Kinetic mechanism of D-amino acid oxidases from Rhodotorula gracilis and Trigonopsis variabilis

Pollegioni, Loredano; Langkau, Bernd; Tischer, Wilhelm; Ghisla, Sandro; Pilone, Mirella S.

doi:10.1016/s0021-9258(19)85181-5

Cited by 93 publications

(25 citation statements)

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“…The kinetic mechanism of hDASPO was investigated using d-Asp as substrate by both the enzyme-monitored turnover method (EMTN) as a function of oxygen concentration and by the rapid kinetic approach using a stopped-flow spectrophotometer (detailed in Supplementary Data 4). 61 In EMTN, the oxidized enzyme was mixed aerobically with increasing amounts of d-Asp and the changes in flavin reduction at increasing d-Asp concentrations is biphasic; the second, slow phase is insensitive to d-Asp concentration and disappears at high d-Asp concentration. The step of IAsp release from the reduced E-FAD red -IAsp complex is not evident in stopped-flow experiments (k 3 in the scheme in Figure 6 top).…”

Section: Kinetic Mechanismmentioning

confidence: 99%

Structure and kinetic properties of human d‐aspartate oxidase, the enzyme‐controlling d‐aspartate levels in brain

et al. 2019

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Savinelli equally contributed to this study.Abbreviations: 5-An, 5-aminonicotinic acid; AMPA, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrate; CBIO, 6-chloro-1,2-benzisoxazol-3(2H)-one; DAAO, d-amino acid oxidase; DASPO or DDO, d-aspartate oxidase; DPPD, pyrido[2,3-b]pyrazine-2,3(1H,4H)-dione; EMTN, enzyme monitored turnover; hDAAO, human d-amino acid oxidase; hDASPO, human d-aspartate oxidase; IAsp, iminoaspartate; MT, meso-tartrate; NMDAR, N-methyl-d-aspartate receptor; OA, oxaloacetate; RgDAAO, d-amino acid oxidase from Rhodotorula gracilis. Abstract d-Amino acids are the "wrong" enantiomers of amino acids as they are not used in proteins synthesis but evolved in selected functions. On this side, d-aspartate (d-Asp) plays several significant roles in mammals, especially as an agonist of N-methyl-daspartate receptors (NMDAR), and is involved in relevant diseases, such as schizophrenia and Alzheimer's disease. In vivo modulation of d-Asp levels represents an intriguing task to cope with such pathological states. As little is known about d-Asp synthesis, the only option for modulating the levels is via degradation, which is due to the flavoenzyme d-aspartate oxidase (DASPO). Here we present the first threedimensional structure of a DASPO enzyme (from human) which belongs to the damino acid oxidase family. Notably, human DASPO differs from human d-amino acid oxidase (attributed to d-serine degradation, the main coagonist of NMDAR)showing peculiar structural features (a specific active site charge distribution), oligomeric state and kinetic mechanism, and a higher FAD affinity and activity. These results provide useful insights into the structure-function relationships of human DASPO: modulating its activity represents now a feasible novel therapeutic target. K E Y W O R D Sd-aspartate, d-aspartate oxidase, flavoprotein, NMDA receptor, structure-function relationships

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Section: Kinetic Mechanismmentioning

confidence: 99%

Structure and kinetic properties of human d‐aspartate oxidase, the enzyme‐controlling d‐aspartate levels in brain

et al. 2019

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“…DAAO's reaction with D-ala is characterized by a Michaelis−Menten constant, K m , in the mM concentration regime. 20 We stimulated cells with a range of H 2 O 2 doses by supplying D-ala at concentrations ranging from 0−50 mM over periods of up to 6.5 h. D-ala alone is not toxic to HeLa cells even at the highest concentration and time combination used (Figure 1c) so it is a viable substrate for these experiments. To validate the function of the tool in the mitochondria, cells were analyzed as a function of time for post-translational protein modifications that provide evidence of H 2 O 2 generation.…”

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Mitochondrial H₂O₂ Generation Using a Tunable Chemogenetic Tool To Perturb Redox Homeostasis in Human Cells and Induce Cell Death

2018

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Among reactive oxygen species (ROS), HO alone acts as a signaling molecule that promotes diverse phenotypes depending on the intracellular concentration. Mitochondria have been suggested as both sources and sinks of cellular HO, and mitochondrial dysfunction has been implicated in diseases such as cancer. A genetically encoded HO generator, d-amino acid oxidase (DAAO), was targeted to the mitochondria of human cells, and its utility in investigating cellular response to a range of HO doses over time was assessed. Organelle-specific peroxiredoxin dimerization and protein S-glutathionylation were measured as indicators of increased HO flux due to the activity of DAAO. Cell death was observed in a concentration- and time-dependent manner, and protein oxidation shifted in localization as the dose increased. This work presents the first systematic study of HO-specific perturbation of mitochondria in human cells, and it reveals a marked sensitivity of this organelle to increases in HO in comparison with prior studies that targeted the cytosol.

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“…A vivid example is D-amino acid oxidase (DAAO), used to generate hydrogen peroxide in cells [ 88 ]. This yeast enzyme catalyzes the conversion of D-amino acids into the respective α-keto derivatives, accompanied by the release of a peroxide molecule [ 89 ]. Hence, almost any D-amino acid can be used to activate the H 2 O 2 generator.…”

Section: Chemogeneticsmentioning

confidence: 99%

Molecular Tools for Targeted Control of Nerve Cell Electrical Activity. Part II

et al. 2021

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In modern life sciences, the issue of a specific, exogenously directed manipulation of a cells biochemistry is a highly topical one. In the case of electrically excitable cells, the aim of the manipulation is to control the cells electrical activity, with the result being either excitation with subsequent generation of an action potential or inhibition and suppression of the excitatory currents. The techniques of electrical activity stimulation are of particular significance in tackling the most challenging basic problem: figuring out how the nervous system of higher multicellular organisms functions. At this juncture, when neuroscience is gradually abandoning the reductionist approach in favor of the direct investigation of complex neuronal systems, minimally invasive methods for brain tissue stimulation are becoming the basic element in the toolbox of those involved in the field. In this review, we describe three approaches that are based on the delivery of exogenous, genetically encoded molecules sensitive to external stimuli into the nervous tissue. These approaches include optogenetics (overviewed in Part I), as well as chemogenetics and thermogenetics (described here, in Part II), which is significantly different not only in the nature of the stimuli and structure of the appropriate effector proteins, but also in the details of experimental applications. The latter circumstance is an indication that these are rather complementary than competing techniques.

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Kinetic mechanism of D-amino acid oxidases from Rhodotorula gracilis and Trigonopsis variabilis

Cited by 93 publications

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Structure and kinetic properties of human d‐aspartate oxidase, the enzyme‐controlling d‐aspartate levels in brain

Structure and kinetic properties of human d‐aspartate oxidase, the enzyme‐controlling d‐aspartate levels in brain

Mitochondrial H₂O₂ Generation Using a Tunable Chemogenetic Tool To Perturb Redox Homeostasis in Human Cells and Induce Cell Death

Molecular Tools for Targeted Control of Nerve Cell Electrical Activity. Part II

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Kinetic mechanism of D-amino acid oxidases from Rhodotorula gracilis and Trigonopsis variabilis

Cited by 93 publications

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Structure and kinetic properties of human d‐aspartate oxidase, the enzyme‐controlling d‐aspartate levels in brain

Structure and kinetic properties of human d‐aspartate oxidase, the enzyme‐controlling d‐aspartate levels in brain

Mitochondrial H2O2 Generation Using a Tunable Chemogenetic Tool To Perturb Redox Homeostasis in Human Cells and Induce Cell Death

Molecular Tools for Targeted Control of Nerve Cell Electrical Activity. Part II

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Mitochondrial H₂O₂ Generation Using a Tunable Chemogenetic Tool To Perturb Redox Homeostasis in Human Cells and Induce Cell Death