Yasuaki Saitoh

Identification of Novel d-Amino Acid Oxidase Inhibitors by in Silico Screening and Their Functional Characterization in Vitro

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.

Comparative characterization of three D-aspartate oxidases and one D-amino acid oxidase from Caenorhabditis elegans.

Previously, we cloned cDNAs for four Caenorhabditis elegans genes (F20 Hp, C47Ap, F18Ep, and Y69Ap genes) that were annotated in the database as encoding D‐amino acid oxidase (DAO) or D‐aspartate oxidase (DDO) proteins. These genes were expressed in Escherichia coli, and the recombinant C47Ap and F18Ep were shown to have functional DDO activities, while Y69Ap had functional DAO activity. In this study, we improved the E. coli culture conditions for the production of recombinant F20 Hp and, following purification of the protein, revealed that it has functional DDO activity. The kinetic properties of recombinant C47Ap (DDO‐1), F18Ep (DDO‐2), F20 Hp (DDO‐3), and Y69Ap (DAO) were also determined and compared with recombinant human DDO and DAO. In contrast to the low catalytic efficiency of human DDO for D‐Glu, all three C. elegans DDOs showed higher catalytic efficiencies for D‐Glu than D‐Asp or N‐methyl‐D‐Asp. The catalytic efficiency of C. elegans DAO for D‐Ser was substantially lower than that of human DAO, while the C. elegans DAO was more efficient at deamination of basic D‐amino acids (D‐Arg and D‐His) than human DAO. Collectively, our results indicate that C. elegans contains at least three genes that encode functional DDOs, and one gene encoding a functional DAO, and that these enzymes have different and distinctive properties.

Biosynthesis of D-aspartate in mammals: the rat and human homologs of mouse aspartate racemase are not responsible for the biosynthesis of D-aspartate.

Abstractd-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.

Spatiotemporal Localization of d-Amino Acid Oxidase and d-Aspartate Oxidases during Development in Caenorhabditis elegans

ABSTRACT Recent investigations have shown that a variety of d-amino acids are present in living organisms and that they possibly play important roles in physiological functions in the body. d-Amino acid oxidase (DAO) and d-aspartate oxidase (DDO) are degradative enzymes stereospecific for d-amino acids. They have been identified in various organisms, including mammals and the nematode Caenorhabditis elegans, although the significance of these enzymes and the relevant functions of d-amino acids remain to be elucidated. In this study, we investigated the spatiotemporal localization of C. elegans DAO and DDOs (DDO-1, DDO-2, and DDO-3) and measured the levels of several d- and l-amino acids in wild-type C. elegans and four mutants in which each gene for DAO and the DDOs was partially deleted and thereby inactivated. Furthermore, several phenotypes of these mutant strains were characterized. The results reported in this study indicate that C. elegans DAO and DDOs are involved in egg-laying events and the early development of C. elegans. In particular, DDOs appear to play important roles in the development and maturation of germ cells. This work provides novel and useful insights into the physiological functions of these enzymes and d-amino acids in multicellular organisms.

Thiolactomycin inhibits D-aspartate oxidase: a novel approach to probing the active site environment.

D-Aspartate oxidase (DDO) and D-amino acid oxidase (DAO) are flavin adenine dinucleotide (FAD)-containing flavoproteins that catalyze the oxidative deamination of D-amino acids. While several functionally and structurally important amino acid residues have been identified in the DAO protein, little is known about the structure-function relationships of DDO. In the search for a potent DDO inhibitor as a novel tool for investigating its structure-function relationships, a large number of biologically active compounds of microbial origin were screened for their ability to inhibit the enzymatic activity of mouse DDO. We discovered several compounds that inhibited the activity of mouse DDO, and one of the compounds identified, thiolactomycin (TLM), was then characterized and evaluated as a novel DDO inhibitor. TLM reversibly inhibited the activity of mouse DDO with a mixed type of inhibition more efficiently than meso-tartrate and malonate, known competitive inhibitors of mammalian DDOs. The selectivity of TLM was investigated using various DDOs and DAOs, and it was found that TLM inhibits not only DDO, but also DAO. Further experiments with apoenzymes of DDO and DAO revealed that TLM is most likely to inhibit the activities of DDO and DAO by competition with both the substrate and the coenzyme, FAD. Structural models of mouse DDO/TLM complexes supported this finding. The binding mode of TLM to DDO was validated further by site-directed mutagenesis of an active site residue, Arg-237. Collectively, our findings show that TLM is a novel, active site-directed DDO inhibitor that will be useful for elucidating the molecular details of the active site environment of DDO.

Characterization of the Enzymatic and Structural Properties of Human D-Aspartate Oxidase and Comparison with Those of the Rat and Mouse Enzymes

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.

Characterization of a homologue of mammalian serine racemase from Caenorhabditis elegans: the enzyme is not critical for the metabolism of serine in vivo

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.

Role of the active site residues arginine-216 and arginine-237 in the substrate specificity of mammalian D-aspartate oxidase.

Abstractd-Aspartate oxidase (DDO) and d-amino acid oxidase (DAO) are flavin adenine dinucleotide-containing flavoproteins that catalyze the oxidative deamination of d-amino acids. Unlike DAO, which acts on several neutral and basic d-amino acids, DDO is highly specific for acidic d-amino acids. Based on molecular modeling and simulated annealing docking analyses, a recombinant mouse DDO carrying two substitutions (Arg-216 to Leu and Arg-237 to Tyr) was generated (R216L-R237Y variant). This variant and two previously constructed single-point mutants of mouse DDO (R216L and R237Y variants) were characterized to investigate the role of Arg-216 and Arg-237 in the substrate specificity of mouse DDO. The R216L-R237Y and R216L variants acquired a broad specificity for several neutral and basic d-amino acids, and showed a considerable decrease in activity against acidic d-amino acids. The R237Y variant, however, did not show any additional specificity for neutral or basic d-amino acids and its activity against acidic d-amino acids was greatly reduced. The kinetic properties of these variants indicated that the Arg-216 residue is important for the catalytic activity and substrate specificity of mouse DDO. However, Arg-237 is, apparently, only marginally involved in substrate recognition, but is important for catalytic activity. Notably, the substrate specificity of the R216L-R237Y variant differed significantly from that of the R216L variant, suggesting that Arg-237 has subsidiary effects on substrate specificity. Additional experiments using several DDO and DAO inhibitors also suggested the involvement of Arg-216 in the substrate specificity and catalytic activity of mouse DDO and that Arg-237 is possibly involved in substrate recognition by this enzyme. Collectively, these results indicate that Arg-216 and Arg-237 play crucial and subsidiary role(s), respectively, in the substrate specificity of mouse DDO.

The antiviral drug acyclovir is a slow-binding inhibitor of (D)-amino acid oxidase.

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.

Identification of Novel d-Aspartate Oxidase Inhibitors by in Silico Screening and Their Functional and Structural Characterization in Vitro

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.