaptic responses. Thus, d -serine is an endogenous modulator of the glycine site of NMDA receptors and fully occupies this site at some functional synapses.

Glutamate racemase (EC 5.1.1.3), an enzyme of microbial origin, shows significant sequence homology with mammalian myoglobins, in particular in the regions corresponding to the E and F helices, which constitute the heme binding pocket of myoglobins. Glutamate racemase binds tightly an equimolar amount of hemin, leading to loss of racemase activity. Although this enzyme shows homology with aspartate racemase, the latter does not bind hemin. The glutamate racemase gene of Pediococcus pentosaceus has a 795-nt open reading frame and encodes 265-amino acid residues, which form a monomeric protein (M(r) 29,000). Neither racemase has cofactors, but they contain essential cysteine residues [Yohda, M., Okada, H. & Kumagai, H. (1991) Biochim. Biophys. Acta 1089, 234-240].

It was believed for long time that d-amino acids are not present in mammals. However, current technological advances and improvements in analytical instruments have enabled studies that now indicate that significant amounts of D-amino acids are present in mammals. The most abundant D-amino acids are D-serine and D-aspartate. D-Serine, which is synthesized by serine racemase and is degraded by D-amino-acid oxidase, is present in the brain and modulates neurotransmission. D-Aspartate, which is synthesized by aspartate racemase and degraded by D-aspartate oxidase, is present in the neuroendocrine and endocrine tissues and testis. It regulates the synthesis and secretion of hormones and spermatogenesis. D-Serine and D-aspartate bind to the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors and function as a coagonist and agonist, respectively. The enzymes that are involved in the synthesis and degradation of these D-amino acids are associated with neural diseases where the NMDA receptors are involved. Knockout mice for serine racemase and D-aspartate oxidase have been generated, and natural mutations in the d-amino-acid oxidase gene are present in mice and rats. These mutant animals display altered behaviors caused by enhanced or decreased NMDA receptor activity. In this article, we review currently available studies on D-amino acid metabolism in mammals and discuss analytical methods used to assay activity of amino acid racemases and D-amino-acid oxidases.

Methods with which to simply and rapidly assay L-aspartate (L-Asp) and D-aspartate (D-Asp) would be highly useful for physiological research and for nutritional and clinical analyses. Levels of L- and D-Asp in food and cell extracts are currently determined using high-performance liquid chromatography. However, this method is time-consuming and expensive. Here we describe a simple and specific method for using an L-aspartate dehydrogenase (L-AspDH) system to colorimetrically assay L-Asp and a system of three hyperthermophilic enzymes--aspartate racemase (AspR), L-AspDH, and L-aspartate oxidase (L-AO)--to assay D-Asp. In the former, the reaction rate of nicotinamide adenine dinucleotide (NAD(+))-dependent L-AspDH was measured based on increases in the absorbance at 438 nm, reflecting formation of formazan from water-soluble tetrazolium-1 (WST-1), using 1-methoxy-5-methylphenazinum methyl sulfate (mPMS) as a redox mediator. In the latter, D-Asp was measured after first removing L-Asp in the sample solution with L-AO. The remaining D-Asp was then changed to L-Asp using racemase, and the newly formed L-Asp was assayed calorimetrically using NAD(+)-dependent aspartate dehydrogenase as described above. This method enables simple and rapid spectrophotometric determination of 1 to 100 μM L- and D-Asp in the assay systems. In addition, methods were applicable to the L- and D-Asp determinations in some living cells and foods.

Free amino acid enantiomers in adult rat brain extracts were analysed as their N,O-pentafluoropropionyl isopropyl derivatives by gas chromatography on a capillary column of Chirasil-L-Val. A peak X, which exhibited the same retention time as the N,O-pentafluoropropionyl isopropyl derivative of authentic D-serine, was detected in the brain extracts. Electron impact and positive chemical ionization mass spectra of the peak X of the brain extracts were identical to those of authentic D-serine. The concentration of free D-serine and the ratio of D-serine/total serine in the brain were estimated to be 0.27 and 0.23, υmol/g of wet weight, respectively. These data provide the first evidence that substantial quantities of free D-serine are present in mammalian brain tissues.

Crystal structure of PH1733, an aspartate racemase homologue, from Pyrococcus horikoshii OT3 Akiko Kita, Shuhei Tasaki, Masafumi Yohda, and Kunio Miki* 1 Research Reactor Institute, Kyoto University, Kumatori-cho, Sannan-gun, Osaka 590-0494, Japan 2Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan 3Department of Biotechnology and Life Science, Faculty of Engineering, Tokyo University of Agriculture and Technology, Tokyo 184-8588, Japan

Previous multilocus sequence typing studies of Campylobacter coli from meat animals identified an unusual cluster of strains, primarily from turkeys, termed "cluster II" and characterized by the presence of the C. jejuni aspA103 allele. To characterize the extent of genomic input from C. jejuni in the aspA region of cluster II C. coli, we sequenced the 6.1 kb genomic region upstream of and including aspA from two turkey-derived cluster II strains (C. coli 6979 and C. coli 7474, of ST-1150 and ST-1161, respectively), as well as from a turkey-derived multidrug-resistant strain (C. coli 6818) representing a major sequence type (ST-1101) outside of cluster II. A gene encoding a putative CRP-family transcriptional regulator (CCO0137) was present in C. coli 6818 and the reference strain C. coli RM2228, whose genome has been sequenced, but not in either cluster II strain evaluated. This gene was also absent from C. jejuni NCTC 11168 and C. jejuni RM1221. Moreover, single nucleotide polymorphism (SNP) analysis revealed that in both cluster II strains, genes encoding subunit II of cytochrome d ubiquinol oxidase (cydB) and a putative aspartate racemase (Cj0085c) harbored numerous C. jejuni-specific SNPs. Interestingly, genes encoding subunit I of cytochrome d ubiquinol oxidase (cydA), uracil-DNA glycosylase (ung), and aspartate ammonia-lyase (aspA) harbored C. coli-specific SNPs in certain portions but C. jejuni-specific SNPs in others, suggesting that these were hybrid genes with C. jejuni-derived segments. Analysis of a ung mutant in C. coli 7474 indicated that the putative hybrid ung of this cluster II strain was functional. Our data suggest the occurrence of recombination events that resulted in genomic import of DNA from C. jejuni in the region between cydA and aspA in cluster II strains of C. coli.

D-Aspartate is one of a few D-amino acids that attracted attention at an early date, since it was detected in various tissues of mammals as a protein component. The occurrence of free D-aspartate in nature was recognized a little later, and raised questions about its physiological functions and metabolism. This amino acid has been gradually accepted, based on various experimental observations, to be a physiological substrate of D-aspartate oxidase, whose role had been considered enigmatic since its early discovery in the 1940s. Mammalian enzymes that serve to liberate D-aspartyl residue in proteins have been identified. One enzyme hydrolyzes peptide bond at the amino side of D-aspartyl residue in a dipeptide and another enzyme hydrolyzes that at the carbonyl side of the residue in proteins. The first pyridoxal 5′-phosphate-dependent aspartate racemase has been purified and cloned from a bivalve species. The enzyme supports the high contents of D-aspartate comparable to those of L-aspartate in the bivalve, and the enantiomers are consumed when hypoxia is imposed on the bivalve. In some yeast species, assimilation of D-aspartate has been found to depend on inducible D-aspartate oxidase, which also serves to detoxify acidic D-amino acids. (c) 2006 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 6: 259–266; 2006: Published online in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/tcr.20089

In the marine mollusk Aplysia limacina, a substantial amount of endogenous D-aspartic acid (D-Asp) was found following its synthesis from L-aspartate by an aspartate racemase. Concentrations of D-Asp between 3.9 and 4.6 μmol/g tissue were found in the cerebral, abdominal, buccal, pleural, and pedal ganglia. In non nervous tissues, D-Asp occurred at a very low concentration compared to the nervous system. Immunohistochemical studies conducted on cultured Aplysia neurons using an anti-D-aspartate antibody demonstrated that D-Asp occurs in the soma, dendrites, and in synaptic varicosities. Synaptosomes and synaptic vesicles from cerebral ganglia were prepared and characterized by electron microscopy. HPLC analysis revealed high concentrations of D-Asp together with L-aspartate and L-glutamate in isolated synaptosomes. In addition, D-Asp was released from synaptosomes by K+ depolarization or by ionomycin. D-Asp was one of the principal amino acids present in synaptic vesicles representing about the 25% of total amino acids present in these cellular organelles. Injection of D-Asp into live animals or addition to the incubation media of cultured neurons, caused an increase in cAMP content. Taken as a whole, these findings suggest a possible role of D-Asp in neurotransmission in the nervous system of Aplysia limacina. © 2005 Wiley-Liss, Inc.

In the green frog, Rana esculenta, a substantial amount Of D-aspartate (D-Asp) is found enclogenously within the Harderian gland (HG) following its synthesis from (L)-aspartate ((L)-Asp) by an aspartate racemase. The frog HG is an orbital seromucoid gland that displays seasonal changes in secretory activity. Our in vivo experiments, consisting of i.p. injection of 2.0 mu mol/g.b.w. (D)-Asp in frogs collected during two periods of differing glandular activity (high or medium-low secretory activity), revealed that HG can to take up and accumulate D-Asp and that this amino acid may modulate the exocrine secretion through a kinase pathway. Atatirnewhenthe gland shows relatively low secretory activity, i.p. administration of (D)-Asp rapidly induced activation of ERK1 and an increase in cells active in RNA synthesis. This increase in transcriptional activity was followed by a significant increase in Mucous secretion. By contrast, administration of exogenous (D)-Asp when HG was showing high activity rapidly induced inhibition of both ERK1 and transcriptional activity. Since D-Asp is known to be recognized by receptors for N-methyl-(D)-aspartic acid (NMDA), it is possible that in the HG, D-Asp mediated NMDA activation may enhance the kinase pathway. The above activation of opposing stimulatory and inhibitory processes could reflect different levels of NMDA-receptor activity, which could vary as a function of the level of gland activity. This study provides the first evidence of a role for this excitatory amino acid in exocrine secretion. The effects of (D)-Asp in HG appear to be specific since they were not seen in frogs treated with other (D)- or (L)-amino acids with known excitatory effects on neurosecretion. (c) 2005 Wiley-Liss, Inc.

This study investigated the involvement of D-aspartic acid (D-Asp) in testicular steroidogenesis of the green frog Rana esculenta and its effect on stimulation of thumb pad morphology and glandular activity, a typical testosterone-dependent secondary sexual characteristic in this amphibian species. In the testis, D-Asp concentrations vary significantly during the reproductive cycle: they are low in pre- and post-reproductive periods, but reach peak levels in the reproductive period (140-236 nmol/g wet tissue). Moreover, the concentrations of D-Asp in the testis through the sexual cycle positively match the testosterone levels in the gonad and the plasma. The racemase activity evaluated during the cycle expresses its peak when D-Asp and testosterone levels are highest, that is, during the reproductive period, confirming the synthesis of D-Asp from L-Asp by an aspartate racemase. Short-term in vivo experiments consisting of a single injection of D-Asp (2·0 μmol/g body weight) demonstrated that this amino acid accumulates significantly in the testis, and after 3 h its uptake is coupled with a testosterone increase in both testis and plasma. Moreover, within 18 h of amino acid administration, the D-Asp concentration in the testis decreased along with the testosterone titer to prestimulation levels. Other amino acids (L-Asp, D-Glu and L-Glu) used instead of D-Asp were ineffective, confirming that the significant increase in testicular testosterone was a specific feature of this amino acid. In long-term experiments, D-Asp had been administered chronically to frogs caught during the three phases of the reproductive cycle, inducing testosterone increase and 17β-estradiol decrease in the gonad during the pre- and post-reproductive period, and vice versa during the reproductive period. The stimulatory effect of D-Asp on testosterone production by the testis is consistent with the stimulation of spermatogenesis and the maturation of thumb pads occurring in D-Asp-treated frogs. In these last animals, there was an increase of seminiferous ampoule area and a higher number of spermatids and sperm. Moreover, in spermatogonia I and II and in spermatocytes, a proliferating cell nuclear antigen (PCNA) intense immunopositivity was observed. In addition, the thumb pads of D-Asp-treated frogs compared with controls showed a significantly thicker epithelial lining, a wider area of their glands with taller secretion cells, and more numerous, PAS-positive-rich secretions. Finally, these results provide functional evidence for a biologic role of D-Asp in amphibian male steroidogenesis; therefore, this unusual amino acid could be considered a modulatory agent for reproductive processes. © 2004 Society for Endocrinology.

High concentrations of d -aspartate occur in blood shell Scapharca broughtonii (Mollusca) tissues. We purified aspartate racemase from the foot muscle of the bivalve to electrophoretic homogeneity. The molecular mass shown by sodium dodecyl sulfate polyacrylamide gel was 39 kDa, while that shown by gel filtration ranged from 51 to 63 kDa. Pyridoxal 5′-phosphate-dependency of the enzyme was demonstrated by its absorption spectrum as well as the effects of amino-oxyacetate and other reagents on the activity and spectrum. The enzyme is highly specific to aspartate and does not racemize l -alanine, l -serine and l -glutamate. It showed the highest activity at pH 8 both in the conversion of l - to d - and d - to l -aspartate, and the optimal temperature was 25 °C. V max and K m values for l -aspartate were 7.39 μmol min −1 mg −1 and 60.4 mM and those for d -aspartate were 22.6 μmol min −1 mg −1 and 159 mM, respectively.

In Alice in Through the Looking-Glass, and What Alice Found There, Alice proposes to her kitten a looking-glass house, whereupon Lewis Carroll fantasizes a backwards world and raises the question of life’s chirality. Homochirality has been thought to be essential to biology— generating distinct enzymes/receptors to cope with multiple isomers which would pose energetic burdens incompatible with life. Hence, we have D-sugars and L-amino acids. D-Amino acids found in mammalian tissues were assumed to be the by-products of dietary consumption, since plants and bacteria do synthesize D-amino acids. A nagging brain-teaser, for over 40 years, has been the existence of mammalian D-amino acid oxidase and D-aspartate oxidase with particularly high levels in the brain. Like Alice, neurobiologists have now stepped through the looking-glass to characterize at least two D-amino acids, D-serine and D-aspartic acid, that may serve as the important signalling molecules in the nervous and endocrine systems. D-Aspartate was first found in the nervous system of marine mollusks and subsequently in nervous and endocrine tissues of many mammals, including humans. In 1992, Nishikawa and his colleagues developed a high performance liquid chromatography (HPLC) system to separate isomers in biological samples. They confirmed the presence of free D-aspartic acid in the brain of rodent, where they also identified free D-serine. Research by the authors and other groups identified the two enzymes, serine racemase and aspartate racemase, that synthesize D-serine and D-aspartate, respectively. Emerging unanticipated roles for these two amino acids challenge fundamental dogma about cell-to-cell signaling. Thus, D-serine fulfils the criteria of a major, if not the sole, ligand for the strychnineinsensitive glycine modulatory binding site of the N-methyl-D-aspartate receptor (NMDA) subtype of glutamate receptor, which mediates excitatory neurotransmission and cognitive function. D-Aspartate mediates diverse neuroendocrine functions. We are just beginning to understand the importance of this novel class of brain messengers that will soon include additional candidates. Indeed, recent studies suggest that other D-amino acids, such as D-glutamate, N-methyl-D-aspartate, D-alanine and D-glutamine, may also serve as messenger molecules. The expanded recognition of D-amino acids as signalling molecules, along with appreciation of gases as neuromodulators, further alters thinking about what should be a neurotransmitter. This special volume of Amino Acids provides an overview of the current status of our understanding on D-amino acid biology, incorporating contributions from leaders in the field. We have assembled eight review articles that convey recent achievements characterizing the metabolism, brain disposition and roles in synaptic plasticity of D-amino acids, including the recent development of biosensors. The issue also incorporates two orignal studies by Prof Wang and his coworkers who report the contribution of spinal D-amino acid oxidase to bone cancer pain, and by Prof. Hamase and colleagues who demonstrate the alteration in D-serine levels in mice lacking serine racemase and D-amino acid oxidase. J.-P. Mothet (&) Aix Marseille University, CRN2M UMR7286 CNRS, 51 Bd Pierre Dramard, 13344 Marseille Cedex 15, France e-mail: jean-pierre.mothet@univ-amu.fr

Although d amino acids are prominent in bacteria, they generally are thought not to occur in mammals. Recently, high levels of d -serine have been found in mammalian brain where it activates glutamate/ N -methyl- d -aspartate receptors by interacting with the “glycine site” of the receptor. Because amino acid racemases are thought to be restricted to bacteria and insects, the origin of d -serine in mammals has been puzzling. We now report cloning and expression of serine racemase, an enzyme catalyzing the formation of d -serine from l -serine. Serine racemase is a protein representing an additional family of pyridoxal-5′ phosphate-dependent enzymes in eukaryotes. The enzyme is enriched in rat brain where it occurs in glial cells that possess high levels of d -serine in vivo . Occurrence of serine racemase in the brain demonstrates the conservation of d -amino acid metabolism in mammals with implications for the regulation of N -methyl- d -aspartate neurotransmission through glia-neuronal interactions.

High levels of d -serine occur in mammalian brain, where it appears to be an endogenous ligand of the glycine site of N -methyl- d -aspartate receptors. In glial cultures of rat cerebral cortex, d -serine is enriched in type II astrocytes and is released upon stimulation with agonists of non- N -methyl- d -aspartate glutamate receptors. The high levels of d -serine in discrete areas of rat brain imply the existence of a biosynthetic pathway. We have purified from rat brain a soluble enzyme that catalyzes the direct racemization of l -serine to d -serine. Purified serine racemase has a molecular mass of 37 kDa and requires pyridoxal 5′-phosphate for its activity. The enzyme is highly selective toward l -serine, failing to racemize any other amino acid tested. Properties such as pH optimum, K m values, and the requirement for pyridoxal phosphate resemble those of bacterial racemases, suggesting that the biosynthetic pathway for d -amino acids is conserved from bacteria to mammalian brain.

Substantial concentrations of D-aspartate were found in several tissues of Scapharca broughtonii together with approximately equal concentrations of L-aspartate. The foot and mantle extracts also contained an aspartate racemase activity. The formation of L-aspartate from the D-enantiomer by the foot extract was apparently slower than the reverse reaction, and this unbalance seemed to be due to the presence of an enzyme activity which rapidly converted L-aspartate to L-alanine. The possible role of D-aspartate in the anaerobiosis was discussed.

The concurrent determination of free amino acid enantiomers and non-chiral amino acids in rat brain and serum was accomplished by high-performance liquid chromatography with fluorimetric detection after derivatization with N-tert.-butyloxycarbonyl-L-cysteine and o-phthaldialdehyde. The method revealed the presence of a large amount of free D-serine (0.22 mumol/g of tissue; D/D + L ratio = 0.25) in the brain whereas D-aspartate and D-alanine were established to be at trace levels. These results further support the presence of D-serine in adult brain tissues as demonstrated by recent work using gas chromatography.

Free D-aspartic acid was measured in fertilized chicken eggs, chicken embryos, and neonatal rats. In each tissue examined a maximum value was found at a characteristic time of development. For the chicken embryo brain, the maximum was 9% D at 11 days of incubation; for the retina, 20% D at 13 days of incubation. In the neonatal rat, as in the chicken embryo, D-aspartic acid continued to increase in the retina after that in the brain and other tissues had begun to decline. The maximum, 29% D, was found 7 days after birth. Thus in two phylogenetically distant species, similar developmental patterns of D-aspartic acid change were observed. Some data on similarities between the D/L aspartic acid ratios of adult chicken and rat tissues are also reported. In addition, the total D-aspartic acid content of the egg, including the embryo, increased from 44 nmol at day 1 to 159 nmol at day 12, showing that release from a bound form or de novo synthesis is a continuing process during development.

Free D-aspartic acid is present in appreciable quantities in the brain and other tissues of rodents and in human blood. In the newborn rat, the highest concentration of D-aspartic acid was found in cerebral hemispheres, where, at 164 nmol/g (8.4% of the total aspartic acid), the level of D-aspartic acid exceeds that of many essential L-amino acids. The highest ratio of D- to total aspartic acid (38%) occurred in neonatal blood cells. In the adult rat, the highest concentration was present in the pituitary gland (127 nmol/g, 3.8%). Within the central nervous system marked regional differences are present and characteristic changes with development take place. In general, the levels of D-aspartic acid fall rapidly with increasing age. In cerebral hemispheres adult values (13 nmol/g, 0.43%) are approached within one week. D-aspartic acid concentrations may also be higher in young humans since fetal blood, taken from placental cord, contains 2.6 nmol/g (4.9%) of D-aspartic acid, a value five times that of adult human blood. These distributional patterns and developmental changes may be the result of differences in the ability of various tissues to dispose of an extraneous metabolite, or, reflect alterations in a specific functional requirement for D-aspartic acid.