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The hyperthermophilic bacterium Thermotoga maritima peptidoglycan contains unusual d-lysine alongside typical d-alanine and d-glutamate. We previously identified lysine racemase and threonine dehydratase, but knowledge of d-amino acid metabolism remains limited. Herein, we identified and characterized T. maritima acetylornithine aminotransferase TM1785. The enzyme was most active towards acetyl-l-ornithine, but also utilized l-glutamate, l-ornithine and acetyl-l-lysine as amino donors, and 2-oxoglutarate was the preferred amino acceptor. TM1785 also displayed racemase activity towards four amino acids and lyase activity towards l-cysteine, but no dehydratase activity towards l-serine, l-threonine or corresponding d-amino acids. Catalytic efficiency (kcat /Km ) was highest for aminotransferase activity and lowest for racemase activity. TM1785 is a novel acetylornithine aminotransferase associated with l-arginine biosynthesis that possesses two additional distinct activities.

Various d-amino acids play important physiological roles in mammals, but the pathways of their production remain unknown except for d-serine, which is generated by serine racemase. Previously, we found that Escherichia coli cystathionine β-lyase possesses amino acid racemase activity in addition to β-lyase activity. In the present work, we evaluated the enzymatic activities of human cystathionine γ-lyase, which shares a relatively high amino acid sequence identity with cystathionine β-lyase. The enzyme did not show racemase activity toward various amino acids including alanine and lyase and dehydratase activities were highest toward l-cystathionine and l-homoserine, respectively. The enzyme also showed weak activity toward l-cysteine and l-serine but no activity toward d-amino acids. Intriguingly, the pH and temperature profiles of lyase activity were distinct from those of dehydratase activity. Catalytic efficiency was higher for lyase activity than for dehydratase activity.

Recent investigations have shown that multiple d-amino acids are present in mammals and these compounds have distinctive physiological functions. Free d-glutamate is present in various mammalian tissues and cells and in particular, it is presumably correlated with cardiac function, and much interest is growing in its unique metabolic pathways. Recently, we first identified d-glutamate cyclase as its degradative enzyme in mammals, whereas its biosynthetic pathway in mammals is unclear. Glutamate racemase is a most probable candidate, which catalyzes interconversion between d-glutamate and l-glutamate. Here, we identified the cDNA encoding l-serine dehydratase-like (SDHL) as the first mammalian clone with glutamate racemase activity. This rat SDHL had been deposited in mammalian databases as a protein of unknown function and its amino acid sequence shares ∼60% identity with that of l-serine dehydratase. Rat SDHL was expressed in Escherichia coli, and the enzymatic properties of the recombinant were characterized. The results indicated that rat SDHL is a multifunctional enzyme with glutamate racemase activity in addition to l-serine/l-threonine dehydratase activity. This clone is hence abbreviated as STDHgr. Further experiments using cultured mammalian cells confirmed that d-glutamate was synthesized and l-serine and l-threonine were decomposed. It was also found that SDHL (STDHgr) contributes to the homeostasis of several other amino acids.

d-Serine (d-Ser) is a coagonist for NMDA-type glutamate receptors and is thus important for higher brain function. d-Ser is synthesized by serine racemase and degraded by d-amino acid oxidase. However, the significance of these enzymes and the relevant functions of d-amino acids remain unclear. Here, we show that in the nematode Caenorhabditis elegans, the serine racemase homolog SERR-1 and d-amino acid oxidase DAAO-1 control an adaptive foraging behavior. Similar to many organisms, C. elegans immediately initiates local search for food when transferred to a new environment. With prolonged food deprivation, the worms exhibit a long-range dispersal behavior as the adaptive foraging strategy. We found that serr-1 deletion mutants did not display this behavior, whereas daao-1 deletion mutants immediately engaged in long-range dispersal after food removal. A quantitative analysis of d-amino acids indicated that d-Ser and d-alanine (d-Ala) are both synthesized and suppressed during food deprivation. A behavioral pharmacological analysis showed that the long-range dispersal behavior requires NMDA receptor desensitization. Long-term pretreatment with d-Ala, as well as with an NMDA receptor agonist, expanded the area searched by wild-type worms immediately after food removal, whereas pretreatment with d-Ser did not. We propose that d-Ser and d-Ala are endogenous regulators that cooperatively induce the long-range dispersal behavior in C. elegans through actions on the NMDA receptor.SIGNIFICANCE STATEMENT In mammals, d-serine (d-Ser) functions as an important neuromodulator of the NMDA-type glutamate receptor, which regulates higher brain functions. In Caenorhabditis elegans, previous studies failed to clearly define the physiological significance of d-Ser, d-alanine (d-Ala), and their metabolic enzymes. In this study, we found that these d-amino acids and their associated enzymes are active during food deprivation, leading to an adaptive foraging behavior. We also found that this behavior involved NMDA receptor desensitization.

Bacteria produce various D-amino acids, including non-canonical D-amino acids, to adapt to environmental changes and overcome a variety of threats. These D-amino acids are largely utilized as components of peptidoglycan, and they promote peptidoglycan remodeling and biofilm disassembly. The biosynthesis, maturation, and recycling of peptidoglycan are catalyzed by penicillin-binding proteins (PBPs). However, although non-canonical D-amino acids are known to be incorporated into peptidoglycan, the maturation and recycling of peptidoglycan containing such residues remain uncharacterized. Therefore, we investigated whether PBP4 and PBP5, low molecular mass (LMM) PBPs from Escherichia coli and Bacillus subtilis, are involved in these events of peptidoglycan metabolism. Enzyme assays using p-nitroaniline (pNA)-derivatized D-amino acids and peptidoglycan-mimicking peptides revealed that PBP4 and PBP5 from both species have peptidase activity toward substrates containing D-Asn, D-His, or D-Trp. These D-amino acids slowed the growth of dacA- or dacB-deficient E. coli (∆dacA or ∆dacB) relative to the wild-type strain. Additionally, these D-amino acids affected biofilm formation by the ∆dacB strain. Collectively, PBP4 and PBP5 are involved in the cleavage of peptidoglycan containing non-canonical D-amino acids, and these properties affect growth and biofilm formation.

The hyperthermophilic bacterium Thermotoga maritima has an atypical peptidoglycan that contains d-lysine alongside the usual d-alanine and d-glutamate. We previously identified a lysine racemase involved in d-lysine biosynthesis, and this enzyme also possesses alanine racemase activity. However, T. maritima has neither alanine racemase nor glutamate racemase enzymes; hence, the precise biosynthetic pathways of d-alanine and d-glutamate remain unclear in T. maritima. In the present study, we identified and characterized a novel d-amino acid aminotransferase (TM0831) in T. maritima. TM0831 exhibited aminotransferase activity towards 23 d-amino acids, but did not display activity towards l-amino acids. It displayed high specific activities towards d-homoserine and d-glutamine as amino donors. The most preferred acceptor was 2-oxoglutarate, followed by glyoxylate. Additionally, TM0831 displayed racemase activity towards four amino acids including aspartate and glutamate. Catalytic efficiency (k

Noncanonical D-amino acids are involved in peptidoglycan and biofilm metabolism in bacteria. Previously, we identified amino acid racemases with broad substrate specificity, including YgeA from Escherichia coli, which strongly prefers homoserine as a substrate. In this study, we investigated the functions of this enzyme in vivo. When wild-type and ygeA-deficient E. coli strains were cultured in minimal medium containing D-homoserine, the D-homoserine level was significantly higher in the ygeA-deficient strain than in the wild-type strain, in which it was almost undetectable. Additionally, D-homoserine was detected in YgeA-expressed E. coli cells cultured in minimal medium containing L-homoserine. The growth of the ygeA-deficient strain was significantly impaired in minimal medium with or without supplemental D-homoserine, while L-methionine, L-threonine or L-isoleucine, which are produced via L-homoserine, restored the growth impairment. Furthermore, the wild-type strain formed biofilms significantly more efficiently than the ygeA-deficient strain. Addition of L- or D-homoserine significantly suppressed biofilm formation in the wild-type strain, whereas this addition had no significant effect in the ygeA-deficient strain. Together, these data suggest that YgeA acts as an amino acid racemase and plays a role in L- and D-homoserine metabolism in E. coli.

The peptidoglycan of the hyperthermophile Thermotoga maritima contains an unusual component, d-lysine (d-Lys), in addition to the typical d-alanine (d-Ala) and d-glutamate (d-Glu). In a previous study, we identified a Lys racemase that is presumably associated with d-Lys biosynthesis. However, our understanding of d-amino acid metabolism in T. maritima and other bacteria remains limited, although d-amino acids in the peptidoglycan are crucial for preserving bacterial cell structure and resistance to environmental threats. Herein, we characterized enzymatic and structural properties of TM0356 that shares a high amino acid sequence identity with serine (Ser) racemase. The results revealed that TM0356 forms a tetramer with each subunit containing a pyridoxal 5′-phosphate as a cofactor. The enzyme did not exhibit racemase activity toward various amino acids including Ser, and dehydratase activity was highest toward l-threonine (l-Thr). It also acted on l-Ser and l-allo-Thr, but not on the corresponding d-amino acids. The catalytic mechanism did not follow typical Michaelis–Menten kinetics; it displayed a sigmoidal dependence on substrate concentration, with highest catalytic efficiency (kcat/K0.5) toward l-Thr. Interestingly, dehydratase activity was insensitive to allosteric regulators l-valine and l-isoleucine (l-Ile) at low concentrations, while these l-amino acids are inhibitors at high concentrations. Thus, TM0356 is a biosynthetic Thr dehydratase responsible for the conversion of l-Thr to α-ketobutyrate and ammonia, which is presumably involved in the first step of the biosynthesis of l-Ile.

d-Glutamate cyclase (DGLUCY) is a unique enzyme that reversibly converts free d-glutamate to 5-oxo-d-proline and H2O. Mammalian DGLUCY is highly expressed in the mitochondrial matrix in the heart, and its downregulation disrupts d-glutamate and/or 5-oxo-d-proline levels, contributing to the onset and/or exacerbation of heart failure. However, detailed characterisation of DGLUCY has not yet been performed. Herein, the structural and enzymatic properties of purified recombinant mouse DGLUCY were examined. The results revealed a dimeric oligomerisation state, and both d-glutamate-to-5-oxo-d-proline and 5-oxo-d-proline-to-d-glutamate reactions were catalysed in a stereospecific manner. Catalytic activity is modulated by divalent cations and nucleotides including ATP and ADP. Interestingly, the presence of Mn2+ completely abolished the 5-oxo-d-proline-to-d-glutamate reaction but stimulated the d-glutamate-to-5-oxo-d-proline reaction. The optimum pH is ∼8.0, similar to that in the mitochondrial matrix, and the catalytic efficiency for d-glutamate is markedly higher than that for 5-oxo-d-proline. These findings suggest that DGLUCY functions as a metalloenzyme that degrades d-glutamate in the mitochondrial matrix in mammalian cells. The results also provide insight into the correlation between DGLUCY enzyme activity and the physiological and pathological roles of d-glutamate and 5-oxo-d-proline in cardiac function, which is of relevance to the risk of onset of heart failure.

The peptidoglycan layer of the bacterial cell wall typically contains D-alanine (D-Ala) and D-glutamic acid (D-Glu), and also various non-canonical D-amino acids that have been linked to peptidoglycan remodeling, inhibition of biofilm formation, and triggering of biofilm disassembly. Bacteria produce D-amino acids when adapting to environmental changes as a common survival strategy. In our previous study, we detected non-canonical D-amino acids in Escherichia coli grown in minimal medium. However, the biosynthetic pathways of non-canonical D-amino acids remain poorly understood. In the present study, we identified amino acid racemases in E. coli MG1655 (YgeA) and Bacillus subtilis (RacX) that produce non-canonical D-amino acids other than D-Ala and D-Glu. We characterized their enzymatic properties, and both displayed broad substrate specificity but low catalytic activity. YgeA preferentially catalyzes the racemization of homoserine, while RacX preferentially racemizes arginine, lysine, and ornithine. RacX is dimeric, and appears not to require pyridoxal 5'-phosphate (PLP) as a coenzyme as is the case with YgeA. To our knowledge, this is the first report on PLP-independent amino acid racemases possessing broad substrate specificity in E. coli and B. subtilis.

Background Autism spectrum disorders (ASD) comprise a heterogeneous group of neurodevelopmental conditions characterized by impairment in social interaction, deviance in communication, and repetitive behaviors. Dysfunctional ionotropic NMDA and AMPA receptors, and metabotropic glutamate receptor 5 activity at excitatory synapses has been recently linked to multiple forms of ASD. Despite emerging evidence showing that d -aspartate and d -serine are important neuromodulators of glutamatergic transmission, no systematic investigation on the occurrence of these D-amino acids in preclinical ASD models has been carried out. Methods Through HPLC and qPCR analyses we investigated d -aspartate and d -serine metabolism in the brain and serum of four ASD mouse models. These include BTBR mice, an idiopathic model of ASD, and Cntnap2−/−, Shank3−/−, and 16p11.2+/− mice, three established genetic mouse lines recapitulating high confidence ASD-associated mutations. Results Biochemical and gene expression mapping in Cntnap2−/−, Shank3−/−, and 16p11.2+/− failed to find gross cerebral and serum alterations in d -aspartate and d -serine metabolism. Conversely, we found a striking and stereoselective increased d -aspartate content in the prefrontal cortex, hippocampus and serum of inbred BTBR mice. Consistent with biochemical assessments, in the same brain areas we also found a robust reduction in mRNA levels of d -aspartate oxidase, encoding the enzyme responsible for d -aspartate catabolism. Conclusions Our results demonstrated the presence of disrupted d -aspartate metabolism in a widely used animal model of idiopathic ASD. General significance Overall, this work calls for a deeper investigation of D-amino acids in the etiopathology of ASD and related developmental disorders.

d-Serine (d-Ser) is a coagonist for NMDA-type glutamate receptors and is thus important for higher brain function. d-Ser is synthesized by serine racemase and degraded by d-amino acid oxidase. However, the significance of these enzymes and the relevant functions of d-amino acids remain unclear. Here, we show that in the nematode Caenorhabditis elegans, the serine racemase homolog SERR-1 and d-amino acid oxidase DAAO-1 control an adaptive foraging behavior. Similar to many organisms, C. elegans immediately initiates local search for food when transferred to a new environment. With prolonged food deprivation, the worms exhibit a long-range dispersal behavior as the adaptive foraging strategy. We found that serr-1 deletion mutants did not display this behavior, whereas daao-1 deletion mutants immediately engaged in long-range dispersal after food removal. A quantitative analysis of d-amino acids indicated that d-Ser and d-alanine (d-Ala) are both synthesized and suppressed during food deprivation. A behavioral pharmacological analysis showed that the long-range dispersal behavior requires NMDA receptor desensitization. Long-term pretreatment with d-Ala, as well as with an NMDA receptor agonist, expanded the area searched by wild-type worms immediately after food removal, whereas pretreatment with d-Ser did not. We propose that d-Ser and d-Ala are endogenous regulators that cooperatively induce the long-range dispersal behavior in C. elegans through actions on the NMDA receptor. SIGNIFICANCE STATEMENT In mammals, d-serine (d-Ser) functions as an important neuromodulator of the NMDA-type glutamate receptor, which regulates higher brain functions. In Caenorhabditis elegans, previous studies failed to clearly define the physiological significance of d-Ser, d-alanine (d-Ala), and their metabolic enzymes. In this study, we found that these d-amino acids and their associated enzymes are active during food deprivation, leading to an adaptive foraging behavior. We also found that this behavior involved NMDA receptor desensitization.

In mammals, metabolism of free d-glutamate is regulated by d-glutamate cyclase (DGLUCY), which reversibly converts d-glutamate to 5-oxo-d-proline and H2O. Metabolism of these d-amino acids by DGLUCY is thought to regulate cardiac function. In this study, we established a simple, accurate, and sensitive colorimetric assay method for measuring DGLUCY activity. To this end, we optimized experimental procedures for derivatizing 5-oxo-d-proline with 2-nitrophenylhydrazine hydrochloride. 5-Oxo-d-proline was derivatized with 2-nitrophenylhydrazine hydrochloride in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as a catalyst to generate the acid hydrazides, whose levels were then determined using a colorimetric method. Under optimized conditions, we examined the sensitivity and accuracy of the colorimetric method and compared our technique with other methods by high-performance liquid chromatography with ultraviolet-visible or fluorescence detection. Moreover, we assessed the suitability of this colorimetric method for measuring DGLUCY activity in biological samples. Our colorimetric method could determine DGLUCY activity with adequate validity and reliability. This method will help to elucidate the relationship among DGLUCY activity, the physiological and pathological roles of d-glutamate and 5-oxo-d-proline, and cardiac function.

d-Aspartate oxidase (DDO) is a flavin adenine dinucleotide (FAD)-containing flavoprotein that stereospecifically acts on acidic d-amino acids (i.e., free d-aspartate and d-glutamate). Mammalian DDO, which exhibits higher activity toward d-aspartate than d-glutamate, is presumed to regulate levels of d-aspartate in the body and is not thought to degrade d-glutamate in vivo. By contrast, three DDO isoforms are present in the nematode Caenorhabditis elegans, DDO-1, DDO-2, and DDO-3, all of which exhibit substantial activity toward d-glutamate as well as d-aspartate. In this study, we optimized the Escherichia coli culture conditions for production of recombinant C. elegans DDO-1, purified the protein, and showed that it is a flavoprotein with a noncovalently but tightly attached FAD. Furthermore, C. elegans DDO-1, but not mammalian (rat) DDO, efficiently and selectively degraded d-glutamate in addition to d-aspartate, even in the presence of various other amino acids. Thus, C. elegans DDO-1 might be a useful tool for determining these acidic d-amino acids in biological samples.

Various d‐amino acids are involved in peptidoglycan and biofilm metabolism in bacteria, suggesting that these compounds are necessary for successful adaptation to environmental changes. In addition to the conventional d‐alanine (d‐Ala) and d‐glutamate, the peptidoglycan of the hyperthermophilic bacterium Thermotoga maritima contains both l‐lysine (l‐Lys) and d‐Lys, but not meso‐diaminopimelate (meso‐Dpm). d‐Lys is an uncommon component of peptidoglycan, and its biosynthetic pathway remains unclear. In this study, we identified and characterized a novel Lys racemase (TM1597) and Dpm epimerase (TM1522) associated with the d‐Lys biosynthetic pathway in T. maritima. The Lys racemase had a dimeric structure containing pyridoxal 5′‐phosphate as a cofactor. Among the amino acids, it exhibited the highest racemase activity toward d‐ and l‐Lys, and also had relatively high activity toward d‐ and l‐enantiomers of ornithine and Ala. The Dpm epimerase had the highest epimerization activity toward ll‐ and meso‐Dpm, and also measurably racemized certain amino acids, including Lys. These results suggest that Lys racemase contributes to production of d‐Lys and d‐Ala for use as peptidoglycan components, and that Dpm epimerase converts ll‐Dpm to meso‐Dpm, a precursor in the l‐Lys biosynthetic pathway.

d-Aspartate oxidase (DDO) is a degradative enzyme that acts stereospecifically on free acidic D-amino acids such as d-aspartate and d-glutamate. d-Aspartate plays an important role in regulating neurotransmission, developmental processes, hormone secretion, and reproductive functions in mammals. In contrast, the physiological role of d-glutamate in mammals remains unclear. In Caenorhabditis elegans, the enzyme responsible for in vivo metabolism of d-glutamate is DDO-3, one of the three DDO isoforms, which is also required for normal self-fertility, hatching, and lifespan. In general, eukaryotic DDOs localize to subcellular peroxisomes in a peroxisomal targeting signal type 1 (PTS1)-dependent manner. However, DDO-3 does not contain a PTS1, but instead has a putative N-terminal signal peptide (SP). In this study, we found that DDO-3 is a secreted DDO, the first such enzyme to be described in eukaryotes. In hermaphrodites, DDO-3 was secreted from the proximal gonadal sheath cells in a SP-dependent manner and transferred to the oocyte surface. In males, DDO-3 was secreted from the seminal vesicle into the seminal fluid in a SP-dependent manner during mating with hermaphrodites. In both sexes, DDO-3 was secreted from the cells where it was produced into the body fluid and taken up by scavenger coelomocytes. Full-length DDO-3 transgene rescued all phenotypes elicited by the deletion of ddo-3, whereas a DDO-3 transgene lacking the putative SP did not. Together, these results indicate that secretion of DDO-3 is essential for its physiological functions.

Mammalian cells possess the molecular apparatus necessary to take up, degrade, synthesize, and release free d-aspartate, which plays an important role in physiological functions within the body. Here, biologically active microbial compounds and pre-existing drugs were screened for their ability to alter the intracellular d-aspartate level in mammalian cells, and several candidate compounds were identified. Detailed analytical studies suggested that two of these compounds, mithramycin A and geldanamycin, suppress the biosynthesis of d-aspartate in cells. Further studies suggested that these compounds act at distinct sites within the cell. These compounds may advance our current understanding of biosynthesis of d-aspartate in mammals, a whole picture of which remains to be disclosed.

In previous report, we detected d -amino acids in the acid hydrolysates of purified recombinant β-galactosidase. Here, we employed a deuterium-hydrogen exchange method to discriminate innate d -amino acids from those generated during hydrolytic incubation. After hydrolysis of β-galactosidase in DCl/D 2 O, amino acids were derivatized with NBD-F and separated on a reverse-phase column, followed by liquid chromatography-tandem mass spectrometry equipped with a chiral column. Our results show an absence of innate d -amino acid residues in the protein and suggest that the protein undergoes isomerization during a very early stage of hydrolytic incubation.

Ovalbumin, a major protein in chicken egg white, is converted into a more thermostable molecular form, known as S-ovalbumin, during the storage of shell eggs. Our previous X-ray crystallographic study indicated that S-ovalbumin contains three D-Ser residues (S164, S236, and S320), which may account for its thermostability. Here, we confirmed the presence of these D-Ser residues in ovalbumin using a technique combining deuterium labeling of α-protons of amino acids and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ovalbumin from chicken egg white and recombinant ovalbumin were incubated for approximately 12 days at pH 9.5 and 37°C. They were then hydrolyzed in DCl/D2O vapor, derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and analyzed by LC-MS/MS. A time-dependent increase in the D-Ser contents in native ovalbumin was observed over a period of 7 days, reaching approximately 8%. This corresponds to a value of three serine residues per molecule, and is consistent with the prediction based on our previous crystallographic analysis. Nearly identical results were obtained with recombinant ovalbumin. We then used this technique to investigate whether D-amino acid residues could arise within other proteins under mild alkaline conditions and detected small but significant amounts of D-Ala and/or D-Ser residues that increased in a time-dependent manner in some proteins.

D-Aspartate oxidase (DDO) is a degradative enzyme that is stereospecific for acidic D-amino acids, including D-aspartate, a potential agonist of the N-methyl-D-aspartate (NMDA) receptor. Dysfunction of NMDA receptor-mediated neurotransmission has been implicated in the onset of various mental disorders, such as schizophrenia. Hence, a DDO inhibitor that increases the brain levels of D-aspartate and thereby activates NMDA receptor function is expected to be a useful compound. To search for potent DDO inhibitor(s), a large number of compounds were screened in silico, and several compounds were identified as candidates. They were then characterized and evaluated as novel DDO inhibitors in vitro (e.g., the inhibitor constant value of 5-aminonicotinic acid for human DDO was 3.80 μM). The present results indicate that some of these compounds may serve as lead compounds for the development of a clinically useful DDO inhibitor and as active site probes to elucidate the structure-function relationships of DDO.

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.

Non-canonical d-amino acids play important roles in bacteria including control of peptidoglycan metabolism and biofilm disassembly. Bacteria appear to produce non-canonical d-amino acids to adapt to various environmental changes, and understanding the biosynthetic pathways is important. We identified novel amino acid racemases possessing the ability to produce non-canonical d-amino acids in Escherichia coli and Bacillus subtilis in our previous study, whereas the biosynthetic pathways of these d-amino acids still remain unclear. In the present study, we demonstrated that two cystathionine β-lyases (MetC and MalY) from E. coli produce non-canonical d-amino acids including non-proteinogenic amino acids. Furthermore, MetC displayed d- and l-serine (Ser) dehydratase activity. We characterised amino acid racemase, Ser dehydratase and cysteine lyase activities, and all were higher for MetC. Interestingly, all three activities were at a comparable level for MetC, although optimal conditions for each reaction were distinct. These results indicate that MetC and MalY are multifunctional enzymes involved in l-methionine metabolism and the production of d-amino acids, as well as d- and l-Ser metabolism. To our knowledge, this is the first evidence that cystathionine β-lyase is a multifunctional enzyme with three different activities.

d -Aspartate oxidase (DDO) is a degradative enzyme that is stereospecific for the acidic amino acid d -aspartate, an endogenous agonist of the N-methyl- d -aspartate (NMDA) receptor. Dysregulation of NMDA receptor-mediated neurotransmission has been implicated in the onset of various neuropsychiatric disorders including schizophrenia, as well as chronic pain. Thus, appropriate regulation of d -aspartate is believed to be important for maintaining proper neural activity in the nervous system. Accordingly, much attention has been paid to the role(s) of DDO in the metabolism of d -aspartate in vivo, and the physiological functions of DDO have been actively investigated using experimental rats and mice. However, detailed characterisation of rat DDO has not yet been performed, and little is known about species-specific differences in the properties of mammalian DDOs. In this study, the structural and enzymatic properties of purified recombinant rat, mouse and human DDOs were examined and compared. The results showed that rat DDO is more similar to human DDO than to mouse DDO. This work provides useful insight into the use of rats as an experimental model for investigating the biological significance of human DDO and/or d -aspartate. This article is part of a Special Issue entitled: d -Amino acids: biology in the mirror, edited by Dr. Loredano Pollegioni, Dr. Jean-Pierre Mothet and Dr. Molla Gianluca.

d-Aspartate oxidase (DDO) is a degradative enzyme that is stereospecific for the acidic amino acid d-aspartate, an endogenous agonist of the N-methyl-d-aspartate (NMDA) receptor. Dysregulation of NMDA receptor-mediated neurotransmission has been implicated in the onset of various neuropsychiatric disorders including schizophrenia and in chronic pain. Thus, appropriate regulation of the amount of d-aspartate is believed to be important for maintaining proper neural activity in the nervous system. Herein, the effects of the non-synonymous single nucleotide polymorphisms (SNPs) R216Q and S308N on several properties of human DDO were examined. Analysis of the purified recombinant enzyme showed that the R216Q and S308N substitutions reduce enzyme activity towards acidic d-amino acids, decrease the binding affinity for the coenzyme flavin adenine dinucleotide and decrease the temperature stability. Consistent with these findings, further experiments using cultured mammalian cells revealed elevated d-aspartate in cultures of R216Q and S308N cells compared with cells expressing wild-type DDO. Furthermore, accumulation of several amino acids other than d-aspartate also differed between these cultures. Thus, expression of DDO genes carrying the R216Q or S308N SNP substitutions may increase the d-aspartate content in humans and alter homeostasis of several other amino acids. This work may aid in understanding the correlation between DDO activity and the risk of onset of NMDA receptor-related diseases.

We have developed and validated a high-performance liquid chromatography method that uses monolithic silica disk-packed spin columns and a monolithic silica column for the simultaneous determination of N(G)-monomethyl-L-arginine, N(G),N(G)-dimethyl-L-arginine, and N(G),N(G')-dimethyl-L-arginine in human plasma. For solid-phase extraction, our method employs a centrifugal spin column packed with monolithic silica bonded to propyl benzenesulfonic acid as a cation exchanger. After pretreatment, the methylated arginines are converted to fluorescent derivatives with 4-fluoro-7-nitro-2,1,3-benzoxadiazole, and then the derivatives are separated on a monolithic silica column. L-arginine concentration was also determined in diluted samples. Standard calibration curves revealed that the assay was linear in the concentration range 0.2-1.0 μM for methylated arginines and 40-200 μM for L-arginine. Linear regression of the calibration curve yielded equations with correlation coefficients of 0.999 (r(2)). The sensitivity was satisfactory, with a limit of detection ranging from 3.75 to 9.0 fmol for all four compounds. The RSDs were 4.3-4.8% (intraday) and 3.0-6.8% (interday). When this method was applied to samples from six healthy donors, the detected concentrations of N(G)-monomethyl-L-arginine, N(G),N(G)-dimethyl-L-arginine, N(G),N(G')-dimethyl-L-arginine and L-arginine were 0.05 ± 0.01, 0.41 ± 0.07, 0.59 ± 0.11, and 83.8 ± 30.43 μM (n = 6), respectively.

d-Amino acid oxidase (DAO) is a degradative enzyme that is stereospecific for d-amino acids, including d-serine and d-alanine, which are believed to be coagonists of the N-methyl-d-aspartate (NMDA) receptor. To identify a new class of DAO inhibitor(s) that can be used to elucidate the molecular details of the active site environment of DAO, manifold biologically active compounds of microbial origin and pre-existing drugs were screened for their ability to inhibit DAO activity, and several compounds were identified as candidates. One of these compounds, acyclovir (ACV), a well-known antiviral drug used for the treatment of herpesvirus infections, was characterized and evaluated as a novel DAO inhibitor in vitro. Analysis showed that ACV acts on DAO as a reversible slow-binding inhibitor, and interestingly, the time required to achieve equilibrium between DAO, ACV, and the DAO/ACV complex was highly dependent on temperature. The binding mechanism of ACV to DAO was investigated in detail by several approaches, including kinetic analysis, structural modeling of DAO complexed with ACV, and site-specific mutagenesis of an active site residue postulated to be involved in the binding of ACV. The results confirm that ACV is a novel, active site-directed inhibitor of DAO that can be a valuable tool for investigating the structure-function relationships of DAO, including the molecular details of the active site environment of DAO. In particular, it appears that ACV can serve as an active site probe to study the structural basis of temperature-induced conformational changes of DAO.

D-amino acid oxidase (DAO) is a degradative enzyme that is stereospecific for D-amino acids, including D-serine and D-alanine, which are potential coagonists of the N-methyl-D-aspartate (NMDA) receptor. Dysfunction of NMDA receptor-mediated neurotransmission has been implicated in the onset of various mental disorders such as schizophrenia. Hence, a DAO inhibitor that augments the brain levels of D-serine and/or D-alanine and thereby activates NMDA receptor function is expected to be an antipsychotic drug, for instance, in the treatment of schizophrenia. In the search for potent DAO inhibitor(s), a large number of compounds were screened in silico, and several compounds were estimated as candidates. These compounds were then characterized and evaluated as novel DAO inhibitors in vitro. The results reported in this study indicate that some of these compounds are possible lead compounds for the development of a clinically useful DAO inhibitor and have the potential to serve as active site probes to elucidate the structure-function relationships of DAO.

The retention mechanisms of nitrobenzofurazan (NBD)-amino acids in reversed-phase ion-pair liquid chromatography were quantitatively analyzed in silico The most contributed interaction for the retention is the Lewis acid-base interaction between an aromatic ring of NBD-amino acids and hydroxyl-group hydrogen of tetra-butyl-ammonium hydroxide coated on the hydrophobic phase. Solvent effects significantly improved the relation between the calculated molecular interaction (MI) energy values using a molecular mechanics program and log k values measured in chromatography. The correlation coefficient between the calculated MI energy values and the log k values was >0.95.

Background Alterations in one-carbon metabolism (OCM) have been observed in patients with schizophrenia (SZ), but a comprehensive study of OCM has not yet been conducted. A carbon atom is transferred from l-serine to methionine during OCM, but the relationship between l-serine and methionine in SZ is not yet known. We investigated the relationship between l-serine and methionine to obtain a comprehensive understanding of OCM in SZ. Methods We recruited forty-five patients with SZ and thirty normal controls (NC). Whole blood, plasma, and DNA specimens were obtained from all participants. Plasma l-serine, d-serine, glycine, methionine, and total homocysteine levels were measured using high-performance liquid chromatography. Plasma vitamin B12 and total folate were measured using a chemiluminescent protein-binding immunoassay. Clinical symptoms were estimated using the positive and negative syndrome scale (PANSS). The methylenetetrahydrofolate reductase (MTHFR) C667T genotype and A298C genotype, which are involved in MTHFR activity, were determined using the TaqMan genotyping assay system. Results Analysis of variance was used to confirm that the SZ cohort has higher plasma homocysteine levels and lower plasma folate levels than the NC group. Multi-regression analysis revealed a relationship between l-serine and methionine in the NC group but not in the SZ group. The MTHFR genotype did not affect the relationship between l-serine and methionine in each group. The total PANSS score was significantly related to d-serine and folate levels and to age. Positive PANSS scores were significantly related to both glycine and sex. In addition, both glycine and d-serine were significantly correlated with negative PANSS scores. Conclusions We found impairment of the relationship between l-serine and methionine in SZ. Clinical symptoms of SZ were partially correlated with the OCM components. These findings contributed to our understanding of OCM alteration in SZ and may explain why the alteration occurs.

In this study, we investigated whether the amino acid residues within peptides were isomerized (and the peptides converted to diastereomers) during the early stages of acid hydrolysis. We demonstrate that the model dipeptides l-Ala-l-Phe and l-Phe-l-Ala are epimerized to produce the corresponding diastereomers at a very early stage, prior to their acid hydrolytic cleavage to amino acids. Furthermore, the sequence-inverted dipeptides were generated via formation of a diketopiperazine during hydrolytic incubation, and these dipeptides were also epimerized. The proportion of diastereomers increased rapidly during incubation for 0.5–2 h. During acid hydrolysis, C-terminal residues of the model dipeptides were isomerized faster than N-terminal residues, consistent with the observation that the d-amino acid values of the C-terminal residues determined by the 0 h-extrapolating method were larger than those of the N-terminal residues. Thus, the artificial d-amino acid contents determined by the 0 h-extrapolating method appear to be products of the isomerization of amino acid residues during acid hydrolysis.

Free d-serine (d-Ser) plays a crucial role in regulating brain function in mammals. In various organisms, including mammals, d-Ser is biosynthesized by Ser racemase, a synthetic enzyme that produces d-Ser from l-Ser. Ser racemase also exhibits dehydratase activity toward several hydroxyamino acids. Thus, this enzyme is unique in that it possesses the capability to both synthesize and degrade d-Ser; however, the physiological significance of its degradative activity remains unclear. In contrast to the physiological roles of d-Ser in mammals, little is known about the role of this amino acid in lower organisms, including the nematode Caenorhabditis elegans. It is known that a mammalian Ser racemase homologue (T01H8.2) from C. elegans exhibits racemase activity. Here, the enzymatic properties of recombinant T01H8.2 were characterized and compared with those of recombinant human Ser racemase. Furthermore, the levels of several d- and l-amino acids were measured in wild-type C. elegans and in a mutant in which the T01H8.2 gene is partially deleted and thereby inactivated. The results indicate that T01H8.2 also shows dehydratase activity toward several hydroxyamino acids, although the enzyme is not critical for Ser metabolism in vivo. The possible physiological roles of T01H8.2 are discussed.

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

Protein l -isoaspartyl/ d -aspartyl o-methyltransferase (PIMT) is a widely expressed protein repair enzyme that restores isomerized aspartyl residues to their normal configuration. Current methods for measuring PIMT activity have limited sensitivity or require radioactivity. We have developed a highly sensitive new assay method to measure PIMT activity in cell lysates. As a substrate, we used a fluorescently labeled delta sleep-inducing peptide (DSIP) that contains an isoaspartyl residue: 7-nitro-2,1,3-benzoxadiazole (NBD)–DSIP(isoAsp). The PIMT-catalyzed transfer of a methyl group onto this substrate can be detected with a simple high-performance liquid chromatography (HPLC) procedure. After the enzyme reaction, the methylated form of the peptide is stable and can be reproducibly separated from the unmethylated form in an acidic solvent and fluorometrically detected by HPLC. The limit of detection was estimated to be approximately 1 pmol of NBD–DSIP(isoAsp) (signal/noise ratio [S/N] = 3), and the quantitation limit of the activity was approximately 18 μg of total cell lysate from HEK293 cells (10.7 pmol/min/mg protein). This assay method is sensitive enough to detect PIMT activity in biological samples without the use of radioisotopes, offering significant advantages over previously reported methods.

New fungal metabolites, designated quinadolines A (1) and B (2), were isolated from culture broth of Aspergillus sp. FKI-1746, and their structures were elucidated by NMR spectroscopy. The complete relative and absolute stereochemistry of 2 was determined by X-ray crystallography and amino acid analysis using a chiral column. Quinadolines moderately inhibited lipid droplet synthesis in mouse macrophages.

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

Functional and structural characterizations of pyridoxal 5'-phosphate-independent aspartate racemase of the acidothermophilic archaeon Picrophilus torridus were performed. Picrophilus aspartate racemase exhibited high substrate specificity to aspartic acid. The optimal reaction temperature was 60 °C, which is almost the same as the optimal growth temperature. Reflecting the low pH in the cytosol, the optimal reaction pH of Picrophilus aspartate racemase was approximately 5.5. However, the activity at the putative cytosolic pH of 4.6 was approximately 6 times lower than that at the optimal pH of 5.5. The crystal structure of Picrophilus aspartate racemase was almost the same as that of other pyridoxal 5'-phosphate -independent aspartate racemases. In two molecules of the dimer, one molecule contained a tartaric acid molecule in the catalytic site; the structure of the other molecule was relatively flexible. Finally, we examined the intracellular existence of D-amino acids. Unexpectedly, the proportion of D-aspartate to total aspartate was not very high. In contrast, both D-proline and D-alanine were observed. Because Picrophilus aspartate racemase is highly specific to aspartate, other amino acid racemases might exist in Picrophilus torridus.

Plasma concentration of three methylated arginines, endogenous nitric oxide synthase inhibitors, is not studied in schizophrenic patients. The purpose of this study was to determine plasma concentrations of N(G)-monomethyl-L-arginine (l-NMMA), N(G),N(G)-dimethyl-L-arginine (ADMA), N(G),N(G')-dimethyl-L-arginine (SDMA), and l-arginine in 56 male and 45 female schizophrenic patients undergoing antipsychotic drug treatment versus those of 39 male and 24 female healthy controls. Plasma concentrations of methylated arginines and l-arginine were measured using newly developed high performance liquid chromatography with fluorescence detection which we previously reported. Methylated arginine levels were slightly but significantly higher in schizophrenic patients. L-Arginine levels and the l-arginine/(ADMA+l-NMMA) ratio were higher in schizophrenic patients than in healthy controls. It is considered that pharmacological treatment of schizophrenic patients may lower methylated arginine levels that are increased by the disease, and increase L-arginine levels, eliciting an improvement in nitric oxide (NO) bioavailability.

The pathophysiology of schizophrenia may involve N-methyl-D-aspartate receptor (NMDAR) hypofunction. D-3serine and glycine are endogenous l-serine-derived NMDAR co-agonists. We hypothesized that the l-serine synthesis pathway could be involved in schizophrenia. We measured the activity of phosphoserine phosphatase (PSP), a rate-limiting enzyme in l-serine synthesis, in peripheral blood mononuclear cells of 54 patients with schizophrenia and 49 normal control subjects. Plasma amino acid (l-serine, d-serine, glycine, glutamine, and glutamate) levels were measured by high performance liquid chromatography. Peripheral blood mRNA expression levels of PHGDH, PSAT1, PSP, and SR, determined by quantitative real-time PCR were compared between patients and controls. PSP activity was higher in patients than in controls, especially in male patients. In male patients, the plasma l-serine concentration was higher than that in controls. In patients, PSP activity was negatively correlated with plasma d-serine and glycine levels. Furthermore, PSP activity was positively correlated with plasma l-serine concentration. These results were statistically significant only in male patients. PSP, PSAT1, and PHGDH mRNA levels were lower in patients than in controls, except when the PHGDH expression level was compared with ACTB expression. In summary, we found the l-serine synthesis system to be altered in patients with schizophrenia, especially in male patients.

D-Aspartate (D-Asp), a free D-amino acid found in mammals, plays crucial roles in the neuroendocrine, endocrine, and central nervous systems. Recent studies have implicated D-Asp in the pathophysiology of infertility and N-methyl-D-Asp receptor-related diseases. D-Asp oxidase (DDO), a degradative enzyme that is stereospecific for acidic D-amino acids, is the sole catabolic enzyme acting on D-Asp in mammals. Human DDO is considered an attractive therapeutic target, and DDO inhibitors may be potential lead compounds for the development of new drugs against the aforementioned diseases. However, human DDO has not been characterized in detail and, although preclinical studies using experimental rodents are prerequisites for evaluating the in vivo effects of potential inhibitors, the existence of species-specific differences in the properties of human and rodent DDOs is still unclear. Here, the enzymatic activity and characteristics of purified recombinant human DDO were analyzed in detail. The kinetic and inhibitor-binding properties of this enzyme were also compared with those of purified recombinant rat and mouse DDOs. In addition, structural models of human, rat, and mouse DDOs were generated and compared. It was found that the differences among these DDO proteins occur in regions that appear involved in migration of the substrate/product in and out of the active site. In summary, detailed characterization of human DDO was performed and provides useful insights into the use of rats and mice as experimental models for evaluating the in vivo effects of DDO inhibitors.

Four cDNA clones that were annotated in the database as encoding d-amino acid oxidase (DAAO) or d-aspartate oxidase (DASPO) were isolated by RT-PCR from Caenorhabditis elegans RNA. The proteins (Y69Ap, C47Ap, F18Ep, and F20Hp) encoded by the cloned cDNAs were expressed in Escherichia coli as recombinant proteins with an N-terminal His-tag. All proteins except F20Hp were recovered in the soluble fractions. The recombinant Y69Ap has functional DAAO activity, as it can deaminate neutral and basic d-amino acids, whereas the recombinants C47Ap and F18Ep have functional DASPO activities, as they can deaminate acidic d-amino acids. Additional experiments using purified recombinant proteins revealed that Y69Ap deaminates d-Arg more efficiently than d-Ala and d-Met, and that C47Ap and F18Ep show distinct kinetic properties against d-Asp, d-Glu, and N-methyl-d-Asp. This is the first time that cDNA cloning of invertebrate DAAO and DASPO genes has been reported. In addition, our study reveals for the first time that C. elegans has at least two genes encoding functional DASPOs and one gene encoding DAAO, although it had previously been thought that organisms only bear one copy each of these genes. The two C. elegans DASPOs differ in their substrate specificities and possibly also in their subcellular localization.

We previously reported for the first time that D-aspartate (D-Asp) is biosynthesized by cultured mammalian cells such as pheochromocytoma (PC)12 cells and its subclone MPT1 (FEBS Lett. 434 (1998) 231, Arch. Biochem. Biophys. 404 (2002) 92). We speculated that D-Asp levels in the intra- and extracellular spaces of the cultured cells are maintained in a dynamic state of homeostasis. To test this here, we utilized a novel and potent L-Glu transporter inhibitor, TFB-TBOA. This inhibitor proved to be a genuine nontransportable blocker of the transporter even during long periods of culture. Use of this inhibitor with MPT1 cells confirmed that D-Asp levels are in a dynamic steady state where it is constantly released into the extracellular space by a yet undefined mechanism as well as being constantly and intensively taken up by the cells via the L-Glu transporter. We estimated the rate with which D-Asp is constitutively released from MPT1 cells is approx. 3.8 pmol/h/1x10(5) cells.

Agonists for muscle contraction in silkworms were screened by injecting test solutions into the hemolymph of decapitated silkworm larvae. Kainic acid, a glutamate receptor agonist, and D-glutamic acid induced muscle contractions, and D-aspartic acid was partially effective, whereas NMDA and AMPA, representative mammalian glutamate receptor agonists, did not induce contraction. L-Glutamic acid inhibited the kainic acid or D-glutamic acid-induced contraction. Amino acid analysis revealed that 3% of the total glutamic acid in the silkworm hemolymph is D-glutamic acid. These results suggest that d-glutamic acid acts physiologically as an agonist for muscle contraction in silkworms, and that L-glutamic acid functions as an inhibitor.

In previous studies [FEBS Lett. 434 (1998) 231, Arch. Biochem. Biophys. 404 (2002) 92], we demonstrated for the first time that D-aspartate (D-Asp) is synthesized in cultured mammalian cell lines, such as pheochromocytoma 12 (PC12) and its subclone, MPT1. Our current focus is analysis of the dynamics of D-Asp homeostasis in these cells. In this communication, we show that L-glutamate (Glu) and L-Glu transporter substrates in the extracellular space regulate the homeostasis of endogenous D-Asp in MPT1 cells. D-Asp is apparently in dynamic homeostasis, whereby endogenous D-Asp is constantly released into the extracellular space by an undefined mechanism, and continuously and intensively taken up into cells by an L-Glu transporter. Under these conditions, L-Glu and its transporter substrates in the medium may competitively inhibit the uptake of D-Asp via the transporter, resulting in accumulation of the amino acid in the extracellular space. We additionally demonstrate that DL-TBOA, a well-established L-Glu transporter inhibitor, is taken up by the transporter during long time intervals, but not on a short time-scale.

The occurrence and biological significance of the d -amino acids, N-methyl- d -aspartate (NMDA) and N-methyl- l -aspartate (NMLA), have been recently studied in a variety of living organisms. In this study, we established a highly sensitive and reliable fluorometric HPLC system for determining levels of N-methyl-aspartate (NMA). The system comprises fluorescent derivatization of NMA with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and two chromatographic steps: one that separates NMA from other primary amino acids in reverse-phase mode and another that enantioseparates NMDA and NMLA in a normal-phase mode. These two steps are linked by an automated column-switching system. A simple pretreatment step with o-phthalaldehyde to remove primary amino acids that can interfere with sensitivity is also described. The detection limit for NMDA is as low as 5 fmol and the correlation between peak heights and concentrations between 5 fmol and 1 pmol is satisfactory (r=0.999). Following sample preparation and separation using the column-switching HPLC system, more than 80% of NMDA was recovered from rat liver homogenates spiked with NMDA. This method was employed to determine the levels of NMDA in tissues from bivalves and the results obtained were consistent with the values reported previously.

D-Aspartate (D-Asp), a free D-amino acid found in mammals, plays crucial roles in the central nervous, neuroendocrine, and endocrine systems. In mammalian tissues, D-Asp oxidase (DDO) is a degradative enzyme that stereospecifically acts on D-Asp. Asp racemase, a synthetic enzyme that produces D-Asp from L-Asp, has been identified in several lower organisms; however, the biosynthetic pathway of D-Asp in mammals remains to be fully clarified. The aim of this study was to establish a simple, accurate, and sensitive enzymatic method for the determination of Asp racemase activity. Using recombinant Streptococcus thermophilus Asp racemase as a model enzyme, two enzymatic methods for the determination of Asp racemase activity were optimized. In these methods, recombinant human DDO was used to degrade D-Asp formed from L-Asp by the Asp racemase reaction to 2-oxo acid, the amounts of which were then determined using a colorimetric assay. In one method, designated the coupling method, DDO was concomitantly included in the Asp racemase reaction mixture, and the Asp racemase reaction was readily coupled to the D-Asp degradative reaction by DDO during the incubation. In the other method, designated the separating method, an aliquot of the Asp racemase reaction mixture was mixed with DDO after the reaction to determine the amounts of D-Asp produced by Asp racemase. Under optimized conditions, the accuracy and sensitivity of these two methods were examined and compared, both to one another and conventional high-performance liquid chromatography (HPLC). The results presented here suggest that the coupling method is more accurate and sensitive than the other two methods and can be used for the determination of Asp racemase activity. The coupling method may help to advance our current understanding of the biosynthetic pathway of D-Asp in mammals.

We have developed and validated a high-performance liquid chromatography method that uses monolithic silica disk-packed spin columns and a monolithic silica column for the simultaneous determination of N(G)-monomethyl-L-arginine, N(G),N(G)-dimethyl-L-arginine, and N(G),N(G')-dimethyl-L-arginine in human plasma. For solid-phase extraction, our method employs a centrifugal spin column packed with monolithic silica bonded to propyl benzenesulfonic acid as a cation exchanger. After pretreatment, the methylated arginines are converted to fluorescent derivatives with 4-fluoro-7-nitro-2,1,3-benzoxadiazole, and then the derivatives are separated on a monolithic silica column. L-arginine concentration was also determined in diluted samples. Standard calibration curves revealed that the assay was linear in the concentration range 0.2-1.0 μM for methylated arginines and 40-200 μM for L-arginine. Linear regression of the calibration curve yielded equations with correlation coefficients of 0.999 (r(2)). The sensitivity was satisfactory, with a limit of detection ranging from 3.75 to 9.0 fmol for all four compounds. The RSDs were 4.3-4.8% (intraday) and 3.0-6.8% (interday). When this method was applied to samples from six healthy donors, the detected concentrations of N(G)-monomethyl-L-arginine, N(G),N(G)-dimethyl-L-arginine, N(G),N(G')-dimethyl-L-arginine and L-arginine were 0.05 ± 0.01, 0.41 ± 0.07, 0.59 ± 0.11, and 83.8 ± 30.43 μM (n = 6), respectively.

Free D-amino acid content in some archaea was investigated and D-forms of several amino acids were found in them. In the acidothermophilic archaeon, Thermoplasma acidophilum, the proportion of D-aspartate (D-Asp) to total Asp was as high as 39.7%. Crude extracts of Thermoplasma acidophilum had Asp-specific racemase activity that was pyridoxal 5'-phosphate (PLP)-dependent. The relative insensitivity to a SH-modifying reagent distinguished this activity from those of the PLP-independent Asp racemases found in other hyperthermophilic archaea (Matsumoto, M., et al., J. Bacteriol. 181, 6560-6563 1999). Thus, high levels of d-Asp should be produced by a new type(s) of Asp-specific racemase in Thermoplasma acidophilum, although the function of d-Asp in this archaeon remains unknown.