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Despite being classified as microaerophilic microorganisms, most Campylobacter species can grow anaerobically, using formate or molecular hydrogen (H 2) as electron donors, and various nitrogenous and sulfurous compounds as electron acceptors. Herein, we showed that both l -asparagine (l -Asn) and l -aspartic acid (l -Asp) bolster H 2 -driven anaerobic growth in several Campylobacter species, whereas the d- enantiomer form of both asparagine (d -Asn) and aspartic acid (d -Asp) only increased anaerobic growth in Campylobacter concisus strain 13826 and Campylobacter ureolyticus strain NCTC10941. A gene annotated as racD encoding for a putative d / l -Asp racemase was identified in the genome of both strains. Disruption of racD in Cc 13826 resulted in the inability of the mutant strain to use either d- enantiomer during anaerobic growth. Hence, our results suggest that the racD gene is required for campylobacters to use either d -Asp or d -Asn. The use of d -Asp by various human opportunistic bacterial pathogens, including C. concisus , C. ureolyticus , and also possibly select strains of Campylobacter gracilis , Campylobacter rectus and Campylobacter showae , is significant, because d -Asp is an important signal molecule for both human nervous and neuroendocrine systems. To our knowledge, this is the first report of pathogens scavenging a d- amino acid essential for human health. [ABSTRACT FROM AUTHOR]

d-Aspartate (d-Asp) is a useful compound for a semisynthetic antibiotic and has potentially beneficial effects on humans. Several lactic acid bacteria (LAB) species produce d-Asp as a component of cell wall peptidoglycan. We previously isolated a LAB strain (named strain WDN19) that can extracellularly produce a large amount of d-Asp. Here, we show the factors that contribute to high d-Asp production ability. Strain WDN19 was most closely related to Latilactobacillus curvatus. The d-Asp production ability of strain WDN19 in a rich medium was 13.7-fold higher than that of L. curvatus DSM 20019. A major part of d-Asp was synthesized from l-Asp contained in the medium by aspartate racemase (RacD). During their cultivation, the RacD activity in strain WDN19 was higher than in strain DSM 20019, especially much higher in the early exponential growth phase because of the higher racD transcription and the higher activity of RacD itself of strain WDN19. In a synthetic medium, the extracellular production of d,l-Asp was observed in strain WDN19 but not in strain DSM 20019. The addition of l-asparagine (l-Asn) to the medium increased and gave d,l-Asp production in strains WDN19 and DSM 20019, respectively, suggesting l-Asp synthesis by l-asparaginase (AsnA). The l-Asn uptake ability of the strains was similar, but the AsnA activity in the middle exponential and early stationary growth phases and intracellular d,l-Asp was much higher in strain WDN19. In their genome sequences, only an aspartate aminotransferase gene was found among l-Asp-metabolizing enzymes, except for RacD, but was disrupted in strain WDN19 by transposon insertion. These observations indicated that the high d-Asp production ability of strain WDN19 was mainly based on high RacD and AnsA activities and l-Asp supply. Key points: • Strain WDN19 was suggested to be a strain of Latilactobacillus curvatus. • Extracellular high d-Asp production ability was not a common feature of L. curvatus. • High d-Asp production was due to high RacD and AnsA activities and l-Asp supply. [ABSTRACT FROM AUTHOR]

D-Aspartate, aspartate racemase activity, and D-aspartate oxidase activity were detected in tissues from several types of starfish. Aspartate racemase activity in male testes of Patiria pectinifera was significantly elevated in the summer months of the breeding season compared with spring months. We also compared aspartate racemase activity with the gonad index and found that activity in individuals with a gonad index ≥6% was four-fold higher than that of individuals with a gonad index <6%. The ratio of the D-form of aspartate to total aspartate was approximately 25% in testes with a gonad index <6% and this increased to approximately 40% in testes with a gonad index ≥6%. However, such changes were not observed in female ovaries. Administration of D-aspartate into male starfish caused testicular growth. These results indicate the possible involvement of aspartate racemase and D-aspartate in testicular maturation in echinoderm starfish. Changes in the ratio of D-aspartate to total aspartate and in aspartate racemase activity during maturation of the starfish testis. [ABSTRACT FROM AUTHOR]

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

Amino acids exist in two chiral forms, namely L and D. Although l -amino acids are predominant in vivo , certain limited circumstances have reported the usage of d -amino acids. d -aspartate (Asp), among them, plays crucial physiological roles in living organisms and is biosynthesized from L-Asp by the enzyme named aspartate racemase (AspRase). D-Asp is known to accumulate in large amounts in the nervous system of cephalopods. To understand the function of D-Asp in nervous system in more detail, it is necessary to elucidate its metabolic pathway; however, AspRase gene has not been identified in cephalopods as in the case of mammals. In this study, we successfully identified a novel gene encoding AspRase from the optic ganglion of Japanese common squid Todarodes pacificus. Our discovery of the squid AspRase challenges the prevailing assumption that AspRases across different animals share similar structures. Surprisingly, the squid AspRase is a unique enzyme that differs significantly from known AspRases, being structurally and phylogenetically related to aspartate aminotransferase (AST) and possessing both AspRase and AST activities. The optimum pH and temperature for AspRase activity using L-Asp as a substrate are approximately 7.0 and 20 °C, respectively. Moreover, we have found that AspRase activity is enhanced in the presence of 2-oxoacids. These findings have far-reaching implications for the understanding of enzymology and suggest that yet-to-be-identified mammalian AspRases may also be phylogenetically related to AST, rather than conventional AspRases. Furthermore, our results provide valuable insights into the evolution of the D-Asp biosynthetic pathway. [Display omitted] • AspRase is an enzyme with many unknowns involved in the biosynthesis of D-Asp. • We have cloned the AspRase gene from the Japanese common squid for the first time in cephalopods. • Squid AspRase is a unique and novel enzyme that possesses both AspRase and AST activities. • Squid AspRase is phylogenetically related to AST, distinct from AspRases from other animals. • Our findings will help elucidate the D-Asp biosynthesis pathway not only in cephalopods, but also in mammals. [ABSTRACT FROM AUTHOR]

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

Recently, we cloned and characterized eleven serine and aspartate racemases (SerR and AspR, respectively) from animals. These SerRs and AspRs are not separated by their racemase functions and form a serine/aspartate racemase family cluster based on phylogenetic analysis. Moreover, we have proposed that the AspR-specific triple serine loop region at amino acid positions 150-152 may be responsible for the large AspR activity. In the present study, to test this hypothesis, we prepared and characterized fourteen mutants in this region of animal SerRs and AspRs. The large AspR activity in Acropora and Crassostrea AspR was reduced to <0.04% of wild-type after substitution of the triple serine loop region. Conversely, introducing the triple serine loop region into Acropora, Crassostrea, and Penaeus SerR drastically increased the AspR activity. Those mutants showed similar or higher substrate affinity for aspartate than serine and showed 11-683-fold higher k and 28-351-fold higher k / K values for aspartate than serine racemization. Furthermore, we introduced serine residues in all combinations at position 150-152 in mouse SerR. These mutants revealed that a change in the enzyme function from SerR to AspR can be caused by introduction of Ser151 and Ser152, and addition of the third serine residue at position 150 further enhances the enzyme specificity for aspartate due to a decrease in the serine racemase and serine dehydratase activity. Here, we provide convincing evidence that the AspR gene has evolved from the SerR gene by acquisition of the triple serine loop region. [ABSTRACT FROM AUTHOR]

We succeeded in expressing the aspartate racemase homolog gene from Thermococcus litoralis DSM 5473 in Escherichia coli Rosetta (DE3) and found that the gene encodes aspartate racemase. The aspartate racemase gene consisted of 687 bp and encoded 228 amino acid residues. The purified enzyme showed aspartate racemase activity with a specific activity of 1590 U/mg. The enzyme was a homodimer with a molecular mass of 56 kDa and did not require pyridoxal 5′-phosphate as a coenzyme. The enzyme showed aspartate racemase activity even at 95 °C, and the activation energy of the enzyme was calculated to be 51.8 kJ/mol. The enzyme was highly thermostable, and approximately 50 % of its initial activity remained even after incubation at 90 °C for 11 h. The enzyme showed a maximum activity at a pH of 7.5 and was stable between pH 6.0 and 7.0. The enzyme acted on l-cysteic acid and l-cysteine sulfinic acid in addition to d- and l-aspartic acids, and was strongly inhibited by iodoacetic acid. The site-directed mutagenesis of the enzyme showed that the essential cysteine residues were conserved as Cys83 and Cys194. d-Forms of aspartic acid, serine, alanine, and valine were contained in T. litoralis DSM 5473 cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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

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. [ABSTRACT FROM AUTHOR]

Aspartate racemase catalyzes the interconversion between L-aspartate and D-aspartate and belongs to the PLP-independent racemases. The enzyme from the lactic acid bacterium Lactobacillus sakei NBRC 15893, isolated from kimoto, is considered to be involved in D-aspartate synthesis during the brewing process of Japanese sake at low temperatures. The enzyme was crystallized at 293 K by the sitting-drop vapour-diffusion method using 25%( v/ v) PEG MME 550, 5%( v/ v) 2-propanol. The crystal belonged to space group P3121, with unit-cell parameters a = b = 104.68, c = 97.29 Å, and diffracted to 2.6 Å resolution. Structure determination is under way. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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

The enzyme aspartate racemase from Pyrococcus horikoshii OT3 catalyzes the interconversion between l- and d-Asp. In this work, we employed the hybrid QM/MM approach with the self-consistent charge-density functional tight binding (SCC-DFTB) model to study the catalytic mechanism for the conversion of l-Asp into d-Asp. The molecular dynamics simulation showed that the substrate l-Asp forms an extensive network of interactions with the active-site residues of the aspartate racemase through its side chain carboxylate, ammonium group, and α-carboxylate. The potential of mean force calculations confirmed that the racemization reaction involves two proton transfers (from the α-carbon to Cys194 and from Cys82 to the α-carbon), which occurs in a concerted way, although highly asynchronous. The calculated free energy of activation is 17.5 kcal/mol, which is consistent with the reaction rate measured from experiment. An electrostatic interaction analysis was performed to estimate the key role played by individual residues in stabilizing the transition state. The docking study on the binding of l-Asp and d-Asp to aspartate racemase indicates that this enzyme employs a 'two-base' mechanism not a 'one-base' mechanism. [ABSTRACT FROM AUTHOR]

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

d-Serine serves as a co-agonist of the N-methyld-aspartate receptor in mammalian brains, and its behavior is probably related to neurological disorders such as schizophrenia, Alzheimer’s disease and amyotrophic lateral sclerosis.d-Serine is synthesized by a pyridoxal 5′-phosphate (PLP)-dependent serine racemase. In this minireview, we provide a detailed discussion on the reaction mechanism of the PLP-dependent amino acid racemase on the basis of its 3D structure. We compared the eukaryotic serine racemase with bacterial alanine racemase, the best-studied enzyme among the PLP-dependent amino acid racemases, and thus suggested a putative reaction mechanism for mammaliand-serine synthesis. [ABSTRACT FROM AUTHOR]

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

Bifidobacterium bifidumis a useful probiotic agent exhibiting health-promoting properties and containsd-aspartate as an essential component of the cross-linker moiety in the peptidoglycan. To help understandd-aspartate biosynthesis inB. bifidumNBRC 14252, aspartate racemase, which catalyzes the racemization ofd- andl-aspartate, was purified to homogeneity and characterized. The enzyme was a monomer with a molecular mass of 27 kDa. This is the first report showing the presence of a monomeric aspartate racemase. Its enzymologic properties, such as its lack of cofactor requirement and susceptibility to thiol-modifying reagents in catalysis, were similar to those of the dimeric aspartate racemase fromStreptococcus thermophilus. The monomeric enzyme, however, showed a novel characteristic, namely, that its thermal stability significantly increased in the presence of aspartate, especially thed-enantiomer. The gene encoding the monomeric aspartate racemase was cloned and overexpressed inEscherichia colicells. The nucleotide sequence of theaspartate racemasegene encoded a peptide containing 241 amino acids with a calculated molecular mass of 26 784 Da. The recombinant enzyme was purified to homogeneity and its properties were almost the same as those of theB. bifidumenzyme. [ABSTRACT FROM AUTHOR]

l-Amino acids are predominant in living organisms, but d-amino acids such as d-alanine and d-glutamate also occur in all eubacterial cell walls. Moreover, even mammals contain endogeneous d-amino acids: d-serine functions as a signaling molecule in mammalian brains, and d-aspartate acts as a mediator in endocrine systems. Various other d-amino acids have been demonstrated in archaea, yeasts, fungi, plants, insects, mollusks and other eucaryotic organisms. These d-amino acids are mostly endogenous and produced in most cases by racemization from their corresponding antipodes by the action of racemases. Therefore, amino acid racemases play a central role in d-amino acid metabolism. Most amino acid racemases require pyridoxal 5′-phosphate (PLP) as a coenzyme, but several others require no coenzymes. Recently, the structures and functions of these two classes of amino acid racemases were clarified on a molecular basis. We here describe recent advances in studies of the functions and mechanisms of PLP-dependent and -independent amino acid racemases. [Copyright &y& Elsevier]

A novel optically active thiol compound, N-(tert-butylthiocarbamoyl)-l-cysteine ethyl ester (BTCC), is synthesized as a chiral derivatization reagent. This compound and o-phthalaldehyde react with amino acid enantiomers to produce fluorescent diastereomers that are readily separable on a reverse-phase column by HPLC. Enantioseparation of acidic amino acids in particular is markedly improved using BTCC. In this study, the HPLC method for enantioseparation with the novel compound is applied to the aspartate (Asp) racemase assay. Derivatized d-Asp is eluted before the l-Asp derivative. Consequently, a small amount of d-Asp produced by the activity of racemase on a large quantity of l-Asp substrate may be quantified accurately, even at very low activity. Since the derivatization reaction proceeds rapidly at room temperature, a fully automated system is established for derivatization and sample injection. The automated method is practical and successfully applied to the archaeal Asp racemase assay. We presume that the procedure is additionally applicable to the enantioseparation of other amino acids, amino alcohols, and catecholamines. [Copyright &y& Elsevier]

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

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

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