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Mirella S. Pilone - One of the best experts on this subject based on the ideXlab platform.
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Characterization of human D-Amino Acid Oxidase.
FEBS letters, 2006Co-Authors: Gianluca Molla, Mirella S. Pilone, Silvia Sacchi, Mariagrazia Bernasconi, Kiyoshi Fukui, Loredano PollegioniAbstract:D-Amino Acid Oxidase (DAAO) has been proposed to be involved in the oxidation of D-serine, an allosteric activator of the NMDA-type glutamate receptor in the brain, and to be associated with the onset of schizophrenia. The recombinant human DAAO was expressed in Escherichia coli and was isolated as an active homodimeric flavoenzyme. It shows the properties of the dehydrogenase-Oxidase class of flavoproteins, possesses a low kinetic efficiency, and follows a ternary complex (sequential) kinetic mechanism. In contrast to the other known DAAOs, the human enzyme is a stable homodimer even in the apoprotein form and weakly binds the cofactor in the free form.
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D-Amino Acid Oxidase: new findings.
Cellular and molecular life sciences : CMLS, 2000Co-Authors: Mirella S. PiloneAbstract:The most recent research on D-Amino Acid Oxidases and D-Amino Acid metabolism has revealed new, intriguing properties of flavoenzymes and enlighted novel biotechnological uses of this catalyst. Concerning the in vivo function of the enzyme, new findings on the physiological role of D-Amino Acid Oxidase point to a detoxifying function of the enzyme in metabolizing exogenous D-Amino Acids in animals. A novel role in modulating the level of D-serine in brain has also been proposed for the enzyme. At the molecular level, site-directed mutagenesis studies on the pig kidney D-Amino Acid Oxidase and, more recently, on the enzyme from the yeast Rhodotorula gracilis indicated that the few conserved residues of the active site do not play a role in Acid-base catalysis but rather are involved in substrate interactions. The three-dimensional structure of the enzyme was recently determined from two different sources: at 2.5–3.0 A resolution for DAAO from pig kidney and at 1.2–1.8 A resolution for R. gracilis. The active site can be clearly depicted: the striking absence of essential residues acting in Acid-base catalysis and the mode of substrate orientation into the active site, taken together with the results of free-energy correlation studies, clearly support a hydrid transfer type of mechanism in which the orbital steering between the substrate and the isoalloxazine atoms plays a crucial role during catalysis.
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Reactivity of histidyl residues in d‐amino Acid Oxidase from Rhodotorula gracilis
FEBS letters, 1995Co-Authors: Giovanni Gadda, Giovanni Luca Beretta, Mirella S. PiloneAbstract:Incubation of D-Amino Acid Oxidase from the yeast Rhodotorula gracilis with excess dansyl chloride at pH 6.6 and 18°C caused an irreversible inactivation of D-Amino Acid Oxidase. Benzoate, a competitive inhibitor of the enzyme, completely protected the enzyme from inactivation. The dansylated-enzyme, isolated by gel-filtration, was in part still active while the substrate specificity was altered substantially. It was completely reduced by d-alanine in anaerobiosic conditions and did stabilize the red anion semiquinone upon photochemical reduction with EDTA. The results provide evidence for the presence of essential histidyl residue(s) in the active center of the yeast enzyme.
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A process for bioconversion of cephalosporin C by Rhodotorula gracilis D-Amino Acid Oxidase
Biotechnology Letters, 1995Co-Authors: Mirella S. Pilone, S. Buto, Loredano PollegioniAbstract:A process for the production (in a stirred tank reactor) of glutaryl-7-ACA from cephalosporin C using immobilized D-Amino Acid Oxidase is described. Results so obtained under optimal conditions (1.2 mg coupled enzyme/L, pH 8.5, 2 mM cephalosporin C) point to a system which shows high conversion efficiency and a remarkable operational stability. No exogenous H2O2 is requested to shift the reaction equilibrium toward glutaryl-7-ACA production, nor any side product is detected. The immobilized system productivity was 54 g/day/mg of enzyme. This process represents the first reported case of a reactor successfully developed with a DAAO for bioconversion of cephalosporin C.
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Stabilization of Yeast D-Amino Acid Oxidase by Matrix Covalent Attachment
Studies in Organic Chemistry, 1993Co-Authors: Mirella S. Pilone, Loredano Pollegioni, S. ButoAbstract:Abstract D-Amino Acid Oxidase from the yeast Rhodotorula gracilis has been immobilized onto Affi-Gel 10 matrix with a 60% yield under optimal conditions and a specific activity comparable to that of the free suspended preparation. No loss of FAD was observed in all experimental conditions; the enzyme-matrix complex exhibited a marked enhancement of stability against inactivation on heating, on organic solvents and on denaturant agents. Activation energies of the thermoinactivation process were 145.2 kJ/mol and 239.0 kJ/mol for immobilized and free form respectively. The thermal stabilization of the immobilized DAAO indicates that a more rigid molecular structure, stabilized against unfolding, resulted from the multipoint attachment of the protein to the support. The coupled system we have developed represents the first case where a marked increase of D-Amino Acid Oxidase stability was obtained by matrix covalent attachment.
Ryuichi Konno - One of the best experts on this subject based on the ideXlab platform.
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D-Amino-Acid Oxidase Is Involved in d-Serine-Induced Nephrotoxicity
Chemical research in toxicology, 2005Co-Authors: Masao Maekawa, Tadashi Okamura, Noriyuki Kasai, Yuuichi Hori, Karl H. Summer, Ryuichi KonnoAbstract:D-serine is nephrotoxic in rats. Based on circumstantial evidence, it has been suspected that D-Amino-Acid Oxidase is involved in this nephrotoxicity. Since we found that LEA/SENDAI rats lacked D-Amino-Acid Oxidase, we examined whether this enzyme was associated with D-serine-induced nephrotoxicity using the LEA/SENDAI rats and control F344 rats. When d-propargylglycine, which is known to have a nephrotoxic effect through its metabolism by D-Amino-Acid Oxidase, was injected intraperitoneally into the F344 rats, it caused glucosuria and polyuria. However, injection of d-propargylglycine into LEA/SENDAI rats did not cause any glucosuria or polyuria, indicating that D-Amino-Acid Oxidase is definitely not functional in these rats. D-serine was then injected into the F344 and LEA/SENDAI rats. It caused glucosuria and polyuria in the F344 rats but not in the LEA/SENDAI rats. These results indicate clearly that D-Amino-Acid Oxidase is responsible for the D-serine-induced nephrotoxicity.
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Role of renal D-Amino-Acid Oxidase in pharmacokinetics of d-leucine
American journal of physiology. Endocrinology and metabolism, 2004Co-Authors: Hiroshi Hasegawa, Ryuichi Konno, Takehisa Matsukawa, Yoshihiko Shinohara, Takao HashimotoAbstract:D-Amino Acids are now recognized to be widely present in mammals. Renal D-Amino-Acid Oxidase (DAO) is associated with conversion of D-Amino Acids to the corresponding α-keto Acids, but its contribu...
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Assignment of D-Amino-Acid Oxidase gene to a human and a mouse chromosome.
Amino acids, 2001Co-Authors: Ryuichi KonnoAbstract:A part of D-Amino-Acid Oxidase gene was amplified in the human and mouse by polymerase chain reaction. The amplified fragments were ligated to plasmids and then cloned. The plasmids containing the parts of D-Amino-Acid Oxidase gene were biotinylated and hybridized to human and mouse metaphase chromosomes. The chromosomal slides were treated with fluorescein isothiocyanate (FITC)-conjugated avidin. The hybridized signals were amplified with biotinylated anti-avidin antibody and FITC-avidin. The chromosomes were counter-stained with diamidino-phenylindole for assignment of the signal to a specific band. Using this fluorescence in situ hybridization (FISH), D-Amino-Acid Oxidase gene was assigned to human chromosome 12q23–24.1 and mouse chromosome 5E3-F. Since these regions are syntenic between human and mouse, the present results indicate that the locus for this enzyme has been conserved through evolution.
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Methods for the Detection of D-Amino-Acid Oxidase.
Biological procedures online, 1998Co-Authors: Ryuichi KonnoAbstract:Four methods (an enzyme activity assay, western blotting, RT-PCR, and northern hybridization) to detect the enzyme D-Amino-Acid Oxidase are described.
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Rat D-Amino-Acid Oxidase cDNA: rat D-Amino-Acid Oxidase as an intermediate form between mouse and other mammalian D-Amino-Acid Oxidases
Biochimica et biophysica acta, 1998Co-Authors: Ryuichi KonnoAbstract:Abstract Nucleotide sequence of cDNA encoding rat d -amino-Acid Oxidase (DAO) was determined. Two species of DAO mRNA were present in rat kidney, liver, and brain. They were probably produced by alternative splicing. Rat DAO cDNA encodes 346 amino Acid residues, indicating that rat DAO is an intermediate form between mouse DAO (345 amino Acids) and DAOs (347 amino Acids) of human, rabbit, and pig. Deduced amino Acid sequence indicates 93% identity between rat and mouse DAO. Northern hybridization and western blotting supported the sequence data.
Loredano Pollegioni - One of the best experts on this subject based on the ideXlab platform.
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Structure–function relationships in human D-Amino Acid Oxidase
Amino acids, 2012Co-Authors: Silvia Sacchi, Loredano Pollegioni, Laura Caldinelli, Pamela Cappelletti, Gianluca MollaAbstract:Since D-Amino Acids were identified in mammals, D-serine has been one of the most extensively studied "unnatural amino Acids". This brain-enriched transmitter-like molecule plays a pivotal role in the human central nervous system by modulating the activity of NMDA receptors. Physiological levels of D-serine are required for normal brain development and function; thus, any alterations in neuromodulator concentrations might result in NMDA receptor dysfunction, which is known to be involved in several pathological conditions, including neurodegeneration(s), epilepsy, schizophrenia, and bipolar disorder. In the brain, the concentration of D-serine stored in cells is defined by the activity of two enzymes: serine racemase (responsible for both the synthesis and degradation) and D-Amino Acid Oxidase (which catalyzes D-serine degradation). Both enzymes emerged recently as new potential therapeutic targets for NMDA receptor-related diseases. In this review we have focused on human D-Amino Acid Oxidase and provide an extensive overview of the biochemical and structural properties of this flavoprotein and their functional significance. Furthermore, we discuss the mechanisms involved in modulating enzyme activity and stability with the aim to substantiate the pivotal role of D-Amino Acid Oxidase in brain D-serine metabolism in physiological and pathological conditions and to highlight its great significance for novel drug design/development.
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Activity of yeast D-Amino Acid Oxidase on aromatic unnatural amino Acids
Journal of Molecular Catalysis B-enzymatic, 2007Co-Authors: Antonio Caligiuri, Gianluca Molla, Paola D'arrigo, Elena Rosini, Giuseppe Pedrocchi-fantoni, Davide Tessaro, Stefano Servi, Loredano PollegioniAbstract:Abstract d -Amino Acid Oxidase is a FAD-dependent enzyme that catalyses the conversion of the d -enantiomer of amino Acids into the corresponding α-keto Acid. Substrate specificity of the enzyme from the yeast Rhodotorula gracilis was investigated towards aromatic amino Acids, and particularly synthetic α-amino Acids. A significant improvement of the activity ( V max,app ) and of the specificity constant (the V max,app / K m,app ratio) on a number of the substrates tested was obtained using a single-point mutant enzyme designed by a rational approach. With R. gracilis d -amino Acid Oxidase the complete resolution of d , l -homo-phenylalanine was obtained with the aim to produce the corresponding pure l -isomer and to use the corresponding α-keto Acid as a precursor of the amino Acid in the l -form.
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Characterization of human D-Amino Acid Oxidase.
FEBS letters, 2006Co-Authors: Gianluca Molla, Mirella S. Pilone, Silvia Sacchi, Mariagrazia Bernasconi, Kiyoshi Fukui, Loredano PollegioniAbstract:D-Amino Acid Oxidase (DAAO) has been proposed to be involved in the oxidation of D-serine, an allosteric activator of the NMDA-type glutamate receptor in the brain, and to be associated with the onset of schizophrenia. The recombinant human DAAO was expressed in Escherichia coli and was isolated as an active homodimeric flavoenzyme. It shows the properties of the dehydrogenase-Oxidase class of flavoproteins, possesses a low kinetic efficiency, and follows a ternary complex (sequential) kinetic mechanism. In contrast to the other known DAAOs, the human enzyme is a stable homodimer even in the apoprotein form and weakly binds the cofactor in the free form.
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A process for bioconversion of cephalosporin C by Rhodotorula gracilis D-Amino Acid Oxidase
Biotechnology Letters, 1995Co-Authors: Mirella S. Pilone, S. Buto, Loredano PollegioniAbstract:A process for the production (in a stirred tank reactor) of glutaryl-7-ACA from cephalosporin C using immobilized D-Amino Acid Oxidase is described. Results so obtained under optimal conditions (1.2 mg coupled enzyme/L, pH 8.5, 2 mM cephalosporin C) point to a system which shows high conversion efficiency and a remarkable operational stability. No exogenous H2O2 is requested to shift the reaction equilibrium toward glutaryl-7-ACA production, nor any side product is detected. The immobilized system productivity was 54 g/day/mg of enzyme. This process represents the first reported case of a reactor successfully developed with a DAAO for bioconversion of cephalosporin C.
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Stabilization of Yeast D-Amino Acid Oxidase by Matrix Covalent Attachment
Studies in Organic Chemistry, 1993Co-Authors: Mirella S. Pilone, Loredano Pollegioni, S. ButoAbstract:Abstract D-Amino Acid Oxidase from the yeast Rhodotorula gracilis has been immobilized onto Affi-Gel 10 matrix with a 60% yield under optimal conditions and a specific activity comparable to that of the free suspended preparation. No loss of FAD was observed in all experimental conditions; the enzyme-matrix complex exhibited a marked enhancement of stability against inactivation on heating, on organic solvents and on denaturant agents. Activation energies of the thermoinactivation process were 145.2 kJ/mol and 239.0 kJ/mol for immobilized and free form respectively. The thermal stabilization of the immobilized DAAO indicates that a more rigid molecular structure, stabilized against unfolding, resulted from the multipoint attachment of the protein to the support. The coupled system we have developed represents the first case where a marked increase of D-Amino Acid Oxidase stability was obtained by matrix covalent attachment.
A Niwa - One of the best experts on this subject based on the ideXlab platform.
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D-Amino-Acid Oxidase is not present in the mouse liver.
Biochimica et biophysica acta, 1997Co-Authors: R Konno, M Sasaki, S Asakura, K Fukui, J Enami, A NiwaAbstract:Since there are conflict reports on the presence of D-Amino-Acid Oxidase in the mouse liver, this problem was examined. D-Amino-Acid Oxidase activity was not detected in the homogenates of the mouse liver, lung, or heart, whereas it was detected in the homogenates of the mouse kidney and brain. Western blotting showed that a protein which reacted with the antiserum against pig D-Amino-Acid Oxidase was present in the homogenates of the mouse kidney and brain but not in those of the liver or heart. Northern hybridization using a D-Amino-Acid Oxidase cDNA probe detected a hybridizing signal in poly(A)+ RNAs extracted from the mouse kidney and brain but not in those from the liver, heart, or lung. Reverse transcription-polymerase chain reaction using three primer pairs always amplified D-Amino-Acid Oxidase cDNA fragments of expected sizes in the mouse kidney and brain but very rarely did so in the liver, heart, or lung. The results indicate that D-Amino-Acid Oxidase is not present in the mouse liver in a measurable amount.
Lutz Fischer - One of the best experts on this subject based on the ideXlab platform.
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Production of a new D-Amino Acid Oxidase from the fungus Fusarium oxysporum
Applied and environmental microbiology, 1999Co-Authors: Matthias Gabler, Lutz FischerAbstract:The fungus Fusarium oxysporum produced a D-Amino Acid Oxidase (EC 1.4.3.3) in a medium containing glucose as the carbon and energy source and ammonium sulfate as the nitrogen source. The specific D-Amino Acid Oxidase activity was increased up to 12.5-fold with various D-Amino Acids or their corresponding derivatives as inducers. The best inducers were d-alanine (2.7 μkat/g of dry biomass) and d-3-aminobutyric Acid (2.6 μkat/g of dry biomass). The addition of zinc ions was necessary to permit the induction of peroxisomal D-Amino Acid Oxidase. Bioreactor cultivations were performed on a 50-liter scale, yielding a volumetric D-Amino Acid Oxidase activity of 17 μkat liter−1 with d-alanine as an inducer. Under oxygen limitation, the volumetric activity was increased threefold to 54 μkat liter−1 (3,240 U liter−1).
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Induction of the D-Amino Acid Oxidase from Trigonopsis variabilis.
Applied and Environmental Microbiology, 1996Co-Authors: Roy Horner, Fritz Wagner, Lutz FischerAbstract:Induction of the D-Amino Acid Oxidase (EC. 1.4.3.3) from the yeast Trigonopsis variabilis was investigated by using a minimal medium containing glucose as the carbon and energy source, (NH(inf4))(inf2)SO(inf4) as the nitrogen source, and various d- and dl-amino Acid derivatives as inducers. The best new inducers found were N-carbamoyl-d-alanine, N-acetyl-d-tryptophan, and N-chloroacetyl-d-(alpha)-aminobutyric Acid; when the induction effects of these compounds were compared with the effects of d-alanine as the nitrogen source and inducer, the resulting activities of D-Amino Acid Oxidase per gram of dried yeast were 4.2, 2.1, and 1.5 times higher, respectively. The optimum concentration of the best inducer, N-carbamoyl-d-alanine, was 5 mM. This inducer could also be used in its racemic form. The induction was pH dependent. After cultivation of the yeast in a 50-liter bioreactor, D-Amino Acid Oxidase activity of about 3,850 (mu)kat (231,000 U) was obtained. In addition, production of the D-Amino Acid Oxidase was found to be significantly dependent on the metal salt composition of the medium. Addition of zinc ions was required to obtain high D-Amino Acid Oxidase levels in the cells. The optimum concentration of ZnSO(inf4) was about 140 (mu)M.