Feasible peripheral biomarker for Alzheimer's disease (AD) is lacking. Dysregulation of N-methyl-D-aspartate (NMDA) receptor is implicated in the pathogenesis of AD. D-amino acid oxidase (DAO) and amino acids can regulate the NMDA receptor function. This study aimed to examine whether peripheral DAO and amino acids levels are characteristic of age-related cognitive decline. We enrolled 397 individuals (including amnestic mild cognitive impairment (MCI), mild AD, moderate to severe AD, and healthy elderly). DAO levels in the serum were measured using ELISA. Amino acids levels in serum were measured by high performance liquid chromatography. Severity of the cognitive deficits in subjects was assessed using Clinical Dementia Rating Scale (CDR). The DAO levels increased with the severity of the cognitive deficits. DAO levels were significantly associated with D-glutamate and D-serine levels. The Receiver Operating Characteristics analysis of DAO levels for AD patients vs. healthy controls determined the optimal cutoff value, 30.10, with high sensitivity (0.842) and specificity (0.889) (area under curve = 0.928). This is the first study indicating that the peripheral DAO levels may increase with age-related cognitive decline. The finding supports the hypofunction of NMDA receptor hypothesis in AD. Whether DAO could serve as a potential surrogate biomarker needs further studies.

The flavoenzyme D-amino acid oxidase (DAAO) is deputed to the degradation of D-enantiomers of amino acids. DAAO plays various relevant physiological roles in different organisms and tissues. Thus, it has been recently suggested that the goblet cells of the mucosal epithelia secrete into the lumen of intestine, a processed and active form of DAAO that uses the intestinal D-amino acids to generate hydrogen peroxide (H2O2), an immune messenger that helps fighting gut pathogens, and by doing so controls the homeostasis of gut microbiota. Here, we show that the DAAO form lacking the 1-16 amino acid residues (the putative secretion signal) is unstable and inactive, and that DAAO is present in the epithelial layer and the mucosa of mouse gut, where it is largely proteolyzed. In silico predicted DAAO-derived antimicrobial peptides show activity against various Gram-positive and Gram-negative bacteria but not on Lactobacilli species, which represent the commensal microbiota. Peptidomic analysis reveals the presence of such peptides in the mucosal fraction. Collectively, we identify a novel mechanism for gut microbiota selection implying DAAO-derived antimicrobial peptides which are generated by intestinal proteases and that are secreted in the gut lumen. In conclusion, we herein report an additional, ancillary role for mammalian DAAO, unrelated to its enzymatic activity.

In the brain, D-amino acid oxidase (DAO/DAAO) mainly oxidizes D-serine, a co-agonist of the N-methyl-D-aspartate (NMDA) receptors. Thus, DAO can regulate the function of NMDA receptors via D-serine breakdown. Furthermore, DAO activator (DAOA)/G72 has been reported as both DAOA and repressor. The co-expression of DAO and DAOA genes and proteins in the human brain is not yet elucidated. The aim of this study was to understand the regional and age span distribution of DAO and DAOA (mRNA and protein) in a concomitant manner. We determined DAO and DAOA mRNA and protein expression across six brain regions in normal human post-mortem brain samples (16 weeks of gestation to 91 years) using quantitative real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. We found higher expression of DAO mRNA in the cerebellum, whereas lower expression of DAO protein in the cerebellum compared to the other brain regions studied, which suggests post-transcriptional regulation. We detected DAOA protein but not DAOA mRNA in all brain regions studied, suggesting a tightly regulated expression. To understand this regulation at the transcriptional level, we analyzed DNA methylation levels at DAO and DAOA CpG sites in the cerebellum and frontal cortex of control human post-mortem brain obtained from Gene Expression Omnibus datasets. Indeed, DAO and DAOA CpG sites in the cerebellum were significantly more methylated than those in the frontal cortex. While investigating lifespan effects, we found that DAO mRNA levels were positively correlated with age <2 years in the cerebellum and amygdala. We also detected a significant positive correlation (controlled for age) between DAO and DAOA protein in all of the brain regions studied except for the frontal cortex. In summary, DAO and DAOA expression in the human brain are both age and brain region dependent.

Neutrophils engulf and kill bacteria using oxidative and nonoxidative mechanisms. Despite robust antimicrobial activity, neutrophils are impaired in directing Salmonella clearance and harbor viable intracellular bacteria during early stages of infection that can subsequently escape to more-permissive cell types. The mechanisms accounting for this immune impairment are not understood. We report that Salmonella limits exposure to oxidative damage elicited by D-amino acid oxidase (DAO) in neutrophils by expressing an ABC importer specific for D-alanine, a DAO substrate found in peptidoglycan stem peptides. A Salmonella dalS mutant defective for D-alanine import was more susceptible to killing by DAO through exposure to greater oxidative stress during infection. This fitness defect was reversed by selective depletion of neutrophils or by inhibition of DAO in vivo with a small-molecule inhibitor. DalS-mediated subversion of neutrophil DAO is a novel host-pathogen interaction that enhances Salmonella survival during systemic infection.

Food quality is also related to safety and prevention of spoilage. Biological antimicrobial agents represent suitable alternatives to clinical preservatives in food industry to increase both safety and stability of aliments. Here, we focused on the enzyme D-amino acid oxidase (DAAO) from the yeast Rhodotorula gracilis, a well-studied protein for biotechnological use based on its stability, high activity, and easy recombinant production. DAAO catalyzes the O2-dependent oxidative deamination of D-enantiomer of amino acids generating α-keto acids, ammonia, and hydrogen peroxide. DAAO shows antibacterial activity on both Gram-positive and Gram-negative bacteria in the presence of D-alanine when tested on plates and reduced by half their growth when tested on liquid cultures. Control experiments performed with alternative amino acid-specific flavoenzymes (able or not to generate H2O2 acting on amino acids), a DAAO inactive variant, catalase (H2O2 scavenger), and L-amino acids instead of D-alanine identified H2O2 as the antibacterial agent. DAAO showed a good ability to decrease the bacterial growth on various food stuffs: e.g., 10-fold less colonies were formed on grated cheese incubated for 16 h at 37°C when a tiny amount (0.01 mg corresponding to 1.2 units) of DAAO was added. No exogenous D-amino acids were added since DAAO used the ones naturally occurring or the ones generated during ripening. Notably, simultaneously to H2O2 generation, DAAO also acts as O2-scavenger thus further hampering food deterioration.

D-amino acid oxidase (DAAO) degrades D-amino acids to produce α-ketoacids, hydrogen peroxide and ammonia. DAAO has often been investigated and engineered for industrial and clinical applications. We combined information from literature with a detailed analysis of the structure to engineer mammalian DAAOs. The structural analysis was complemented with molecular dynamics simulations to characterize solvent accessibility and product release mechanisms. We identified non-obvious residues located on the loops on the border between the active site and the secondary binding pocket essential for pig and human DAAO substrate specificity and activity. We engineered DAAOs by mutating such critical residues and characterised the biochemical activity of the resulting variants. The results highlight the importance of the selected residues in modulating substrate specificity, product egress and enzyme activity, suggesting further steps of DAAO re-engineering towards desired clinical and industrial applications.

The schizophrenia-related protein G72 plays a unique role in the regulation of D-amino acid oxidase (DAO) in great apes. Several psychiatric diseases, including schizophrenia and bipolar disorder, are linked to overexpression of DAO and G72. Whether G72 plays a positive or negative regulatory role in DAO activity, however, has been controversial. Exploring the molecular basis of the relationship between G72 and DAO is thus important to understand how G72 regulates DAO activity. We performed yeast two-hybrid experiments and determined enzymatic activity to identify potential sites in G72 involved in binding DAO. Our results demonstrate that residues 123-153 and 138-153 in the long isoform of G72 bind to DAO and enhance its activity by 22% and 32%, respectively. A docking exercise indicated that these G72 peptides can interact with loops in DAO that abut the entrance of the tunnel that substrate and cofactor must traverse to reach the active site. We propose that a unique gating mechanism underlies the ability of G72 to increase the activity of DAO. Because upregulation of DAO activity decreases d-serine levels, which may lead to psychiatric abnormalities, our results suggest a molecular mechanism involving interaction between DAO and the C-terminal region of G72 that can regulate N-methyl-d-aspartate receptor-mediated neurotransmission.

In the central nervous system, the flavoprotein D-amino acid oxidase is responsible for catabolizing D-serine, the main endogenous coagonist of N-methyl-D-aspartate receptor. Dysregulation of D-serine brain levels in humans has been associated with neurodegenerative and psychiatric disorders. This D-amino acid is synthesized by the enzyme serine racemase, starting from the corresponding L-enantiomer, and degraded by both serine racemase (via an elimination reaction) and the flavoenzyme D-amino acid oxidase. To shed light on the role of human D-amino acid oxidase (hDAAO) in D-serine metabolism, the structural/functional relationships of this enzyme have been investigated in depth and several strategies aimed at controlling the enzymatic activity have been identified. Here, we focused on the effect of post-translational modifications: by using a combination of structural analyses, biochemical methods, and cellular studies, we investigated whether hDAAO is subjected to nitrosylation, sulfhydration, and phosphorylation. hDAAO is S-nitrosylated and this negatively affects its activity. In contrast, the hydrogen sulfide donor NaHS seems to alter the enzyme conformation, stabilizing a species with higher affinity for the flavin adenine dinucleotide cofactor and thus positively affecting enzymatic activity. Moreover, hDAAO is phosphorylated in cerebellum; however, the protein kinase involved is still unknown. Taken together, these findings indicate that D-serine levels can be also modulated by post-translational modifications of hDAAO as also known for the D-serine synthetic enzyme serine racemase.

To elucidate the molecular mechanisms underlying switching from asexual to sexual reproduction, namely sexual induction, we developed an assay system for sexual induction in the hermaphroditic planarian species Dugesia ryukyuensis. Ovarian development is the initial and essential step in sexual induction, and it is followed by the formation of other reproductive organs, including the testes. Here, we report a function of a planarian D-amino acid oxidase, Dr-DAO, in the control of ovarian development in planarians. Asexual worms showed significantly more widespread expression of Dr-DAO in the parenchymal space than did sexual worms. Inhibition of Dr-DAO by RNAi caused the formation of immature ovaries. In addition, we found that feeding asexual worms 5 specific D-amino acids could induce the formation of immature ovaries that are similar to those observed in Dr-DAO knockdown worms, suggesting that Dr-DAO inhibits the formation of immature ovaries by degrading these D-amino acids. Following sexual induction, Dr-DAO expression was observed in the ovaries. The knockdown of Dr-DAO during sexual induction delayed the maturation of the other reproductive organs, as well as ovary. These findings suggest that Dr-DAO acts to promote ovarian maturation and that complete sexual induction depends on the production of mature ovaries. We propose that Dr-DAO produced in somatic cells prevents the onset of sexual induction in the asexual state, and then after sexual induction, the female germ cells specifically produce Dr-DAO to induce full maturation. Therefore, Dr-DAO produced in somatic and female germline cells may play different roles in sexual induction.

Diabetic retinopathy is characterised by retinal neurodegeneration and retinal vascular abnormalities, affecting one third of diabetic patients with disease duration of more than 10 years. Accumulated evidence suggests that serine racemase (SR) and D-serine are correlated with the pathogenesis of diabetic retinopathy and the deletion of the Srr gene reverses neurovascular pathologies in diabetic mice. Since D-serine content is balanced by SR synthesis and D-amino acid oxidase (DAAO) degradation, we examined the roles of DAAO in diabetic retinopathy and further explored relevant therapy.

Application of nanoenzymes, based on D-amino acid oxidase (DAAO) conjugated to magnetic nanoparticles (NPs), as anticancer system requires improvement of the synthesis protocol and in vivo distribution evaluation.

Dysfunction of D-amino acid oxidase (DAO) and DAO activator (DAOA)/G72 genes have been linked to neuropsychiatric disorders. The glutamate hypothesis of schizophrenia has proposed that increased DAO activity leads to decreased D-serine, which subsequently may lead to N-methyl-D-aspartate (NMDA) receptor hypofunction. It has been shown that DAOA binds to DAO and increases its activity. However, there are also studies showing DAOA decreases DAO activity. Thus, the effect of DAOA on DAO is controversial. We aimed to understand the effect of DAOA on DAO activity in neuron-like (SH-SY5Y), astrocyte-like (1321N1) and kidney-like (HEK293) human cell lines. DAO activity was measured based on the release of hydrogen peroxide and its interaction with Amplex Red reagent. We found that DAOA increases DAO activity only in HEK293 cells, but has no effect on DAO activity in SH-SY5Y and 1321N1 cells. This might be because of different signaling pathways, or due to lower DAO and DAOA expression in SH-SY5Y and 1321N1 cells compared to HEK293 cells, but also due to different compartmentalization of the proteins. The lower DAO and DAOA expression in neuron-like SH-SY5Y and astrocyte-like 1321N1 cells might be due to tightly regulated expression, as previously reported in the human post-mortem brain. Our simulation experiments to demonstrate the interaction between DAOA and human DAO (hDAO) showed that hDAO holoenzyme [hDAO with flavine adenine dinucleotide (FAD)] becomes more flexible and misfolded in the presence of DAOA, whereas DAOA had no effect on hDAO apoprotein (hDAO without FAD), which indicate that DAOA inactivates hDAO holoenzyme. Furthermore, patch-clamp analysis demonstrated no effect of DAOA on NMDA receptor activity in NR1/NR2A HEK293 cells. In summary, the interaction between DAO and DAOA seems to be cell type and its biochemical characteristics dependent which still needs to be elucidated.

A series of thiophene-2-carboxylic acids and thiophene-3-carboxylic acids were identified as a new class of DAO inhibitors. Structure-activity relationship (SAR) studies revealed that small substituents are well-tolerated on the thiophene ring of both the 2-carboxylic acid and 3-carboxylic acid scaffolds. Crystal structures of human DAO in complex with potent thiophene carboxylic acids revealed that Tyr224 was tightly stacked with the thiophene ring of the inhibitors, resulting in the disappearance of the secondary pocket observed with other DAO inhibitors. Molecular dynamics simulations of the complex revealed that Tyr224 preferred the stacked conformation irrespective of whether Tyr224 was stacked or not in the initial state of the simulations. MM/GBSA indicated a substantial hydrophobic interaction between Tyr244 and the thiophene-based inhibitor. In addition, the active site was tightly closed with an extensive network of hydrogen bonds including those from Tyr224 in the stacked conformation. The introduction of a large branched side chain to the thiophene ring markedly decreased potency. These results are in marked contrast to other DAO inhibitors that can gain potency with a branched side chain extending to the secondary pocket due to Tyr224 repositioning. These insights should be of particular importance in future efforts to optimize DAO inhibitors with novel scaffolds.

d-amino acid oxidase (DAO) is a flavin adenine dinucleotide (FAD)-dependent oxidase metabolizing neutral and polar d-amino acids. Unlike l-amino acids, the amounts of d-amino acids in mammalian tissues are extremely low, and therefore, little has been investigated regarding the physiological role of DAO. We have recently identified DAO to be up-regulated in cellular senescence, a permanent cell cycle arrest induced by various stresses, such as persistent DNA damage and oxidative stress. Because DAO produces reactive oxygen species (ROS) as byproducts of substrate oxidation and the accumulation of ROS mediates the senescence induction, we explored the relationship between DAO and senescence. We found that inhibition of DAO impaired senescence induced by DNA damage, and ectopic expression of wild-type DAO, but not enzymatically inactive mutant, enhanced it in an ROS-dependent manner. Furthermore, addition of d-amino acids and riboflavin, a metabolic precursor of FAD, to the medium potentiated the senescence-promoting effect of DAO. These results indicate that DAO promotes senescence through the enzymatic ROS generation, and its activity is regulated by the availability of its substrate and coenzyme.

d-Serine plays an important role in modulating N-methyl-d-aspartate receptor (NMDAR) neurotransmission in the mammalian brain by binding to the receptor's glycine modulatory site (GMS). The cytosolic enzyme serine racemase (SR) converts L-serine to d-serine, while the peroxisomal enzyme d-amino acid oxidase (DAAO) catalyzes the breakdown of d-serine. Although it is important to understand how the activities of SR and DAAO regulate d-serine levels, very little is known about the mechanisms that regulate the expression of SR and DAAO. In this study, we investigated whether the different centrally active drugs affect the expression of SR and DAAO in adult mouse brain. We found that the NMDAR antagonist, MK801, and cocaine, psychotropic drugs that both augment glutamate release, reduce the expression of SR and DAAO. This regulation is brain region selective, and in the case of cocaine, is reversed in part byα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX). However, d-serine and antipsychotics do not regulate SR and DAAO protein levels. In a genetic model of SR disruption, we found that DAAO expression was unaltered in SR conditional knockout mice, in which tissue d-serine content remains fairly stable despite marked reduction in SR expression. This study reveals a new mechanism by which AMPAR activity could regulate NMDAR function via d-serine availability.

It has been proposed that D-amino acid oxidase (DAO) plays an essential role in degrading D-serine, an endogenous coagonist of N-methyl-D-aspartate (NMDA) glutamate receptors. DAO shows genetic association with amyotrophic lateral sclerosis (ALS) and schizophrenia, in whose pathophysiology aberrant metabolism of D-serine is implicated. Although the pathology of both essentially involves the forebrain, in rodents, enzymatic activity of DAO is hindbrain-shifted and absent in the region. Here, we show activity-based distribution of DAO in the central nervous system (CNS) of humans compared with that of mice. DAO activity in humans was generally higher than that in mice. In the human forebrain, DAO activity was distributed in the subcortical white matter and the posterior limb of internal capsule, while it was almost undetectable in those areas in mice. In the lower brain centers, DAO activity was detected in the gray and white matters in a coordinated fashion in both humans and mice. In humans, DAO activity was prominent along the corticospinal tract, rubrospinal tract, nigrostriatal system, ponto-/olivo-cerebellar fibers, and in the anterolateral system. In contrast, in mice, the reticulospinal tract and ponto-/olivo-cerebellar fibers were the major pathways showing strong DAO activity. In the human corticospinal tract, activity-based staining of DAO did not merge with a motoneuronal marker, but colocalized mostly with excitatory amino acid transporter 2 and in part with GFAP, suggesting that DAO activity-positive cells are astrocytes seen mainly in the motor pathway. These findings establish the distribution of DAO activity in cerebral white matter and the motor system in humans, providing evidence to support the involvement of DAO in schizophrenia and ALS. Our results raise further questions about the regulation of D-serine in DAO-rich regions as well as the physiological/pathological roles of DAO in white matter astrocytes.

The NMDAR (N-methyl-D-aspartate receptor) is a central regulator of synaptic plasticity and learning and memory. hDAAO (human D-amino acid oxidase) indirectly reduces NMDAR activity by degrading the NMDAR co-agonist D-serine. Since NMDAR hypofunction is thought to be a foundational defect in schizophrenia, hDAAO inhibitors have potential as treatments for schizophrenia and other nervous system disorders. Here, we sought to identify novel chemicals that inhibit hDAAO activity. We used computational tools to design a focused, purchasable library of compounds. After screening this library for hDAAO inhibition, we identified the structurally novel compound, 'compound 2' [3-(7-hydroxy-2-oxo-4-phenyl-2H-chromen-6-yl)propanoic acid], which displayed low nM hDAAO inhibitory potency (Ki=7 nM). Although the library was expected to enrich for compounds that were competitive for both D-serine and FAD, compound 2 actually was FAD uncompetitive, much like canonical hDAAO inhibitors such as benzoic acid. Compound 2 and an analog were independently co-crystalized with hDAAO. These compounds stabilized a novel conformation of hDAAO in which the active-site lid was in an open position. These results confirm previous hypotheses regarding active-site lid flexibility of mammalian D-amino acid oxidases and could assist in the design of the next generation of hDAAO inhibitors.

The D-amino acid oxidase activator (DAOA) protein regulates the function of D-amino oxidase (DAO), an enzyme that catalyzes the oxidative deamination of D-3,4-dihydroxyphenylalanine (D-DOPA) and D-serine. D-DOPA is converted to L-3,4-DOPA, a precursor of dopamine, whereas D-serine participates in glutamatergic transmission. We hypothesized that DAOA polymorphisms are associated with dopamine, serotonin and noradrenaline turnover in the human brain. Four single-nucleotide polymorphisms, previously reported to be associated with schizophrenia, were genotyped. Cerebrospinal fluid (CSF) samples were drawn by lumbar puncture, and the concentrations of the major dopamine metabolite homovanillic acid (HVA), the major serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) and the major noradrenaline metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG) were measured. Two of the investigated polymorphisms, rs3918342 and rs1421292, were significantly associated with CSF HVA concentrations. Rs3918342 was found to be nominally associated with CSF 5-HIAA concentrations. None of the polymorphisms were significantly associated with MHPG concentrations. Our results indicate that DAOA gene variation affects dopamine turnover in healthy individuals, suggesting that disturbed dopamine turnover is a possible mechanism behind the observed associations between genetic variation in DAOA and behavioral phenotypes in humans.

Amyotrophic lateral sclerosis is a neuromuscular disease characterized by selective loss of motor neurons leading to fatal paralysis. We previously reported a coding mutation in D-amino acid oxidase (R199W DAO) associated with familial amyotrophic lateral sclerosis. DAO metabolizes D-serine, a co-agonist at the N-methyl-D-aspartic acid receptor. We investigated the mechanisms mediating the pathogenic effects of R199W DAO on motor neuron survival and showed that expression of glial R199W DAO is sufficient to induce apoptosis in cocultured motor neurons and this is sensitive to 5,7-dichloro-4-hydroxyquinoline-2-carboxylic acid, an N-methyl-d-aspartic acid receptor antagonist selective for the D-serine/glycine site. R199W DAO activates protein aggregation and autophagy, which is also sensitive to this antagonist. Using immunocytochemistry, we showed that D-serine and DAO were abundant in spinal cord motor neurons and depleted in amyotrophic lateral sclerosis. In summary, the toxic effects of R199W DAO on motor neurons can be mediated directly by expression in motor neurons or by astrocytes in coculture, R199W DAO promotes autophagy and its pathogenic effects are at least in part mediated via the N-methyl-d-aspartic acid receptor.

D-Amino acid oxidase (DAAO) is a valuable flavoenzyme capable of being used in various practical applications, such as in determining D-amino acids and producing a material for semisynthetic cephalosporins, requiring higher thermal stability, higher catalytic activity, and broad substrate specificity. In this study, we isolated the thermophilic fungus Rasamsonia emersonii strain YA, which can grow on several D-amino acids as the sole nitrogen source, from a compost and characterized DAAO (ReDAAO) of the fungus. ReDAAO expressed in Escherichia coli exhibited significant oxidase activity against various neutral and basic D-amino acids, in particular hydrophobic D-amino acids. In addition, the enzyme also significantly acted on cephalosporin C, a starting material for semisynthetic antibiotics, and D-Glu, a general substrate for D-aspartate oxidase but not for DAAO, showing its unique and practically useful substrate specificity. The apparent kcat and Km values of the enzyme toward good substrates were comparable to those of higher catalytic fungal DAAOs, and the thermal stability (T50 value of ~60 °C) was comparable to that of a thermophilic bacterial DAAO and significantly higher than that of other eukaryotic DAAOs. These results highlight the great potential of ReDAAO for use in practical applications.