d-Amino acids in the brain and mutant rodents lacking d-amino-acid oxidase activity

d-Amino acids are stereoisomers of l-amino acids. They are often called unnatural amino acids, but several d-amino acids have been found in mammalian brains. Among them, d-serine is abundant in the forebrain and functions as a co-agonist of NMDA receptors to enhance neurotransmission. d-Amino-acid oxidase (DAO), which degrades neutral and basic d-amino acids, is mainly present in the hindbrain. DAO catabolizes d-serine and, therefore, modulates neurotransmission. In the brains of mutant mice and rats lacking DAO activity, the amounts of d-serine and other d-amino acids are markedly increased. Mutant mice manifested behavioral changes characteristic of altered NMDA receptor activity, likely due to increased levels of d-serine. d-Serine and DAO have been demonstrated to play important roles in cerebellar development and synaptic plasticity. They have also implicated in amyotrophic lateral sclerosis and pain response. There have also been several lines of evidence correlating DAO with schizophrenia. Taken together, the experiments indicate that d-amino acids and DAO have pivotal functions in the central nervous system.     

d-Amino Acid-Induced Expression of d-Amino Acid Oxidase in the Yeast Schizosaccharomyces pombe

We investigated d-amino acid oxidase (DAO) induction in the popular model yeast Schizosaccharomyces pombe. The product of the putative DAO gene of the yeast expressed in E.?coli displayed oxidase activity to neutral and basic d-amino acids, but not to an l-amino acid or acidic d-amino acids, showing that the putative DAO gene encodes catalytically active DAO. DAO activity was weakly detected in yeast cells grown on a culture medium without d-amino acid, and was approximately doubled by adding d-alanine. The elimination of ammonium chloride from culture medium induced activity by up to eight-fold. l-Alanine also induced the activity, but only by about half of that induced by d-alanine. The induction by d-alanine reached a maximum level at 2?h cultivation; it remained roughly constant until cell growth reached a stationary phase. The best inducer was d-alanine, followed by d-proline and then d-serine. Not effective were N-carbamoyl-d,l-alanine (a better inducer of DAO than d-alanine in the yeast Trigonopsis variabilis), and both basic and acidic d-amino acids. These results showed that S. pombe DAO could be a suitable model for analyzing the regulation of DAO expression in eukaryotic organisms.     

Alteration of intrinsic amounts of d-serine in the mice lacking serine racemase and d-amino acid oxidase

For elucidation of the regulation mechanisms of intrinsic amounts of d-serine (d-Ser) which modulates the neuro-transmission of N-methyl-d-aspartate receptors in the brain, mutant animals lacking serine racemase (SRR) and d-amino acid oxidase (DAO) were established, and the amounts of d-Ser in the tissues and physiological fluids were determined. d-Ser amounts in the frontal brain areas were drastically decreased followed by reduced SRR activity. On the other hand, a moderate but significant decrease in d-Ser amounts was observed in the cerebellum and spinal cord of SRR knock-out (SRR^?/?) mice compared with those of control mice, although the amounts of d-Ser in these tissues were low. The amounts of d-Ser in the brain and serum were not altered with aging. To clarify the uptake of exogenous d-Ser into the brain tissues, we have determined the d-Ser of SRR^?/? mice after oral administration of d-Ser for the first time, and a drastic increase in d-Ser amounts in all the tested tissues was observed. Because both DAO and SRR are present in some brain areas, we have established the double mutant mice lacking SRR and DAO for the first time, and the contribution of both enzymes to the intrinsic d-Ser amounts was investigated. In the frontal brain, most of the intrinsic d-Ser was biosynthesized by SRR. On the other hand, half of the d-Ser present in the hindbrain was derived from the biosynthesis by SRR. These results indicate that the regulation of intrinsic d-Ser amounts is different depending on the tissues and provide useful information for the development of treatments for neuronal diseases.     

Serine racemase: an unconventional enzyme for an unconventional transmitter

The discovery of large amounts of d-serine in the brain challenged the dogma that only l-amino acids are relevant for eukaryotes. The levels of d-serine in the brain are higher than many l-amino acids and account for as much as one-third of l-serine levels. Several studies in the last decades have demonstrated a role of d-serine as an endogenous agonist of N-methyl-d-aspartate receptors (NMDARs). d-Serine is required for NMDAR activity during normal neurotransmission as well as NMDAR overactivation that takes place in neurodegenerative conditions. Still, there are many unanswered questions about d-serine neurobiology, including regulation of its synthesis, release and metabolism. Here, we review the mechanisms of d-serine synthesis by serine racemase and discuss the lessons we can learn from serine racemase knockout mice, focusing on the roles attributed to d-serine and its cellular origin.     

Structure–function relationships in human d-amino acid oxidase

Since d-amino acids were identified in mammals, d-serine has been one of the most extensively studied “unnatural amino acids”. This brain-enriched transmitter-like molecule plays a pivotal role in the human central nervous system by modulating the activity of NMDA receptors. Physiological levels of d-serine are required for normal brain development and function; thus, any alterations in neuromodulator concentrations might result in NMDA receptor dysfunction, which is known to be involved in several pathological conditions, including neurodegeneration(s), epilepsy, schizophrenia, and bipolar disorder. In the brain, the concentration of d-serine stored in cells is defined by the activity of two enzymes: serine racemase (responsible for both the synthesis and degradation) and d-amino acid oxidase (which catalyzes d-serine degradation). Both enzymes emerged recently as new potential therapeutic targets for NMDA receptor-related diseases. In this review we have focused on human d-amino acid oxidase and provide an extensive overview of the biochemical and structural properties of this flavoprotein and their functional significance. Furthermore, we discuss the mechanisms involved in modulating enzyme activity and stability with the aim to substantiate the pivotal role of d-amino acid oxidase in brain d-serine metabolism in physiological and pathological conditions and to highlight its great significance for novel drug design/development.     

Amperometric microbiosensor as an alternative tool for investigation of d-serine in brain

This paper discusses the application of a reagentless, selective microbiosensor as a useful alternative tool for monitoring d-serine in neural samples. The main components of the 125-μm-diameter disk biosensor were d-amino acid oxidase for d-serine sensitivity (linear region slope, 61?±?7?μA?cm^–2?mM^–1; limit of detection, 20?nM), and poly-phenylenediamine for rejection of electroactive interference. The response time of the biosensor was of the order of 1?s, ideal for ‘real-time’ monitoring, and detection of systemically administered d-serine in brain extracellular fluid is demonstrated. Exploitation of this probe might resolve queries involving regulation of d-serine in excitotoxicity, and modulation of N-methyl-d-aspartate receptor function by d-serine and glycine in the central nervous system.     

New insights on the role of free d-aspartate in the mammalian brain

Free d-aspartate (d-Asp) occurs in substantial amounts in the brain at the embryonic phase and in the first few postnatal days, and strongly decreases in adulthood. Temporal reduction of d-Asp levels depends on the postnatal onset of d-aspartate oxidase (DDO) activity, the only enzyme able to selectively degrade this d-amino acid. Several results indicate that d-Asp binds and activates N-methyl-d-aspartate receptors (NMDARs). Accordingly, recent studies have demonstrated that deregulated, higher levels of d-Asp, in knockout mice for Ddo gene and in d-Asp-treated mice, modulate hippocampal NMDAR-dependent long-term potentiation (LTP) and spatial memory. Moreover, similarly to d-serine, administration of d-Asp to old mice is able to rescue the physiological age-related decay of hippocampal LTP. In agreement with a neuromodulatory action of d-Asp on NMDARs, increased levels of this d-amino acid completely suppress long-term depression at corticostriatal synapses and attenuate the prepulse inhibition deficits produced in mice by the psychotomimetic drugs, amphetamine and MK-801. Based on the evidence which points to the ability of d-Asp to act as an endogenous agonist on NMDARs and considering the abundance of d-Asp during prenatal and early life, future studies will be crucial to address the effect of this molecule in the developmental processes of the brain controlled by the activation of NMDARs.     

D-Amino acid oxidase of Streptomyces coelicolor and the effect of D-amino acids on the bacterium

The homologous gene of D-amino acid oxidase (DAO) in prokaryotic organisms is predominantly found in a group of bacteria called the Actinobacteria. We have analyzed the DAO of the model actinomycete Streptomyces coelicolor and the effect of D-amino acids on this bacterium. When expressed in Escherichia coli, the translated product of the putative dao gene of this bacterium exhibited oxidase activity against neutral and basic D-amino acids, with a higher activity toward D-valine and D-isoleucine, but not to their corresponding L-amino acids. This substrate specificity was largely different from that of the DAO of the actinobacterium Arthrobacter protophormiae. The gene message and DAO activity were constitutively detected in S. coelicolor cells, and unlike eukaryotic DAOs, the presence of a D-amino acid did not significantly induce expression. The D-amino acids that were a good substrate for S. coelicolor DAO inhibited cell growth, delayed morphological development and affected cell morphology, but they did not inhibit biofilm formation. Disruption of the dao gene had no effect on the morphology and morphological development of S. coelicolor cells, the assimilation of D-valine or the sensitivity to growth inhibition by D-valine under the experimental conditions, showing that in this bacterium DAO does not play a significant role in either morphological development or the assimilation and detoxification of D-amino acids.     

d-Serine and Serine Racemase are Localized to Neurons in the Adult Mouse and Human Forebrain

d-Serine, a co-agonist at the NMDA receptor (NMDAR), is synthesized from l-serine by the enzyme serine racemase (SR), which is heavily expressed in the forebrain. Although SR was originally reported to be localized exclusively to astrocytes, recent conditional knock out results demonstrate that little SR is expressed in forebrain astrocytes. As a consequence, the cellular location of its product, d-serine, in the brain is also uncertain. Immunocytochemistry now indicates that SR is expressed primarily in forebrain glutamatergic neurons with the remainder in GABAergic interneurons. We utilized SR deficient (SR?/?) mice, which have <15 % of normal d-serine levels, to validate and optimize a d-serine immunohistochemical method. Nearly all of the d-serine in neocortex and hippocampus (HP) is found in neurons, with virtually no d-serine co-localizing with two astrocyte markers. Interestingly, only a subset of the d-serine positive neurons contained SR in the neocortex and HP. Greater than half of the d-serine positive neurons were GABAergic interneurons, with a majority of these neurons containing parvalbumin and/or somatostatin. Only ~25–40 % of interneurons expressed SR in the neocortex and HP. Finally, we demonstrate in human post-mortem neocortex that SR is found in both excitatory and inhibitory neurons, but not in S100β-containing astrocytes. In sum, these findings conclusively demonstrate that the majority of d-serine is both synthesized and stored in neurons. It will be important to determine the functional significance for the separation of synthesis and storage of d-serine in neurons, as well as the presence of this NMDAR co-agonist in GABAergic interneurons.     

Detection of d-amino acids in purified proteins synthesized in Escherichia coli

It has long been believed that amino acids comprising proteins of all living organisms are only of the l-configuration, except for Gly. However, peptidyl d-amino acids were observed in hydrolysates of soluble high molecular weight fractions extracted from cells or tissues of various organisms. This strongly suggests that significant amounts of d-amino acids are naturally present in usual proteins. Thus we analyzed the d-amino acid contents of His-tag-purified β-galactosidase and human urocortin, which were synthesized by Escherichia coli grown in controlled synthetic media. After acidic hydrolysis for various times at 110°C, samples were derivatized with 4-fluoro-7-nitro-2, 1, 3-benzoxadiazole (NBD-F) and separated on a reverse-phase column followed by a chiral column into d- and l-enantiomers. The contents of d-enantiomers of Ala, Leu, Phe, Val, Asp, and Glu were determined by plotting index d/(d + l) against the incubation time for hydrolysis and extrapolating the linear regression line to 0 h to eliminate the effect of racemization of amino acids during the incubation. Significant contents of d-amino acids were reproducibly detected, the d-amino acid profile being specific to an individual protein. This finding indicated the likelihood that d-amino acids are in fact present in the purified proteins. On the other hand, the d-amino acid contents of proteins were hardly influenced by the addition of d- or l-amino acids to the cultivation medium, whereas intracellular free d-amino acids sensitively varied according to the extracellular conditions. The origin of these d-amino acids detected in proteins was discussed.     

Evaluation of the E. coli d-serine ammonia lyase gene (Ec. dsdA) for use as a selectable marker in maize transformation

In this study, the d-serine ammonia lyase (dsdA) gene from Escherichia coli was evaluated as a selectable marker for maize transformation. Plants are incapable of utilizing the D-form of most amino acids, and d-serine has recently been demonstrated to be phytoinhibitory to plant growth. d-Serine ammonia lyase detoxifies d-serine via a substrate-specific reaction to pyruvate, ammonia, and water. d-Serine inhibits germination of isolated maize immature embryos and growth of embryogenic callus from wild-type plants at concentrations about approx. 2?C15 mM. Transgenic plants were recovered in the presence of d-serine in tissue culture media with dsdA as the selection marker at efficiencies comparable to using a mutated acetohydroxy acid synthase selection marker gene and selection in the presence of imidazolinone herbicides. Immature embryos infected with an Agrobacterium strain containing an acetohydroxy acid synthase gene construct without dsdA did not yield any transgenic events on the selection medium with 10 mM d-serine, indicating that d-serine provided selection tight enough to prevent escapes. Molecular analysis confirmed the integration of the dsdA gene into the genome of the transgenic plants. No adverse phenotypes were observed in the greenhouse, and expression of the dsdA marker had no affect on agronomic characteristics or grain yield in multi-location field trials. Seed compositional analysis demonstrated no significant differences in the contents of seed protein, starch, fatty acids, fiber, phytic acid, and free amino acids between transgenic and non-transgenic control plants. These data indicate that the dsdA gene is properly expressed in maize and the d-serine ammonia lyase (DSDA) enzyme functions appropriately to metabolize d-serine during in vitro selection. Preliminary safety assessments indicated that no adverse affects would be expected if humans were exposed to the DSDA protein in the diet from an allergenicity or toxicity perspective. The dsdA gene in combination with phytoinhibitory levels of d-serine represents a new and effective selectable marker system for maize transformation.     

Intrathecal Delivery of ssAAV9-DAO Extends Survival in SOD1<Superscript>G93A</Superscript> ALS Mice

Amyotrophic lateral sclerosis (ALS) is an adult-onset, irreversible neurodegenerative disease that leads to progressive paralysis and inevitable death 3–5 years after diagnosis. The mechanisms underlying this process remain unknown, but new evidence indicates that accumulating levels of d-serine result from the downregulation of d-amino acid oxidase (DAO) and that this is a novel mechanism that leads to motoneuronal death in ALS via N-methyl-d-aspartate receptor-mediated cell toxicity. Here, we explored a new therapeutic approach to ALS by overexpressing DAO in the lumbar region of the mouse spinal cord using a single stranded adeno-associated virus serotype 9 (ssAAV9) vector. A single intrathecal injection of ssAAV9-DAO was made in SOD1^G93A mice, a well-established mouse model of ALS. Treatment resulted in moderate expression of exogenous DAO in motorneurons in the lumbar spinal cord, reduced immunoreactivity of d-serine, alleviated motoneuronal loss and glial activation, and extended survival. The potential mechanisms underlying these effects were associated with the down-regulation of NF-κB and the restoration of the phosphorylation of Akt. In conclusion, administering ssAAV9-DAO may be an effective complementary approach to gene therapy to extend lifespans in symptomatic ALS.     

Enantiomer-specific selection of amino acids

Dietary intake of l-amino acids impacts on several physiological functions, including the control of gastrointestinal motility, pancreatic secretion, and appetite. However, the biological mechanisms regulating behavioral predilections for certain amino acid types remain poorly understood. We tested the hypothesis that, in mice, the potency with which a given glucogenic amino acid increases glucose utilization reflects its rewarding properties. We have found that: (1) during long-, but not short-, term preference tests, l-alanine and l-serine were preferred over their d-enantiomer counterparts, while no such effect was observed for l-threonine vs. d-threonine; (2) these behavioral patterns were closely associated with the ability of l-amino acids to promote increases in respiratory exchange ratios such that those, and only those, l-amino acids able to promote increases in respiratory exchange ratios were preferred over their d-isomers; (3) these behavioral preferences were independent of gustatory influences, since taste-deficient Trpm5 knockout mice displayed ingestive responses very similar to those of their wild-type counterparts. We conclude that the ability to promote increases in respiratory exchange ratios enhances the reward value of nutritionally relevant amino acids and suggest a mechanistic link between substrate utilization and amino acid preferences.     

Synthesis of 3-O-β-d-xylopyranosyl-l-serine (xylosylserine) andO-β-d-galactopyranosyl-(1-4)-O-β-d-xylopyranosyl-l-serine (galactosylxylosylserine) and use of the synthetic products for detection of galactosyltransferase I activity in rat liver

3-O-β-d-Xylopyranosyl-l-serine (xylosylserine) was synthesized by the following three-step procedure: 1) 2,3,4-tri-O-benzoyl-α-d-xylopyranosyl bromide (benzobromoxylose) was condensed withN-carbobenzoxy-l-serine benzyl ester using the silver triflate-collidine complex as promoter; 2) theN-carbobenzoxy and benzyl ester groups in the resultant glycoside were cleaved by transfer hydrogenation with palladium black as catalyst and ammonium formate as hydrogen donor; and 3) the benzoyl groups were removed with methanolic ammonia. Xylosylserine was obtained in an overall yield of 70%. O-β-d-Galactopyranosyl-(1-4)-O-β-d-xylopyranosyl-(1-3)-l-serine (galactosylxylosylserine) was also synthesized by this methodology and was characterized by 2-dimensional (2D) NMR spectroscopy techniques. The two serine glycosides (xylosylserine and galactosylxylosylserine) were used in detection and partial purification of galactosyltransferase I (UDP-d-galactose:d-xylose galactosyltransferase) from adult rat liver.     

Catalytic mechanism of serine racemase from Dictyostelium discoideum

The eukaryotic serine racemase from Dictyostelium discoideum is a fold-type II pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes racemization and dehydration of both isomers of serine. In the present study, the catalytic mechanism and role of the active site residues of the enzyme were examined by site-directed mutagenesis. Mutation of the PLP-binding lysine (K56) to alanine abolished both serine racemase and dehydrase activities. Incubation of d- and l-serine with the resultant mutant enzyme, K56A, resulted in the accumulation of PLP-serine external aldimine, while less amounts of pyruvate, α-aminoacrylate, antipodal serine and quinonoid intermediate were formed. An alanine mutation of Ser81 (S81) located on the opposite side of K56 against the PLP plane converted the enzyme from serine racemase to l-serine dehydrase; S81A showed no racemase activity and had significantly reduced d-serine dehydrase activity, but it completely retained its l-serine dehydrase activity. Water molecule(s) at the active site of the S81A mutant enzyme probably drove d-serine dehydration by abstracting the α-hydrogen in d-serine. Our data suggest that the abstraction and addition of α-hydrogen to l- and d-serine are conducted by K56 and S81 at the si- and re-sides, respectively, of PLP.     

d-Amino acids in brain neurotransmission and synaptic plasticity

Far from our initial view of d-amino acids as being limited to invertebrates, they are now considered active molecules at synapses of mammalian central and peripheral nervous systems, capable of modulating synaptic communication within neuronal networks. In particular, experimental data accumulated in the last few decades show that through the regulation of glutamatergic neurotransmission, d-serine influences the functional plasticity of cerebral circuitry throughout life. In addition, the modulation of NMDA-R-dependent signalling by d-aspartate has been demonstrated by pharmacological studies and after the targeted deletion of the d-aspartate-degrading enzyme. Considering the major contribution of the glutamatergic system to a wide range of neurological disorders such as schizophrenia, Alzheimer’s disease and amyotrophic lateral sclerosis, an improved understanding of the mechanisms of d-amino-acid-dependent neuromodulation will certainly offer new insights for the development of relevant strategies to treat these neurological diseases.     

Increasing effects of S-methyl-l-cysteine on the extracellular d-serine concentrations in the rat medial frontal cortex

In an in vivo dialysis experiment, the intra-medial frontal cortex infusion of a system A and Asc-1 transporter inhibitor, S-methyl-l-cysteine, caused a concentration-dependent increase in the dialysate contents of an endogenous coagonist for the N-methyl-d-aspartate (NMDA) type glutamate receptor, d-serine, in the cortical portion. These results suggest that these neutral amino acid transporters could control the extracellular d-serine signaling in the brain and be a target for the development of a novel threapy for neuropsychiatric disorders with an NMDA receptor dysfunction.     

Active site model of (R)-selective ω-transaminase and its application to the production of d-amino acids

ω-Transaminase (ω-TA) is one of the important biocatalytic toolkits owing to its unique enzyme property which enables the transfer of an amino group between primary amines and carbonyl compounds. In addition to preparation of chiral amines, ω-TA reactions have been exploited for the asymmetric synthesis of l-amino acids using (S)-selective ω-TAs. However, despite the availability of (R)-selective ω-TAs, catalytic utility of the ω-TAs has not been explored for the production of d-amino acids. Here, we investigated the substrate specificity of (R)-selective ω-TAs from Aspergillus terreus and Aspergillus fumigatus and demonstrated the asymmetric synthesis of d-amino acids from α-keto acids. Substrate specificity toward d-amino acids and α-keto acids revealed that the two (R)-selective ω-TAs possess strict steric constraints in the small binding pocket that precludes the entry of a substituent larger than an ethyl group, which is reminiscent of (S)-selective ω-TAs. Molecular models of the active site bound to an external aldimine were constructed and used to explain the observed substrate specificity and stereoselectivity. α-Methylbenzylamine (α-MBA) showed the highest amino donor reactivity among five primary amines (benzylamine, α-MBA, α-ethylbenzylamine, 1-aminoindan, and isopropylamine), leading us to employ α-MBA as an amino donor for the amination of 5 reactive α-keto acids (pyruvate, 2-oxobutyrate, fluoropyruvate, hydroxypyruvate, and 2-oxopentanoate) among 17 ones tested. Unlike the previously characterized (S)-selective ω-TAs, the enzyme activity of the (R)-selective ω-TAs was not inhibited by acetophenone (i.e., a deamination product of α-MBA). Using racemic α-MBA as an amino donor, five d-amino acids (d-alanine, d-homoalanine, d-fluoroalanine, d-serine, and d-norvaline) were synthesized with excellent product enantiopurity (enantiomeric excess >99.7 %).     

Cyclic tetrapeptides with –SS– bridging between amino acid side chains for potent histone deacetylases’ inhibition

Cyclic depsipeptide FK228 with an intramolecular disulfide bond is a potent inhibitor of histone deacetylases (HDAC). FK228 is stable in blood because of its prodrug function, whose –SS– bond is reduced within the cell. Here, cyclic peptides with –SS– bridges between a variety of amino acids were synthesized and assayed for HDAC inhibition. Cyclic peptide 3, cyclo(-l-amino acid-l-amino acid-l-Val-d-Pro-), with an –SS– bridge between the first and second amino acids, was found to be a potent HDAC inhibitor. Cyclic peptide 7, cyclo(-l-amino acid-d-amino acid-l-Val-d-Pro-), with an –SS– bridge between the first and second amino acids, was also a potent HDAC inhibitor.     

Effects of d-Amino Acids on the EPS Production and Cell Aggregation of Alteromonas macleodii Strain JL2069

Exopolysaccharide (EPS) is produced by many marine bacteria and is important for cell aggregation in the ocean. d-amino acids are important components in bacteria and are recently recognized as signal molecules for regulation of bacterial growth. In this study, the effects of d-amino acids on EPS production, cell aggregation, and metabolic activity were investigated using an EPS-producing bacterium Alteromonas macleodii strain JL2069. EPS produced by JL2069 was inhibited by 1 mM of d-Ala and d-Ser, but not by d-Glu. The formation of particulate organic matter (POM) was promoted by the three amino acids. A new technique of microcalorimetry analysis indicated that the metabolic activity of the JL2069 cells was inhibited by these d-amino acids. Our results suggested that d-amino acids may reduce the bacterial metabolism by changing bacterial lifestyle from planktonic to cell aggregation growth which occurs independent of the production of EPS.