1,720,974 research outputs found
Structure of D-amino acid oxidase: new insights from an old enzyme
No full textD-amino acid oxidase is the prototype of flavin-dependent oxidases. The recent resolution of its 3D structure has provided an explanation for several of its properties and has led to a substantial revision of the mechanism of D-amino acid dehydrogenation, with significant implications for the general understanding of flavin-dependent catalysis
“ Syntesis and biological evaluation of new amino acids structurally related to antitumor agent acivicin”.
No full textAbstract
A set of racemic conformationally constrained analogues of the antitumor antibiotic acivicin (+)-1 has been prepared through a strategy based on 1,3-dipolar cycloaddition of bromonitrile oxide to suitable dipolarophiles. The bromo analogue (2) of acivicin was also synthesized and tested as a reference compound, together with its stereoisomer 3. The antitumor properties of novel amino acids 4-7 were evaluated in vitro against human tumor cell lines. Their efficacy to inhibit glutamate synthase (GltS) from Azospirillum brasilense was also assayed. None of the studied compounds, but 2, showed significant activity
A cross-talk and amonia channeling between active in the unexpected domain arrangement of glutamate synthase.
No full textINTRODUCTION: The complex iron-sulfur flavoprotein glutamate synthase catalyses the reductive synthesis of L-glutamate from 2-oxoglutarate and L-glutamine, a reaction in the plant and bacterial pathway for ammonia assimilation. The enzyme functions through three distinct active centers carrying out L-glutamine hydrolysis, conversion of 2-oxoglutarate into L-glutamate, and electron uptake from an electron donor. RESULTS: The 3.0 A crystal structure of the dimeric 324 kDa core protein of a bacterial glutamate synthase was solved by the MAD method, using the very weak anomalous signal of the two 3Fe-4S clusters present in the asymmetric unit. The 1,472 amino acids of the monomer fold into a four-domain architecture. The two catalytic domains have canonical Ntn-amidotransferase and FMN binding (beta/alpha)8 barrel folds, respectively. The other two domains have an unusual "cut (beta/alpha)8 barrel" topology and an unexpected novel beta-helix structure. Channeling of the ammonia intermediate is brought about by an internal tunnel of 31 A length, which runs from the site of L-glutamine hydrolysis to the site of L-glutamate synthesis. CONCLUSIONS: The outstanding property of glutamate synthase is the ability to coordinate the activity of its various functional sites to avoid wasteful consumption of L-glutamine. The structure reveals two polypeptide segments that connect the catalytic centers and embed the ammonia tunnel, thus being ideally suited to function in interdomain signaling. Depending on the enzyme redox and ligation states, these signal-transducing elements may affect the active site geometry and control ammonia diffusion through a gating mechanism
Crystal structure of D-amino acid oxidase: a case of active site mirror-image convergent evolution with flavocytochrome b2
No full textD-amino acid oxidase is the prototype of the FAD-dependent oxidases. It catalyses the oxidation of D-amino acids to the corresponding alpha-ketoacids. The reducing equivalents are transferred to molecular oxygen with production of hydrogen peroxide. We have solved the crystal structure of the complex of D-amino acid oxidase with benzoate, a competitive inhibitor of the substrate, by single isomorphous replacement and eightfold averaging. Each monomer is formed by two domains with an overall topology similar to that of p-hydroxybenzoate hydroxylase. The benzoate molecule lays parallel to the flavin ring and is held in position by a salt bridge with Arg-283. Analysis of the active site shows that no side chains are properly positioned to act as the postulated base required for the catalytic carboanion mechanism. On the contrary, the benzoate binding mode suggests a direct transfer of the substrate alpha-hydrogen to the flavin during the enzyme reductive half-reaction.The active site Of D-amino acid oxidase exhibits a striking similarity with that of flavocytochrome b2, a structurally unrelated FMN-dependent flavoenzyme. The active site groups (if these two enzymes are in fact superimposable once the mirror-image of the flavocytochrome b2 active site is generated with respect to the flavin plane. Therefore, the catalytic sites of D-amino acid oxidase and flavocytochrome b2 appear to have converged to a highly similar but enantiomeric architecture in order to catalvze similar reactions (oxidation of alpha-amino acids or alpha-hydroxy acids), although with opposite stereochemistry
Crystal structure of D-amino acis oxidase: a case of active site mirror-image convergent evolution with flavocytochrome b2
No full textEnergy matters: Mitochondrial proteomics for biomedicine
No full textThis review compiles results of medical relevance from mitochondrial proteomics, grouped either according to the type of disease - genetic or degenerative - or to the involved mechanism - oxidative stress or apoptosis. The findings are commented in the light of our current understanding of uniformity/variability in cell responses to different stimuli. Specificities in the conceptual and technical approaches to human mitochondrial proteomics are also outlined
Limited proteolysis and X-ray crystallography reveal the origin of substrate specificity and of the rate-limiting product release during oxidation of D-amino acids catalyzed by mammalian D-amino acid oxidase
No full textLimited proteolysis of D-amino acid oxidase holoenzyme with trypsin cleaves the protein at Arg 221 and near the C-terminus, producing stable 25, 13.4, and 2 kDa polypeptides [Torri-Tarelli, G., Vanoni, M. A., Negri, A., & Curti, B. (1990) J. Biol. Chem. 265, 21242-21246]. The 25 and 13.4 kDa polypeptides remain associated to form a nicked D-amino acid oxidase species. This nicked protein form maintains the ability to bind FAD, but exhibits altered catalytic efficiency toward the oxidation of various D-amino acids when compared to native DAAO. Changes in substrate specificity were first monitored by measuring the activity in the presence of different amino acid substrates at various times during proteolysis. Three amino acid substrates were then selected for further analysis of the properties of the nicked D-amino acid oxidase species produced by limited tryptic proteolysis: D-serine, D-arginine, and D-alanine. The three D-amino acids represented limiting cases of the observed changes of enzyme activity on nicking: loss of activity, increase of activity, and minor activity changes, respectively. D-serine was found to be no longer a substrate of D-amino acid oxidase. D-arginine exhibited a 2.5-fold increased apparent maximum velocity although its Km value increased 2-fold with the nicked enzyme in comparison to the native species. D-alanine was oxidized 1.5-fold faster by the nicked D-amino acid oxidase at infinite substrate concentration, and its Km value increased approximately 4-fold. The Kd for benzoate, which was determined kinetically with D-alanine as the enzyme substrate, increased 17-fold in the nicked species. Primary deuterium kinetic isotope effects on V and V/K during the oxidation of D-alanine were also measured. (D)V/K increased from 1.4 +/- 0.2 to 1.8 +/- 0.3 on nicking, while (D)V increased from 1.04 +/- 0.1 to 2.53 +/- 0.5. All the observed changes of the values of the kinetic parameters and of the observed isotope effects are consistent with the hypothesis that nicking of D-amino acid oxidase at position 221 decreases the strength of binding of both substrates and products to the enzyme active site. The information obtained by limited tryptic proteolysis nicely complements that gathered from the analysis of the three-dimensional structure of D-amino acid oxidase in complex with benzoate, which was recently determined [Mattevi, A., Vanoni, M. A., Todone, F., Rizzi, M., Teplyakov, A., Coda, A., Bolognesi, M., & Curti, B. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 7496-7501]. Arginine 221 is part of the 216-228 loop that covers the active site and contributes residues to substrate binding and catalysis. The limited proteolysis data support the hypothesis that this loop acts as a lid on the active site and controls both substrate specificity and the rate of turnover of D-amino acid oxidase
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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