Biochemical characterization of d-aspartate oxidase from Caenorhabditis elegans: its potential use in the determination of free d-glutamate in biological samples.

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.

Distinctive Roles of D-Amino Acids in the Homochiral World: Chirality of Amino Acids Modulates Mammalian Physiology and Pathology.

Living organisms enantioselectively employ L-amino acids as the molecular architecture of protein synthesized in the ribosome. Although L-amino acids are dominantly utilized in most biological processes, accumulating evidence points to the distinctive roles of D-amino acids in non-ribosomal physiology. Among the three domains of life, bacteria have the greatest capacity to produce a wide variety of D-amino acids. In contrast, archaea and eukaryotes are thought generally to synthesize only two kinds of D-amino acids: D-serine and D-aspartate. In mammals, D-serine is critical for neurotransmission as an endogenous coagonist of N-methyl D-aspartate receptors. Additionally, D-aspartate is associated with neurogenesis and endocrine systems. Furthermore, recognition of D-amino acids originating in bacteria is linked to systemic and mucosal innate immunity. Among the roles played by D-amino acids in human pathology, the dysfunction of neurotransmission mediated by D-serine is implicated in psychiatric and neurological disorders. Non-enzymatic conversion of L-aspartate or L-serine residues to their D-configurations is involved in age-associated protein degeneration. Moreover, the measurement of plasma or urinary D-/L-serine or D-/L-aspartate levels may have diagnostic or prognostic value in the treatment of kidney diseases. This review aims to summarize current understanding of D-amino-acid-associated biology with a major focus on mammalian physiology and pathology.

The presence of free d-aspartate in marine macroalgae is restricted to the Sargassaceae family.

The presence of d-aspartate (d-Asp), a biologically rare amino acid, was evaluated in 38 species of marine macroalgae (seaweeds). Despite the ubiquitous presence of free l-Asp, free d-Asp was detected in only 5 species belonging to the Sargassaceae family of class Phaeophyceae (brown algae) but not in any species of the phyla Chlorophyta (green algae) and Rhodophyta (red algae). All other members of Phaeophyceae, including 3 species classified into the section Teretia of Sargassaceae did not contain d-Asp. These results indicate that the presence of free d-Asp in marine macroalgae is restricted only to the Sargassaceae family, excluding the species in the section Teretia.

Prerequisites of Isopeptide Bond Formation in Microcystin Biosynthesis.

The biosynthesis of the potent cyanobacterial hepatotoxin microcystin involves isopeptide bond formation through the carboxylic acid side chains of d-glutamate and ß-methyl d-aspartate. Analysis of the in vitro activation profiles of the two corresponding adenylation domains, McyE-A and McyB-A2 , either in a didomain or a tridomain context with the cognate thiolation domain and the upstream condensation domain revealed that substrate activation of both domains strictly depended on the presence of the condensation domains. We further identified two key amino acids in the binding pockets of both adenylation domains that could serve as a bioinformatic signature of isopeptide bond-forming modules incorporating d-glutamate or d-aspartate. Our findings further contribute to the understanding of the multifaceted role of condensation domains in nonribosomal peptide synthetase assembly lines.

An overview on D-amino acids.

More than half a century ago researchers thought that D-amino acids had a minor function compared to L-enantiomers in biological processes. Many evidences have shown that D-amino acids are present in high concentration in microorganisms, plants, mammals and humans and fulfil specific biological functions. In the brain of mammals, D-serine (D-Ser) acts as a co-agonist of the N-methyl-D-aspartate (NMDA)-type glutamate receptors, responsible for learning, memory and behaviour. D-Ser metabolism is relevant for disorders associated with an altered function of the NMDA receptor, such as schizophrenia, ischemia, epilepsy and neurodegenerative disorders. On the other hand, D-aspartate (D-Asp) is one of the major regulators of adult neurogenesis and plays an important role in the development of endocrine function. D-Asp is present in the neuroendocrine and endocrine tissues and testes, and regulates the synthesis and secretion of hormones and spermatogenesis. Also food proteins contain D-amino acids that are naturally originated or processing-induced under conditions such as high temperatures, acid and alkali treatments and fermentation processes. The presence of D-amino acids in dairy products denotes thermal and alkaline treatments and microbial contamination. Two enzymes are involved in the metabolism of D-amino acids: amino acid racemase in the synthesis and D-amino acid oxidase in the degradation.

Chiral separations for d-amino acid analysis in biological samples.

It is widely accepted that some of the free d-amino acids play important biological role. d-Aspartate and d-serine formed in the central nervous system of higher vertebrates have neurotransmitter/neuromodulator function. Together with d-alanine they are distributed in various tissues and biological fluids. Studying their physiological and pathological significance requires their sensitive and accurate determination in biological samples. The various separation and detection methods used for their analysis are overviewed in the present paper. Our focus is mainly the quantitative performance and the analysis of real biospecimens.

A rapid and sensitive detection of D-Aspartic acid in Crystallin by chiral derivatized liquid chromatography mass spectrometry.

A method for the determination of D-Aspartic acid (D-Asp) and its D/L ratio in peptides and proteins has been developed. This method was carried out with good separation of the D/L chiral peptide pairs by combination of a chiral derivatization and an ADME column separation. Furthermore, a cationic derivatization reagent, DBD-Py-NCS, increased the sensitivity of the ESI-MS/MS detection. To confirm the comprehensive peptide analysis, synthesized α-Crystallin tryptic peptides, which included D-Asp residues, were analyzed. The 5 pairs of D/L-Asp that included peptide diastereomers were well separated. Their peak resolutions were more than 1.5 and the results were reproducible (RSD<0.05, n=5). As an application of this method, we analyzed the α-Crystallin standard and UV irradiated α-Crystallin. After trypsin digestion and DBD-Py-NCS derivatization, the tryptic peptide derivatives were applied to LC-MS/MS. Based on the results of peptide sequence identification, almost all the tryptic peptides of the αA- and αB-Crystallin homologous subunits of α-Crystallin were detected as DBD-Py NCS derivatives. However, there was no D-Asp residue in the standard proteins. In the case of the UV irradiated α-Crystallin, Asp76 and Asp84 in the αA-Crystallin and Asp96 in αB-Crystallin were racemized to D-Asp. These results show that this proposed chiral peptide LC-MS/MS method using chiral derivatization provides a rapid and sensitive analysis for post translational Asp racemization sites in aging proteins.

D-ß-aspartyl residue exhibiting uncommon high resistance to spontaneous peptide bond cleavage.

Although L-amino acids were selected as main constituents of peptides and proteins during chemical evolution, D-aspartyl (Asp) residue is found in a variety of living tissues. In particular, D-ß-Asp is thought to be stable than any other Asp isomers, and this could be a reason for gradual accumulation in abnormal proteins and peptides to modify their structures and functions. It is predicted that D-ß-Asp shows high resistance to biomolecular reactions. For instance, less reactivity of D-ß-Asp is expected to bond cleavage, although such information has not been provided yet. In this work, the spontaneous peptide bond cleavage was compared between Asp isomers, by applying real-time solution-state NMR to eye lens αΑ-crystallin 51-60 fragment, S(51)LFRTVLD(58)SG(60) and αΒ-crystallin 61-67 analog, F(61)D(62)TGLSG(67) consisting of L-α- and D-ß-Asp 58 and 62, respectively. Kinetic analysis showed how tough the uncommon D-ß-Asp residue was against the peptide bond cleavage as compared to natural L-α-Asp. Differences in pKa and conformation between L-α- and D-ß-Asp side chains were plausible factors to determine reactivity of Asp isomers. The present study, for the first time, provides a rationale to explain less reactivity of D-ß-Asp to allow abnormal accumulation.

Possible role of a histidine residue in the substrate specificity of yeast d-aspartate oxidase.

D-Aspartate oxidase (DDO) catalyzes the oxidative deamination of acidic D-amino acids, whereas neutral and basic D-amino acids are substrates of D-amino acid oxidase (DAO). DDO of the yeast Cryptococcus humicola (ChDDO) has much higher substrate specificity to D-aspartate, but the structural features that confer this specificity have not been elucidated. A three-dimensional model of ChDDO suggested that a histidine residue (His56) in the active site might be involved in the unique substrate specificity, possibly through the interaction with the substrate side chain in the active site. His56 mutants with several different amino acid residues (H56A, H56D, H56F, H56K and H56N) exhibited no significant activity toward acidic D-amino acids, but H56A and H56N mutants gained the ability to utilize neutral D-amino acids as substrates, such as D-methionine, D-phenylalanine and D-glutamine, showing the conversion of ChDDO to DAO by these mutations. This conversion was also demonstrated by the sensitivity of these mutants to competitive inhibitors of DAO. These results and kinetic properties of the mutants show that His56 is involved in the substrate specificity of ChDDO and possibly plays a role in the higher substrate specificity toward D-aspartate.

The D-isoAsp-25 variant of histone H2B is highly enriched in active chromatin: potential role in the regulation of gene expression?

Approximately 12 % of histone H2B in mammalian brain contains an unusual D-aspartate residue in its N-terminal tail. Most of this D-aspartate is linked to the C-flanking glycine via an isopeptide bond. To explore the possible significance of these modifications, we generated an antibody to the D-isoaspartyl form of H2B, and used it to assess its levels in H2B associated with "active" vs. "silent" chromatin. We found that the D-isoaspartyl form of H2B appears to be highly enriched in the former. This irreversible modification could serve a novel regulatory function in gene expression.

Peptidyl prolyl isomerase Pin1-inhibitory activity of D-glutamic and D-aspartic acid derivatives bearing a cyclic aliphatic amine moiety.

Pin1 is a peptidyl prolyl isomerase that specifically catalyzes cis-trans isomerization of phosphorylated Thr/Ser-Pro peptide bonds in substrate proteins and peptides. Pin1 is involved in many important cellular processes, including cancer progression, so it is a potential target of cancer therapy. We designed and synthesized a novel series of Pin1 inhibitors based on a glutamic acid or aspartic acid scaffold bearing an aromatic moiety to provide a hydrophobic surface and a cyclic aliphatic amine moiety with affinity for the proline-binding site of Pin1. Glutamic acid derivatives bearing cycloalkylamino and phenylthiazole groups showed potent Pin1-inhibitory activity comparable with that of known inhibitor VER-1. The results indicate that steric interaction of the cyclic alkyl amine moiety with binding site residues plays a key role in enhancing Pin1-inhibitory activity.

The possible involvement of D-amino acids or their metabolites in Arabidopsis cysteine proteinase/cystatin N-dependent proteolytic pathway.

Cysteine proteinases and their inhibitors 'cystatins' play essential roles in plant growth and development. They are involved in various signaling pathways and in the response to wide ranges of biotic and abiotic environmental stresses. To investigate their possible influence from D-amino acids or their metabolism in vivo, Arabidopsis seedlings were allowed to grow under four physicochemically different D-amino acids including D-aspartate, D-serine, D-alanine and D-phenylalanine containing media. The reverse transcription polymerase chain reaction (R T-PCR) analysis of cysteine proteinase and cystatin gene expressions showed that the addition of D-amino acid to the plant growth media considerably induce the expression of proteinase transcript while decrease the expression level of inhibitor gene in the leaf and root tissues of the test plant in overall. Based on the obtained results the potential impact of D-amino acids or their metabolism on the activity of cysteine proteinase/cystatin-dependent proteolytic apparatus as well as their possible cooperation were predicted and discussed in the plant system.

Alpha B- and ßA3-crystallins containing d-aspartic acids exist in a monomeric state.

Crystallin stability and subunit-subunit interaction are essential for eye lens transparency. There are three types of crystallins in lens, designated as α-, ß-, and γ-crystallins. Alpha-crystallin is a hetero-polymer of about 800kDa, consisting of 35-40 subunits of two different αA- and αB-subunits, each of 20kDa. The ß/γ-crystallin superfamily comprises oligomeric ß-crystallin (2-6 subunits) and monomeric γ-crystallin. Since lens proteins have very long half-lives, they undergo numerous post-translational modifications including racemization, isomerization, deamidation, oxidation, glycation, and truncation, which may decrease crystallin solubility and ultimately cause cataract formation. Racemization and isomerization of aspartyl (Asp) residues have been detected only in polymeric α- and oligomeric ß-crystallin, while the situation in monomeric γ-crystallin has not been studied. Here, we investigated the racemization and isomerization of Asp in the γ-crystallin fraction of elderly donors. The results show that Asp residues of γS-, γD- and γC-crystallins were not racemized and isomerized. However, strikingly, we found that a portion of αB-crystallin and ßA3-crystallin moved to the lower molecular weight fraction which is the same size of γ-crystallin. In those fractions, Asp-96 of αB-crystallin and Asp-37 of ßA3-crystallin were highly inverted, which do not occur in the native lens higher molecular weight fraction. Our results indicate the possibility that the inversion of Asp residues may induce dissociation of αB- and ßA3-crystallins from the polymeric and oligomeric states. This is the first report that stereoinversion of amino acids disturbs lens protein assembly in aged human lens.

Discriminating D-amino acid-containing peptide epimers by radical-directed dissociation mass spectrometry.

The presence of a single D-amino acid in a peptide is very difficult to detect. Mass spectrometry-based approaches rely on differences in fragmentation between all L-amino acid-containing peptides and single D-amino acid-containing peptides (which are epimers) for identification. The success of this approach is dependent on the structural sensitivity of the fragmentation method. Recently, experiments have demonstrated that fragmentation initiated by radical chemistry, or radical-directed dissociation (RDD), is particularly sensitive to the structure of the ion being fragmented. Herein, RDD is used to identify the presence of D-serine, D-alanine, or D-aspartic acid in eight biologically relevant peptides. It is demonstrated that chiral disambiguation by RDD is dependent on both the initial radical site and subsequent radical migration. Fortuitously, RDD can be initiated by a variety of different radical precursors which can be associated with the peptide via covalent or noncovalent means, and RDD can be examined in all observable charge states (both positive and negative). This diversity enables numerous initial radical sites and migration pathways to be explored. For all but one of the peptides that were examined, RDD provides significantly better chiral discrimination than CID. Quantitation of peptide epimers by RDD is also described.

Oostatic peptides containing D-amino acids: synthesis, oostatic activity, degradation, accumulation in ovaries and NMR study.

Analogs of the H-Tyr-Asp-Pro-Ala-Pro-OH pentapeptide with D-amino acid residues either in differing or in all of the positions of the sequences were prepared and their oostatic potency was compared with that of the parent pentapeptide. The D-amino acid residue containing analogs exhibited an equal or even higher oostatic effect in the flesh fly Neobellieria bullata than the parent peptide. Contrary to the rapid incorporation of radioactivity from the labeled H-Tyr-Asp-[3H]Pro-Ala-Pro-OH pentapeptide into the ovaries of N. bullata in vitro, the radioactivity incorporation from the labeled pentapeptides with either D-aspartic acid or D-alanine was significantly delayed. As compared to the parent pentapeptide, also the degradation of both the D-amino acid-containing analogs mentioned above proceeded at a significantly lower rate. The decreased intake of radioactivity, the lower degradation and finally also the high oostatic effect may be ascribed to the decreased enzymatic degradation of the peptide bonds neighboring the D-amino acid residues in the corresponding peptides. The introduction of the non-coded D: -amino acids thus enhances the oostatic effect in N. bullata owing to the prolonged half-life of the corresponding pentapeptides, which can thus affect more ovarian cells.

Substrate stereoselectivity of mammalian D-aspartyl endopeptidase.

The formation and accumulation of D-aspartate residue (D-Asp) in proteins caused by oxidative stress leads to dysfunction and/or denaturation of proteins, and is consequently responsible for aging-related misfolding diseases such as cataracts, prion disease, and Alzheimer's disease. We sought to identify that an unknown protease selectively degrades the noxious D-Asp-containing protein, namely D-aspartyl endopeptidase (DAEP), and finally purified it from the inner mitochondrial membrane of mouse liver. In order to analyze the substrate stereoselectivity of DAEP, we synthesized a peptide corresponding to 55-65 (Thr-Val-Leu-Asp-Ser-Gly-Ile-Ser-Glu-Val-Arg) of human αA-crystallin and its corresponding diastereoisomers in which L-α-Asp was replaced with L-ß-, D-α- or D-ß-Asp residue at position 58. Following incubation of that peptide with purified DAEP, it was only degraded at D-α-Asp(58), independent of ATP or NAD. This result indicates that DAEP stereoselectively recognizes and degrades its substrate at the internal D-α-Asp residue. DAEP therefore seems to physiologically serve as the quality control system against the noxious D-Asp-containing protein in the long life span of mammals.

Comparison of molecular dynamics simulation methods for amyloid ß(1-42) monomers containing D-aspartic acid residues for predicting retention times in chromatography.

Molecular dynamics simulations of amyloid ß(1-42) containing D-aspartic acid residues were performed using several continuous solvent models to investigate the usefulness of simulation methods for D-amino acid-containing proteins and peptides. Normal molecular dynamics simulations and replica exchange molecular dynamics simulations, which are one of the generalized-ensemble algorithms, were performed. Because the ß-structure contents of amyloid ß(1-42) peptides obtained by replica exchange molecular dynamics simulations with Onufriev-Bashford-Case generalized Born implicit solvent were qualitatively consistent with experimental data, replica exchange molecular dynamics rather than other methods appeared to be more reasonable for calculations of amyloid ß(1-42) containing D-aspartic acid residues. Computational results revealed that peptides with stereoinversion of Asp23 tend to form ß-sheet structures by themselves, in contrast to the wild-type peptides that form ß-sheet structures only after aggregation. These results are expected to be useful for computational investigations of proteins and peptides such as prediction of retention time of peptides and proteins containing D-aspartic acid residues.

Computational investigation of the substrate recognition mechanism of protein D-aspartyl (L-isoaspartyl) O-methyltransferase by docking and molecular dynamics simulation studies and application to interpret size exclusion chromatography data.

Unusual amino acid residues such as L-ß-aspartyl (Asp), D-α-Asp, and D-ß-Asp have been detected in proteins and peptides such as α-crystallin in the lens and ß-amyloid in the brain. These residues increase with age, and hence they are associated with age-related diseases. The enzyme protein D-aspartyl (L-isoaspartyl) O-methyltransferase (PIMT) can revert these residues back to the normal L-α-Asp residue. PIMT catalyzes transmethylation of S-adenosylmethionine to L-ß-Asp and D-α-Asp residues in proteins and peptides. In this work, the substrate recognition mechanism of PIMT was investigated using docking and molecular dynamics simulation studies. It was shown that the hydrogen bonds of Ser60 and Val214 to the carboxyl group of Asp are important components during substrate recognition by PIMT. In addition, specific hydrogen bonds were observed between the main chains of the substrates and those of Ala61 and Ile212 of PIMT when PIMT recognized L-ß-Asp. Hydrophobic interactions between the (n-1) residue of the substrates and Ile212 and Val214 of PIMT may also have an important effect on substrate binding. Volume changes upon substrate binding were also evaluated in the context of possible application to interpretation of size exclusion chromatography data.

D-Amino acids in aged proteins: analysis and biological relevance.

Homochirality is essential for life. L-Amino acids are exclusively used as substrates for the polymerization and formation of peptides and proteins in living systems. However, d-amino acids, which are enantiomers of L-amino acids, were recently detected in various living organisms in the form of free D-amino acids and D-amino acid residues in peptides and proteins. In particular, D-aspartyl (Asp) residues have been detected in various proteins from diverse tissues of elderly individuals. Here, we describe three important aspects of our research: (i) a method for detecting D-ß-Asp at specific sites in particular proteins, (ii) a likely spontaneous mechanism by which Asp residues in proteins invert and isomerize to the D-ß-form with age under physiological conditions, (iii) a discussion of factors that favor such a reaction.