1,720,957 research outputs found
Biochemical and structural analysis of peroxisomal enzymes involved in ether phospholipid synthesis
No full textEther phospholipids are essential components of eukaryotic cell membrane and are involved in many fundamental functions such as bone development, signal transduction and intracellular transport. They are characterised by an alkyl or alkenyl substituent at sn-1 position of the glycerol backbone. Dihydroxyacetone-phosphate acyltransferase (DHAPAT) and the flavoenzyme alkyldihydroxyacetone-phosphate synthase (ADPS) work in complex to introduce an ether linkage into a phospholipid-precursor molecule. Deficit in ether phospholipids has very serious pathological consequences as observed in patients affected by genetic diseases such as Zellweger syndrome and rhizomelic chondrodysplasia punctata (RCDP). Here, we present the crystal structure of the mammalian Cavia porcellus ADPS, which reveals a dimeric organization. ADPS consists of a FAD-binding domain, a cap domain, and a substrate-binding domain. This third domain exhibits a beta-sheet and a gating alpha-helix, which is involved in the closure of a substrate tunnel specific for aliphatic chains of 16 carbon atoms. Superposition of C. porcellus and Dictyostelium discoideum ADPS structures shows that the FAD-binding domain is highly conserved between the two species whereas the substrate-binding domain is rotated by 20 degrees. Mutations in the amino acids Arg419, Tyr515, and Tyr578 have been found in patients affected by RCDP. Mutagenesis studies indicate that each of these mutations lead to the complete inactivation of the enzyme. Structural data confirmed the role of these side chains as they are located in the active site and are likely to directly take part in catalysis. Our data are discussed in the context of a proposed catalytic mechanism that involves formation of a covalent adduct between the flavin and the substrate, which enables the enzyme to catalyze the acyl-alkyl exchange reaction through a most unusual non-redox mechanism
Crystal structure of human renalase, a novel flavoenzyme involved in the pathogenesis of cardiovascular diseases
No full textRenalase is a recently identified flavoprotein (1), highly conserved in vertebrates, with orthologs in other organisms, including lower eukaryotes and bacteria. In humans, renalase is synthetized in kidneys, heart, skeletal muscles, brain and small intestine, being present in blood and urine (2). In mammals, renalase has been shown to regulate blood pressure, sodium and phosphate excretion, and to exert a cardioprotectant action (2). Despite its medical relevance, the mechanism of renalase action is not known at the molecular level (3); based on its moderate sequence similarity to monoamine oxidases (MAOs), it has been proposed to be a catecholamine degrading enzyme. To gain insight into its catalytic activity, we produced human renalase in Escherichia coli and showed that it contains non-covalently bound FAD (4), slightly stabilizes the neutral flavin semiquinone, and reacts slowly with sodium dithionite to yield a flavin adduct, with a Kd of ca. 2 mM. We found that renalase is devoid of any measurable catecholamine oxidase or dehydrogenase activities. Further, we solved the crystal structure of human renalase at 2.5 Å resolution. The protein adopts the p-hydroxybenzoate hydroxylase fold, and is structurally related to MAO-like enzymes. However, renalase is composed of two domains, thus lacking the third domain conserved in the other members of the family. A cavity (228 Å3), facing the re-face of the isoalloxazine, likely representing the active site, opens to the molecular surface. Compared to mono- or poly-amine oxidases, the renalase putative active-site lacks both a conserved Lys, that would interact via a water molecule with the N5 atom of the flavin ring, and the ‘aromatic cage’ expected to bind the substrate amino-group. Although these data do not allow to assign a catalytic activity to renalase yet, our studies represent a reference framework to test hypotheses on the enzyme molecular mechanism of action.
References
1)Xu, J., Li, G., Wang, P., Velazquez, H., Yao, X., Li, Y., Wu, Y., Peixoto, A., Crowley, S., Desir, G.V. (2005) J. Clin. Invest. 115, 1275-1280.
2)Desir, G. V. (2011) Curr. Opin. Nephrol. Hypertens. 20, 31-36.
3)Eikelis, N., Hennebry, S.C., Lambert, G.W., Schlaich, M.P. (2011) Kidney Int. 79, 1380.
4)Pandini, V., Ciriello, F., Tedeschi, G., Rossoni, G., Zanetti, G., Aliverti, A. (2010) Protein Expr. Purif. 72, 244-253
A plant-type enzyme as a putative target for novel antimalarial drugs : properties of the Plasmodium falciparum ferredoxin-NADP+ reductase
No full textApicomplexan parasites harbor a specific organelle, named apicoplast, which is related to plant non-photosynthetic plastids and displays a plant-like metabolism. The apicoplast has been shown to contain typical vegetal proteins, such as ferredoxin–NADP+ reductase (FNR) and ferredoxin (Fd) (1-2). Both proteins from Plasmodium falciparum (PfFNR and PfFd) have been produced in recombinant form and characterized (3). The PfFNR/PfFd couple was shown to be catalytically active in vitro yielding reducing power to support the activity of LytB (4), the last enzyme of the biosynthetic pathway for isoprenoid precursors, a known site of action of antiplasmodial compounds. On this basis, PfFNR has been proposed as a possible target for new antimalarial drugs (2).
The three-dimensional structure of PfFNR has been determined by X-ray crystallography (3). Compared to other plastidic-type FNRs, PfFNR displays a significantly lower catalytic efficiency and lower selectivity against NADH. These functional features are probably the consequence of the lack of protein positively-charges stabilizing the 2’-phosphate of the bound substrate. NADP(H) binding to PfFNR occurs through an induced-fit mechanism never observed in other FNRs. The conformational changes induced by binding to the enzyme of 2’-P-AMP, a NADP+ analogue, includes the partial unwinding of an α-helix localized in the NADP+-binding domain. Furthermore, the binding of NADP+ triggers the formation of a disulfide-stabilized homodimer resulting in the inactivation of PfFNR. This process, observed in vitro, could represent a physiologic mechanism regulating the enzyme activity.
Structure-based design of PfFNR inhibitors is in progress and has already yielded some active compounds, with inhibitory constants in the range of micromolar or lower.
1. Pandini V. et al. (2002) J. Biol. Chem. 277, 48463-48471
2. Seeber F. et al. (2005) Curr. Farm. Des. 11, 3159-7312
3. Milani et al. (2007) J. Mol. Biol. 367, 501-513
4. Röhrich R.C. et al. (2005) FEBS lett. 579, 6433-643
Involvement of serine 96 in the catalytic mechanism of ferredoxin-NADP+ reductase: structure--function relationship as studied by site-directed mutagenesis and X-ray crystallography
No full textThe crystal structure of ferredoxin-NADP+ reductase (FNR) suggests that Ser96 is directly involved in hydride transfer between the isoalloxazine moiety of FAD and the nicotinamide ring of NADP(H). To probe its role, Ser96 has been mutated to valine (S96V) and glycine (S96G). These mutations primarily affected the interaction of the nicotinamide ring with the flavin. Absorbance, fluorescence, and circular dichroism spectra and the crystal structure of FNR-S96V indicate that this mutant folds properly. FNR-S96V shows only 0.05% of wild-type activity, while the affinities for both ferredoxin and NADP+ are virtually unchanged. However, spectral perturbations induced by NADP+ binding to FNR-S96V strongly resemble those elicited by the binding of 2'-monophosphoadenosine-5'-diphosphoribose, a substrate analog lacking the nicotinamide ring, both to the mutant and wild-type enzymes. Rapid reaction studies on the valine mutant failed to detect charge-transfer intermediates during flavin reduction by NADPH. In addition, no semiquinone formation was seen during photoreduction of FNR-S96V. The three-dimensional structure of the valine mutant shows small, albeit definite, changes only in the isoalloxazine microenvironment. The glycine mutant of FNR displays behavior intermediate between that of wild-type enzyme and that of the valine mutant. It maintains ca. 2% of the wild-type activity as well as the ability to form the charge-transfer species between reduced FNR and NADP+. In photoreduction experiments, the same degree of flavin semiquinone stabilization was observed with FNR-S96G and with the wild-type enzyme. NADP+ binding to the glycine mutant was very similar to that observed in the case of the valine mutant
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
High-resolution studies of hydride transfer in the ferredoxin:NADP+ reductase superfamily
Full text linkFerredoxin: NADP+ reductase (FNR) is an FAD-containing enzyme best known for catalysing the transfer of electrons from ferredoxin (Fd) to NADP+ to make NADPH during photosynthesis. It is also the prototype for a broad enzyme superfamily, including the NADPH oxidases (NOXs) that all catalyse similar FAD-enabled electron transfers between NAD(P)H and one-electron carriers. Here, we define further mechanistic details of the NAD(P)H ⇌ FAD hydride-transfer step of the reaction based on spectroscopic studies and high-resolution (~ 1.5 Å) crystallographic views of the nicotinamide-flavin interaction in crystals of corn root FNR Tyr316Ser and Tyr316Ala variants soaked with either nicotinamide, NADP+ , or NADPH. The spectra obtained from FNR crystal complexes match those seen in solution and the complexes reveal active site packing interactions and patterns of covalent distortion of the FAD that imply significant active site compression that would favour catalysis. Furthermore, anisotropic B-factors show that the mobility of the C4 atom of the nicotinamide in the FNR:NADP+ complex has a directionality matching that expected for boat-like excursions of the nicotinamide ring thought to enhance hydride transfer. Arguments are made for the relevance of this binding mode to catalysis, and specific consideration is given to how the results extrapolate to provide insight to structure-function relations for the membrane-bound NOX enzymes for which little structural information has been available
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
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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