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.     

Current knowledge of d-aspartate in glandular tissues

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.     

Central administration of l- and d-aspartate attenuates stress behaviors by social isolation and CRF in neonatal chicks

Intracerebroventricular (i.c.v.) administration of l-aspartate (l-Asp) attenuates stress responses in neonatal chicks, but the mechanism has not been clarified. In the present study, three behavioral experiments were carried out under socially isolated stressful conditions exacerbated by the use of corticotrophin-releasing factor (CRF). In Experiment 1, i.c.v. injection of l-Asp attenuated behavioral stress responses (distress vocalization and active wakefulness) in a dose-dependent manner. Furthermore, l-Asp increased time spent standing/sitting motionless with eyes open and sitting motionless with head dropped (sleeping posture) in comparison with the group receiving CRF alone. In Experiment 2, i.c.v. injection of d-Asp dose-dependently decreased the number of distress vocalizations and the amount of time spent in active wakefulness. d-Asp increased the time spent standing/sitting motionless with eyes open compared with the group receiving CRF alone. In Experiment 3, we directly compared the effect of l-Asp with that of d-Asp. Both l- and d-Asp induced sedative effects under an acutely stressful condition. However, l-Asp, but not d-Asp, increased the time spent in a sleeping posture. These results indicate that both l- and d-Asp, when present in the brain, could induce a sedative effect, while the mechanism for hypnosis in neonatal chicks may be different for l-Asp in comparison with d-Asp.     

Principal component analysis of the relationship between the d-amino acid concentrations and the taste of the sake

We performed sensory evaluations on 141 bottles of sake and analyzed the relationship between the d-amino acid concentrations, and the taste of the sake using principal component analysis, which yielded seven principal components (PC1–7) that explained 100 % of the total variance in the data. PC1, which explains 33.6 % of the total variance, correlates most positively with strong taste and most negatively with balanced tastes. PC2, which explains 54.4 % of the total variance, correlates most positively with a sweet taste and most negatively with bitter and sour tastes. Sakes brewed with “Kimoto yeast starter” and “Yamahaimoto” had high scores for PC1 and PC2, and had strong taste in comparison with sakes brewed with “Sokujo-moto”. When present at concentrations below 50 μM, d-Ala did not affect the PC1 score, but all the sakes showed a high PC1 score, when the d-Ala was above 100 μM. Similar observations were found for the d-Asp and d-Glu concentrations with regard to PC1, and the threshold concentrations of d-Asp and d-Glu that affected the taste were 33.8 and 33.3 μM, respectively. Certain bacteria present in sake, especially lactic acid bacteria, produce d-Ala, d-Asp and d-Glu during storage, and these d-amino acids increased the PC1 score and produced a strong taste (Nojun). When d- and l-Ala were added to the sakes, the value for the umami taste in the sensory evaluation increased, with the effect of d-Ala being much stronger than that of l-Ala. The addition of 50–5,000 μM dl-Ala did not effect on the aroma of the sakes at all.     

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.     

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.     

d-Aspartate acts as a signaling molecule in nervous and neuroendocrine systems

d-Aspartate (d-Asp) is an endogenous amino acid in the central nervous and reproductive systems of vertebrates and invertebrates. High concentrations of d-Asp are found in distinct anatomical locations, suggesting that it has specific physiological roles in animals. Many of the characteristics of d-Asp have been documented, including its tissue and cellular distribution, formation and degradation, as well as the responses elicited by d-Asp application. d-Asp performs important roles related to nervous system development and hormone regulation; in addition, it appears to act as a cell-to-cell signaling molecule. Recent studies have shown that d-Asp fulfills many, if not all, of the definitions of a classical neurotransmitter—that the molecule’s biosynthesis, degradation, uptake, and release take place within the presynaptic neuron, and that it triggers a response in the postsynaptic neuron after its release. Accumulating evidence suggests that these criteria are met by a heterogeneous distribution of enzymes for d-Asp’s biosynthesis and degradation, an appropriate uptake mechanism, localization within synaptic vesicles, and a postsynaptic response via an ionotropic receptor. Although d-Asp receptors remain to be characterized, the postsynaptic response of d-Asp has been studied and several l-glutamate receptors are known to respond to d-Asp. In this review, we discuss the current status of research on d-Asp in neuronal and neuroendocrine systems, and highlight results that support d-Asp’s role as a signaling molecule.     

The role of d-amino acids in amyotrophic lateral sclerosis pathogenesis: a review

A potential role for d-amino acids in motor neuron disease/amyotrophic lateral sclerosis (ALS) is emerging. d-Serine, which is an activator/co-agonist at the N-methyl-d-aspartate glutamate receptor subtype, is elevated both in spinal cord from sporadic cases of ALS and in an animal model of ALS. Furthermore, we have shown that a mutation in d-amino acid oxidase (DAO), an enzyme strongly localized to spinal cord motor neurons and brain stem motor nuclei, is associated with familial ALS. DAO plays an important role in regulating levels of d-serine, and its function is impaired by the presence of this mutation and this may contribute to the pathogenic process in ALS. In sporadic ALS cases, elevated d-serine may arise from induction of serine racemase, its synthetic enzyme, caused by cell stress and inflammatory processes thought to contribute to disease progression. Both these abnormalities in d-serine metabolism lead to an increase in synaptic d-serine which may contribute to disease pathogenesis.     

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.     

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.     

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.     

Transportable and Non-transportable Inhibitors of L-glutamate Uptake Produce Astrocytic Stellation and Increase EAAT2 Cell Surface Expression

Astrocytic excitatory amino acid transporters (EAATs) regulate excitatory transmission and limit excitotoxicity. Evidence for a functional interface between EAATs and glial fibrillary acidic protein (GFAP) relevant to astrocytic morphology led to investigations of actions of transportable (d-Aspartate (d-Asp) and (2S,3S,4R)-2-(carboxycyclopropyl)glycine (l-CCG-III)) and non-transportable (dl-threo-β-benzyloxyaspartate (dl-TBOA)) inhibitors of Glu uptake in murine astrocytes. d-Asp (1 mM), l-CCG-III (0.5 mM) and dl-TBOA (0.5 mM) produced time-dependent (24–72 h) reductions in ³[H]d-Asp uptake (approximately 30–70%) with little or no gliotoxicity. All drugs induced a profound change in phenotype from cobblestone to stellate morphology and image analysis revealed increases in the intensity of GFAP immunolabelling for l-CCG-III and dl-TBOA. Cytochemistry indicated localized changes in F-actin distribution. Cell surface expression of EAAT2, but not EAAT1, was elevated at 72 h. Blockade of Glu uptake by both types of EAAT inhibitor exerts longer-term effects on astrocytic morphology and a compensatory homeostatic rise in EAAT2 abundance.     

D-amino acids in the cell pool of bacteria

About 30 different bacterial species were tested for the possible presence of freed-amino acids in their cell pool. Gram-positive bacteria particularly the species of the genusBacillus have a fairly large pool of freely extractabled-amino acids. Varied quantities of freed-amino acids were detected inBacillus subtilis B₃,Bacillus subtilis Marburg,Bacillus licheniformis, Bacillus brevis, Bacillus stearothermophilus, Lactobacillus fermenti, Lactobacillus delbrueckii, Staphylococcus aureus andClostridium acetobutylicum. The individual components ofd-amino acids were identified in 5Bacillus species referred to above,d-alanine is the major component; the otherd-amino acids identified are aspartic acid, glutamic acid, histidine, leucines, proline, serine and tyrosine. Thed-amino acid pool size inBacillus subtilis B₃ varies with different culture conditions. The pool size is maximum when growth temperature is 30°C and it fluctuates with change in pH of the medium. The maximum quantity ofd-amino acids could be recovered when the culture was at mid log phase. O₂ supply to the medium has little effect ond-amino acid pool size. The starvation of cells leads to depletion of thed-amino acid pool which is exhausted almost completely within 4 hours by incubation in nutrient-free medium.     

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.     

Characterization of uptake and release processes ford- andl-aspartate in primary cultures of astrocytes and cerebellar granule cells

The uptake ofl-andd-aspartate was studied in astrocytes cultured from prefrontal cortex and in granule cells cultured from cerebellum. A high affinity uptake system forl- andd-aspartate was found in both cell types, and the two stereoisomers exhibited essentially the sameK _m - andV _max -values in bouth astrocytes (l-aspartate:K _m 77 μM;V _max 11.8 nmol×min^?1×mg^?1;d-aspartate:K _m 83 μM;V _max 14.0 nmol×min^?1×mg^?1) and granule cells (l-aspartate:K _m 32 μM;V _max 2.8 nmol ×min^?1×mg^?1;d-aspartate:K _m 26 μM;V _max 3.0 nmol×min^?1×mg^?1). To investigate whetherl-glutamate,l-aspartate andd-aspartate use the same uptake system a detailed kenetic analysis was performed. The uptake kinetics of each one of the three amino acids was studied in the presence of the two other amino acids, and no essential differences between the uptake characteristics of the amino acids were found. In addition to the uptake studies the release ofD-aspartate from cerebellar granule cells was investigated and compared withl-glutamate release. A Ca²⁺-dependent, K⁺-induced release was found for both amino acids.     

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.     

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.     

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.     

The human L-pipecolic acid oxidase is similar to bacterial monomeric sarcosine oxidases rather than D-amino acid oxidases

L-Pipecolic acid oxidase activity is deficient in patients with peroxisome biogenesis disorders (PBDs). Because its role, if any, in these disorders is unknown, the authors cloned the human gene to order to further study its functions. BLAST search of the translated sequence showed greatest homology to Bacillus sp. NS-129 monomeric sarcosine oxidase. The purified enzyme could use either L-pipecolic acid or sarcosine as a substrate. No homology was found to the peroxisomal D-amino acid oxidases. A further comparison of L-pipecolic acid oxidase to the two D-amino acid oxidases in peroxisomes showed that the proteins differed in many ways. First, both D-amino acid oxidase and L-pipecolic acid oxidase showed no enzyme activity in liver from Zell-weger syndrome patients; D-aspartate oxidase activity was unchanged from control levels. Although all were targeted to peroxisomes, their targeting signals differed. No L-pipecolic acid oxidase was found in brain or other tissues outside of liver and kidney. The D-amino acid oxidases were similarly and more widely distributed. Finally, although D-amino acid degradation is limited to peroxisomes in mammals, L-pipecolic acid can be oxidized in either mitochondria or peroxisomes, or both.     

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.