L-serine deficiency: on the properties of the Asn133Ser variant of human phosphoserine phosphatase.

The non-essential amino acid L-serine is involved in a number of metabolic pathways and in the brain its level is largely due to the biosynthesis from the glycolytic intermediate D-3-phosphoglycerate by the phosphorylated pathway (PP). This cytosolic pathway is made by three enzymes proposed to generate a reversible metabolon named the "serinosome". Phosphoserine phosphatase (PSP) catalyses the last and irreversible step, representing the driving force pushing L-serine synthesis. Genetic defects of the PP enzymes result in strong neurological phenotypes. Recently, we identified the homozygous missense variant [NM_004577.4: c.398A > G p.(Asn133Ser)] in the PSPH, the PSP encoding gene, in two siblings with a neurodevelopmental syndrome and a myelopathy. The recombinant Asn133Ser enzyme does not show significant alterations in protein conformation and dimeric oligomerization state, as well as in enzymatic activity and functionality of the reconstructed PP. However, the Asn133Ser variant is less stable than wild-type PSP, a feature also apparent at cellular level. Studies on patients' fibroblasts also highlight a strong decrease in the level of the enzymes of the PP, a partial nuclear and perinuclear localization of variant PSP and a stronger perinuclear aggregates formation. We propose that these alterations contribute to the formation of a dysfunctional serinosome and thus to the observed reduction of L-serine, glycine and D-serine levels (the latter playing a crucial role in modulating NMDA receptors). The characterization of patients harbouring the Asn133Ser PSP substitution allows to go deep into the molecular mechanisms related to L-serine deficit and to suggest treatments to cope with the observed amino acids alterations.

Biochemical and cellular studies of three human 3-phosphoglycerate dehydrogenase variants responsible for pathological reduced L-serine levels.

In the brain, the non-essential amino acid L-serine is produced through the phosphorylated pathway (PP) starting from the glycolytic intermediate 3-phosphoglycerate: among the different roles played by this amino acid, it can be converted into D-serine and glycine, the two main co-agonists of NMDA receptors. In humans, the enzymes of the PP, namely phosphoglycerate dehydrogenase (hPHGDH, which catalyzes the first and rate-limiting step of this pathway), 3-phosphoserine aminotransferase, and 3-phosphoserine phosphatase are likely organized in the cytosol as a metabolic assembly (a "serinosome"). The hPHGDH deficiency is a pathological condition biochemically characterized by reduced levels of L-serine in plasma and cerebrospinal fluid and clinically identified by severe neurological impairment. Here, three single-point variants responsible for hPHGDH deficiency and Neu-Laxova syndrome have been studied. Their biochemical characterization shows that V261M, V425M, and V490M substitutions alter either the kinetic (both maximal activity and Km for 3-phosphoglycerate in the physiological direction) and the structural properties (secondary, tertiary, and quaternary structure, favoring aggregation) of hPHGDH. All the three variants have been successfully ectopically expressed in U251 cells, thus the pathological effect is not due to hindered expression level. At the cellular level, mistargeting and aggregation phenomena have been observed in cells transiently expressing the pathological protein variants, as well as a reduced L-serine cellular level. Previous studies demonstrated that the pharmacological supplementation of L-serine in hPHGDH deficiencies could ameliorate some of the related symptoms: our results now suggest the use of additional and alternative therapeutic approaches.

Phosphoserine Aminotransferase Pathogenetic Variants in Serine Deficiency Disorders: A Functional Characterization.

In humans, the phosphorylated pathway (PP) converts the glycolytic intermediate D-3-phosphoglycerate (3-PG) into L-serine through the enzymes 3-phosphoglycerate dehydrogenase, phosphoserine aminotransferase (PSAT) and phosphoserine phosphatase. From the pathogenic point of view, the PP in the brain is particularly relevant, as genetic defects of any of the three enzymes are associated with a group of neurometabolic disorders known as serine deficiency disorders (SDDs). We recombinantly expressed and characterized eight variants of PSAT associated with SDDs and two non-SDD associated variants. We show that the pathogenetic mechanisms in SDDs are extremely diverse, including low affinity of the cofactor pyridoxal 5'-phosphate and thermal instability for S179L and G79W PSAT, loss of activity of the holo form for R342W PSAT, aggregation for D100A PSAT, increased Km for one of the substrates with invariant kcats for S43R PSAT, and a combination of increased Km and decreased kcat for C245R PSAT. Finally, we show that the flux through the in vitro reconstructed PP at physiological concentrations of substrates and enzymes is extremely sensitive to alterations of the functional properties of PSAT variants, confirming PSAT dysfunctions as a cause of SSDs.

Serine metabolism during differentiation of human iPSC-derived astrocytes.

Astrocytes are essential players in development and functions, being particularly relevant as regulators of brain energy metabolism, ionic homeostasis and synaptic transmission. They are also the major source of l-serine in the brain, which is synthesized from the glycolytic intermediate 3-phosphoglycerate through the phosphorylated pathway. l-Serine is the precursor of the two main co-agonists of the N-methyl-d-aspartate receptor, glycine and d-serine. Strikingly, dysfunctions in both l- and d-serine metabolism are associated with neurological and psychiatric disorders. Here, we exploited a differentiation protocol, based on the generation of human mature astrocytes from neural stem cells, and investigated the modification of the proteomic and metabolomic profile during the differentiation process. We show that differentiated astrocytes are more similar to mature rather than to reactive ones, and that axogenesis and pyrimidine metabolism increase up to 30 days along with the folate cycle and sphingolipid metabolism. Consistent with the proliferation and cellular maturation processes that are taking place, also the intracellular levels of l-serine, glycine, threonine, l- and d-aspartate (which level is unexpectedly higher than that of d-serine) show the same biosynthetic time course. A significant utilization of l-serine from the medium is apparent while glycine is first consumed and then released with a peak at 30 days, parallel to its intracellular level. These results underline how metabolism changes during astrocyte differentiation, highlight that d-serine synthesis is restricted in differentiated astrocytes and provide a valuable model for developing potential novel therapeutic approaches to address brain diseases, especially the ones related to serine metabolism alterations.

The human phosphorylated pathway: a multienzyme metabolic assembly for l-serine biosynthesis.

De novo l-serine biosynthesis in the mammalian astrocytes proceeds via a linear, three-step pathway (the phosphorylated pathway) catalysed by 3-phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase (PSAT) and phosphoserine phosphatase (PSP). The first reaction, catalysed by PHGDH and using the glycolytic intermediate 3-phosphoglycerate, is strongly shifted towards the reagents, and coupling to the following step by PSAT is required to push the equilibrium towards l-serine formation; the last step, catalysed by PSP, is virtually irreversible and inhibited by the final product l-serine. Very little is known about the regulation of the human phosphorylated pathway and the ability of the three enzymes to organise in a complex with potential regulatory functions. Here, the complex formation was investigated in differentiated human astrocytes, by proximity ligation assay, and in vitro on the human recombinant enzymes. The results indicate that the three enzymes co-localise in cytoplasmic clusters that more stably engage PSAT and PSP. Although in vitro analyses based on native PAGE, size exclusion chromatography and cross-linking experiments do not show the formation of a stable complex, kinetic studies of the reconstituted pathway using physiological enzyme and substrate concentrations support cluster formation and indicate that PHGDH catalyses the rate-limiting step while PSP reaction is the driving force for the whole pathway. The enzyme agglomerate assembly of the phosphorylated pathway (the putative 'serinosome') delivers a relevant level of sophistication to the control of l-serine biosynthesis in human cells, a process strictly related to the modulation of the brain levels of d-serine and glycine, the main co-agonists of N-methyl-d-aspartate receptors and various pathological states.

L-serine biosynthesis in the human central nervous system: Structure and function of phosphoserine aminotransferase.

Organisms from all kingdoms of life synthesize L-serine (L-Ser) from 3-phosphoglycerate through the phosphorylated pathway, a three-step diversion of glycolysis. Phosphoserine aminotransferase (PSAT) catalyzes the intermediate step, the pyridoxal 5'-phosphate-dependent transamination of 3-phosphohydroxypyruvate and L-glutamate to O-phosphoserine (OPS) and α-ketoglutarate. PSAT is particularly relevant in the central nervous system of mammals because L-Ser is the metabolic precursor of D-serine, cysteine, phospholipids, and nucleotides. Several mutations in the human psat gene have been linked to serine deficiency disorders, characterized by severe neurological symptoms. Furthermore, PSAT is overexpressed in many tumors and this overexpression has been associated with poor clinical outcomes. Here, we report the detailed functional and structural characterization of the recombinant human PSAT. The reaction catalyzed by PSAT is reversible, with an equilibrium constant of about 10, and the enzyme is very efficient, with a kcat /Km of 5.9 × 106  M-1  s-1 , thus contributing in driving the pathway towards the products despite the extremely unfavorable first step catalyzed by 3-phosphoglycerate dehydrogenase. The 3D X-ray crystal structure of PSAT was solved in the substrate-free as well as in the OPS-bound forms. Both structures contain eight protein molecules in the asymmetric unit, arranged in four dimers, with a bound cofactor in each subunit. In the substrate-free form, the active site of PSAT contains a sulfate ion that, in the substrate-bound form, is replaced by the phosphate group of OPS. Interestingly, fast crystal soaking used to produce the substrate-bound form allowed the trapping of different intermediates along the catalytic cycle.

D-Amino Acids and Cancer: Friends or Foes?

α-amino acids exist in two configurations, named D-(dextro) and L-(levo) enantiomers. L-amino acids are used in protein synthesis and play a central role in cell metabolism. The effects of the L-amino acid composition of foods and the dietary modifications of this composition on the efficacy of cancer therapies have been widely investigated in relation to the growth and reproduction of cancerous cells. However, less is known about the involvement of D-amino acids. In recent decades, D-amino acids have been identified as natural biomolecules that play interesting and specific roles as common components of the human diet. Here, we focus on recent investigations showing altered D-amino acid levels in specific cancer types and on the various roles proposed for these biomolecules related to cancer cell proliferation, cell protection during therapy, and as putative, innovative biomarkers. Notwithstanding recent progress, the relationship between the presence of D-amino acids, their nutritional value, and cancer cell proliferation and survival represents an underrated scientific issue. Few studies on human samples have been reported to date, suggesting a need for routine analysis of D-amino acid content and an evaluation of the enzymes involved in regulating their levels in clinical samples in the near future.

Insulin and serine metabolism as sex-specific hallmarks of Alzheimer's disease in the human hippocampus.

Healthy aging is an ambitious aspiration for humans, but neurodegenerative disorders, such as Alzheimer's disease (AD), strongly affect quality of life. Using an integrated omics approach, we investigate alterations in the molecular composition of postmortem hippocampus samples of healthy persons and individuals with AD. Profound differences are apparent between control and AD male and female cohorts in terms of up- and downregulated metabolic pathways. A decrease in the insulin response is evident in AD when comparing the female with the male group. The serine metabolism (linked to the glycolytic pathway and generating the N-methyl-D-aspartate [NMDA] receptor coagonist D-serine) is also significantly modulated: the D-Ser/total serine ratio represents a way to counteract age-related cognitive decline in healthy men and during AD onset in women. These results show how AD changes and, in certain respects, almost reverses sex-specific proteomic and metabolomic profiles, highlighting how different pathophysiological mechanisms are active in men and women.

Biochemical Properties and Physiological Functions of pLG72: Twenty Years of Investigations.

In 2002, the novel human gene G72 was associated with schizophrenia susceptibility. This gene encodes a small protein of 153 amino acids, named pLG72, which represents a rare case of primate-specific protein. In particular, the rs2391191 single nucleotide polymorphism (resulting in in the R30K substitution) was robustly associated to schizophrenia and bipolar disorder. In this review, we aim to summarize the results of 20 years of biochemical investigations on pLG72. The main known role of pLG72 is related to its ability to bind and inactivate the flavoenzyme d-amino acid oxidase, i.e., the enzyme that controls the catabolism of d-serine, the main NMDA receptor coagonist in the brain. pLG72 was proposed to target the cytosolic form of d-amino acid oxidase for degradation, preserving d-serine and protecting the cell from oxidative stress generated by hydrogen peroxide produced by the flavoenzyme reaction. Anyway, pLG72 seems to play additional roles, such as affecting mitochondrial functions. The level of pLG72 in the human body is still a controversial issue because of its low expression and challenging detection. Anyway, the intriguing hypothesis that pLG72 level in blood could represent a suitable marker of Alzheimer's disease progression (a suggestion not sufficiently established yet) merits further investigations.

D-amino Acids as Novel Blood-based Biomarkers.

BACKGROUND: D-amino acids are present in the human body originating from diet, bacterial flora, and endogenous synthesis (at least for D-serine and, probably, D-aspartate). D-amino acids are involved in important physiological processes (e.g., D-serine and D-aspartate act on the N-methyl-D-aspartate receptor as co-agonist and agonist, respectively) and increasing evidence links D-amino acids to different pathological states. METHODS: Determination of D-amino acids levels in blood is mainly based on enantiomeric separations by high performance liquid chromatography. Because of the low amount of D-enantiomers compared to the corresponding L-amino acids and the high background noise associated with biological matrices, positive and negative controls are absolutely required to obtain reliable values. RESULTS: Altered levels of D-serine in blood have been reported in several neurological and psychiatric disorders: it has been proposed as promising biomarker in schizophrenia, Alzheimer's disease, and amyotrophic lateral sclerosis. Indeed, D-serine levels seem an appropriate predictor of anti-depressant response in major depressive disorder and posttraumatic stress disorder, as well as a prognostic biomarker of early cognitive decline, especially when considering D-serine and D-proline levels simultaneously. Furthermore, D-amino acids seem useful biomarkers for pathologies not related to the central nervous system, such as pancreatic cancer and chronic kidney diseases. CONCLUSION: This is the first review focusing on the determination of blood levels of Damino acids as diagnostic and prognostic biomarkers. The experimental evidence of involvement of D-amino acids in various physiological pathways suggest investigating their levels in additional pathologies too, such as diabetes mellitus. In conclusion, the levels of D-amino acids in blood may represent novel diagnostic peripheral biomarkers for various disorders. Further studies are required to standardize/automatize the determinations and for confirming their clinical effectiveness.

Human D-aspartate Oxidase: A Key Player in D-aspartate Metabolism.

In recent years, the D-enantiomers of amino acids have been recognized as natural molecules present in all kingdoms, playing a variety of biological roles. In humans, d-serine and d-aspartate attracted attention for their presence in the central nervous system. Here, we focus on d-aspartate, which is involved in glutamatergic neurotransmission and the synthesis of various hormones. The biosynthesis of d-aspartate is still obscure, while its degradation is due to the peroxisomal flavin adenine dinucleotide (FAD)-containing enzyme d-aspartate oxidase. d-Aspartate emergence is strictly controlled: levels decrease in brain within the first days of life while increasing in endocrine glands postnatally and through adulthood. The human d-aspartate oxidase (hDASPO) belongs to the d-amino acid oxidase-like family: its tertiary structure closely resembles that of human d-amino acid oxidase (hDAAO), the enzyme that degrades neutral and basic d-amino acids. The structure-function relationships of the physiological isoform of hDASPO (named hDASPO_341) and the regulation of gene expression and distribution and properties of the longer isoform hDASPO_369 have all been recently elucidated. Beyond the substrate preference, hDASPO and hDAAO also differ in kinetic efficiency, FAD-binding affinity, pH profile, and oligomeric state. Such differences suggest that evolution diverged to create two different ways to modulate d-aspartate and d-serine levels in the human brain. Current knowledge about hDASPO is shedding light on the molecular mechanisms underlying the modulation of d-aspartate levels in human tissues and is pushing novel, targeted therapeutic strategies. Now, it has been proposed that dysfunction in NMDA receptor-mediated neurotransmission is caused by disrupted d-aspartate metabolism in the nervous system during the onset of various disorders (such as schizophrenia): the design of suitable hDASPO inhibitors aimed at increasing d-aspartate levels thus represents a novel and useful form of therapy.

Biochemical and Biophysical Characterization of Recombinant Human 3-Phosphoglycerate Dehydrogenase.

The human enzyme D-3-phosphoglycerate dehydrogenase (hPHGDH) catalyzes the reversible dehydrogenation of 3-phosphoglycerate (3PG) into 3-phosphohydroxypyruvate (PHP) using the NAD+/NADH redox cofactor, the first step in the phosphorylated pathway producing L-serine. We focused on the full-length enzyme that was produced in fairly large amounts in E. coli cells; the effect of pH, temperature and ligands on hPHGDH activity was studied. The forward reaction was investigated on 3PG and alternative carboxylic acids by employing two coupled assays, both removing the product PHP; 3PG was by far the best substrate in the forward direction. Both PHP and α-ketoglutarate were efficiently reduced by hPHGDH and NADH in the reverse direction, indicating substrate competition under physiological conditions. Notably, neither PHP nor L-serine inhibited hPHGDH, nor did glycine and D-serine, the coagonists of NMDA receptors related to L-serine metabolism. The investigation of NADH and phosphate binding highlights the presence in solution of different conformations and/or oligomeric states of the enzyme. Elucidating the biochemical properties of hPHGDH will enable the identification of novel approaches to modulate L-serine levels and thus to reduce cancer progression and treat neurological disorders.

The Role of D-Amino Acids in Alzheimer's Disease.

Alzheimer's disease (AD), the main cause of dementia worldwide, is characterized by a complex and multifactorial etiology. In large part, excitatory neurotransmission in the central nervous system is mediated by glutamate and its receptors are involved in synaptic plasticity. The N-methyl-D-aspartate (NMDA) receptors, which require the agonist glutamate and a coagonist such as glycine or the D-enantiomer of serine for activation, play a main role here. A second D-amino acid, D-aspartate, acts as agonist of NMDA receptors. D-amino acids, present in low amounts in nature and long considered to be of bacterial origin, have distinctive functions in mammals. In recent years, alterations in physiological levels of various D-amino acids have been linked to various pathological states, ranging from chronic kidney disease to neurological disorders. Actually, the level of NMDA receptor signaling must be balanced to promote neuronal survival and prevent neurodegeneration: this signaling in AD is affected mainly by glutamate availability and modulation of the receptor's functions. Here, we report the experimental findings linking D-serine and D-aspartate, through NMDA receptor modulation, to AD and cognitive functions. Interestingly, AD progression has been also associated with the enzymes related to D-amino acid metabolism as well as with glucose and serine metabolism. Furthermore, the D-serine and D-/total serine ratio in serum have been recently proposed as biomarkers of AD progression. A greater understanding of the role of D-amino acids in excitotoxicity related to the pathogenesis of AD will facilitate novel therapeutic treatments to cure the disease and improve life expectancy.

Antimicrobial D-amino acid oxidase-derived peptides specify gut microbiota.

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.

Enhancing Electrochemical Biosensor Selectivity with Engineered d-Amino Acid Oxidase Enzymes for d-Serine and d-Alanine Quantification.

d-Amino acid oxidase (DAAO) enzymes bind a range of d-amino acids with variable affinity. As such, the design of selective DAAO-based enzymatic biosensors remains a challenge for real-world biosensor application. Herein, a methodology for developing biosensors with varying substrate selectivity is presented. First, we address DAAO-based biosensor selectivity toward d-serine by introducing point mutations into DAAO using rational design. Next, the wild-type yeast DAAO (RgDAAO WT) and variants human DAAO W209R and yeast M213G are characterized for their selectivity and activity toward d-serine and d-alanine, the preferred DAAO substrates. The DAAO enzymes have been immobilized for final biosensor design, where they demonstrate selectivity comparable to free DAAO. The cross-linking procedure impacts on DAAO structure and function and the use of a regeneration strategy allows the biosensor response to be improved.

Synthesis and preliminary evaluation of 4-hydroxy-6-(3-[11C]methoxyphenethyl)pyridazin-3(2H)-one, a 11C-labeled d-amino acid oxidase (DAAO) inhibitor for PET imaging.

Selective DAAO inhibitors have demonstrated promising therapeutic effects in clinical studies, including clinically alleviating symptoms of schizophrenic patients and ameliorating cognitive function in Alzheimer's patients with early phase. Herein we report the synthesis and preliminary evaluation of a 11C-labeled positron emission tomography ligand based on a DAAO inhibitor, DAO-1903 (8). 11C-Isotopologue of 8 was prepared in high radiochemical yield with high radiochemical purity (>99%) and high molar activity (>37 GBq/µmol). In vitro autoradiography studies indicated that the ligand possessed high in vitro specific binding to DAAO, while in vivo dynamic PET studies demonstrated that [11C]8 failed to cross the blood-brain barrier possibly due to moderate brain efflux mechanism. Further chemical scaffold optimization is necessary to overcome limited brain permeability and improve specific binding.

Is the primate-specific protein pLG72 affecting SOD1 functionality and superoxide formation?

pLG72 is a primate-specific protein of enigmatic function that was proposed to modulate mitochondria fragmentation and the activity of the peroxisomal enzyme D-amino acid oxidase (DAAO). DAAO is deputed to degradation of the NMDA receptor co-agonist D-serine in human brain and the R199W substitution in DAAO was identified in a familial case of amyotrophic lateral sclerosis (ALS). A recent work reported that U87 glioblastoma cells ectopically expressing pLG72 showed a lower proliferation, produced superoxide radicals, induced SOD1 aggregation and decreased its activity. Because of the role of SOD1 in eliminating ROS species and its relevance in ALS we evaluated the link between pLG72 and SOD1 using both wild-type pLG72 and its R30K variant related to schizophrenia susceptibility. In vitro studies on recombinant proteins excluded the establishment of a stable complex and that pLG72 could affect SOD1 activity and stability. At cellular level, ectopic expression of pLG72 in glioblastoma U87 cells did not affect cell viability and ROS/superoxide production: only caspase activity (a marker of apoptosis) was slightly increased in cells expressing the R30K pLG72 variant. SOD1 and pLG72 did not colocalize in transfected U87 glioblastoma cells: pLG72 largely localised to mitochondria and SOD1 was largely cytosolic. Moreover, the ectopic expression of pLG72 appeared not to alter the expression of SOD1 and its aggregation. Altogether, the combination of biochemical and cellular studies allow to exclude that pLG72 modulates SOD1 function and aggregation, thus that it could play a role in ALS susceptibility.

L-serine synthesis via the phosphorylated pathway in humans.

L-serine is a nonessential amino acid in eukaryotic cells, used for protein synthesis and in producing phosphoglycerides, glycerides, sphingolipids, phosphatidylserine, and methylenetetrahydrofolate. Moreover, L-serine is the precursor of two relevant coagonists of NMDA receptors: glycine (through the enzyme serine hydroxymethyltransferase), which preferentially acts on extrasynaptic receptors and D-serine (through the enzyme serine racemase), dominant at synaptic receptors. The cytosolic "phosphorylated pathway" regulates de novo biosynthesis of L-serine, employing 3-phosphoglycerate generated by glycolysis and the enzymes 3-phosphoglycerate dehydrogenase, phosphoserine aminotransferase, and phosphoserine phosphatase (the latter representing the irreversible step). In the human brain, L-serine is primarily found in glial cells and is supplied to neurons for D-serine synthesis. Serine-deficient patients show severe neurological symptoms, including congenital microcephaly, psychomotor retardation, and intractable seizures, thus highlighting the relevance of de novo production of this amino acid in brain development and morphogenesis. Indeed, the phosphorylated pathway is strictly linked to cancer. Moreover, L-serine has been suggested as a ready-to-use treatment, as also recently proposed for Alzheimer's disease. Here, we present our current state of knowledge concerning the three mammalian enzymes of the phosphorylated pathway and known mutations related to pathological conditions: although the structure of these enzymes has been solved, how enzyme activity is regulated remains largely unknown. We believe that an in-depth investigation of these enzymes is crucial to identify the molecular mechanisms involved in modulating concentrations of the serine enantiomers and for studying the interplay between glial and neuronal cells and also to determine the most suitable therapeutic approach for various diseases.

Substitution of Arginine 120 in Human D-Amino Acid Oxidase Favors FAD-Binding and Nuclear Mistargeting.

The peroxisomal enzyme human D-amino acid oxidase (hDAAO) is attracting attention owing to its role in degrading D-serine, the main co-agonist of N-methyl D-aspartate receptors in brain, and its involvement in brain functions and diseases. Here, we focused on arginine 120, a residue located at the protein interface, 20 Å from the assumed second ligand-binding site, showing a different orientation of the side chain in the hDAAO-benzoate complex, and corresponding to Ser119 in rat DAAO, which is part of a putative nuclear translocation signal (NTS). By substituting Arg120 in hDAAO with a glutamate (to mimic the active NTS) or a leucine (to eliminate the positive charge) the protein conformation, thermal stability, and kinetic properties are slightly altered, while the dimeric structure and the ligand-binding properties are unchanged. The most relevant alteration in Arg120 variants is the strongest interaction with FAD. Nevertheless, the activity assayed at low D-serine and FAD concentrations (resembling physiological conditions) was quite similar for wild-type and Arg120 hDAAO variants. These results resemble the ones obtained substituting another residue located at the interface region (i.e., the W209R variant), indicating that substitutions at the monomer-monomer interface mainly affects the FAD binding in hDAAO. Indeed, U87 glioblastoma cells transiently transfected for hDAAO variants show that substitution of Arg120 favors mistargeting: the increase in cytosolic localization observed for the variants promotes nuclear targeting, especially for the R120E hDAAO, without affecting cell viability. Notably, mistargeting to the nucleus is an innate process as it is apparent for the wild-type hDAAO, too: whether such a process is related to specific pathologic processes is still unknown.

Human D-Amino Acid Oxidase: Structure, Function, and Regulation.

D-Amino acid oxidase (DAAO) is an FAD-containing flavoenzyme that catalyzes with absolute stereoselectivity the oxidative deamination of all natural D-amino acids, the only exception being the acidic ones. This flavoenzyme plays different roles during evolution and in different tissues in humans. Its three-dimensional structure is well conserved during evolution: minute changes are responsible for the functional differences between enzymes from microorganism sources and those from humans. In recent years several investigations focused on human DAAO, mainly because of its role in degrading the neuromodulator D-serine in the central nervous system. D-Serine is the main coagonist of N-methyl D-aspartate receptors, i.e., excitatory amino acid receptors critically involved in main brain functions and pathologic conditions. Human DAAO possesses a weak interaction with the FAD cofactor; thus, in vivo it should be largely present in the inactive, apoprotein form. Binding of active-site ligands and the substrate stabilizes flavin binding, thus pushing the acquisition of catalytic competence. Interestingly, the kinetic efficiency of the enzyme on D-serine is very low. Human DAAO interacts with various proteins, in this way modulating its activity, targeting, and cell stability. The known properties of human DAAO suggest that its activity must be finely tuned to fulfill a main physiological function such as the control of D-serine levels in the brain. At present, studies are focusing on the epigenetic modulation of human DAAO expression and the role of post-translational modifications on its main biochemical properties at the cellular level.