Isoprenoid biosynthesis via the methylerythritol phosphate pathway: the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (LytB/IspH) from Escherichia coli is a [4Fe–4S] protein

AbstractThe last enzyme (LytB) of the methylerythritol phosphate pathway for isoprenoid biosynthesis catalyzes the reduction of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate into isopentenyl diphosphate and dimethylallyl diphosphate. This enzyme possesses a dioxygen-sensitive [4Fe–4S] cluster. This prosthetic group was characterized in the Escherichia coli enzyme by UV/visible and electron paramagnetic resonance spectroscopy after reconstitution of the purified protein. Enzymatic activity required the presence of a reducing system such as flavodoxin/flavodoxin reductase/reduced nicotinamide adenine dinucleotide phosphate or the photoreduced deazaflavin radical

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Uluslararası Bakalorya Programı, A1 dersi Türk Dili ve Edebiyatı alanında ele alınan bu tezde, Orhan Kemal'in Gurbet Kuşları adlı yapıtında göç olgusu nedenleri ve sonuçlarıyla beraber incelenmiştir. Göç olgusuyla değişen toplumsal yapı, ekonomik ve kültürel farklılıklar çerçevesinde değerlendirilmiştir. Bu tezin amacı, göç olgusunun toplumsal yapıda alt sınıf ve üst sınıflardaki bireyler üzerindeki etkilerini ortaya koymaktır. Üç ana bölümden oluşan tezin ilk bölümünde yapıta adını veren Gurbet Kuşları kavramı üzerinde durulmuştur. Köylülerin aidiyetsizliği ve uyum sorunu bu bölümde aktarılmıştır. Tezin ikinci bölümünde ise köylülerin köyden kente göç sürecinde yaşadıkları kadın ve erkek figürler üzerinden neden ve sonuçlarıyla işlenmiştir. Tezin üçüncü bölümünde şehirliler başlığı altından genel olarak şehirde – İstanbul – yaşayan insanların göç sürecinde köylülerle yaşadıkları uyumsuzluk ve çatışmalara yer verilmektedir. Çalışmada göç sürecinde şehre yerleşen figürlerin şehirlilerle aralarındaki ekonomik ve kültürel farklılıkların sınıflar arasında geçişe olanak tanımadığı sonucuna varılmıştır

Biosynthèse des isoprénoïdes bactériens (Inhibiteurs potentiels de la désoxyxylulose phosphate synthase et caractérisation des étapes de réduction dans la formation des hopanïdes)

La désoxyxylulose phosphate synthase (DXS), première enzyme de la voie du MEP présente chez de nombreux microorganismes mais absente chez les animaux, représente une cible potentielle pour le développement de nouveaux médicaments contre les maladies causées par des bactéries pathogènes ou par des parasites. Dans cet objectif, l élaboration d inhibiteurs potentiels de cette enzyme a été envisagée. Plusieurs accepteurs de Michael, un analogue phosphoré du pyruvate et des dérivés di- et trihalogénés ont été synthétisés. Certains de ces composés montrent une faible activité inhibitrice de la DXS. De plus, afin de mieux comprendre la biosynthèse des isoprénoïdes, nous nous sommes plus particulièrement intéressés à la protonation in vivo d un cation allylique intermédiaire dans la réaction catalysée par la dernière enzyme de la voie du MEP (LytB). Le donneur de proton serait le cofacteur (FADH2) de réduction utilisé à la fois comme donneur d électrons et de protons. Pour mettre en évidence la signature de ces étapes de réduction, le métabolisme de Zymomonas mobilis est utilisé, car il permet la formation d un pool de NADPH deutérié en incubant du [1-2H]glucose. Le deutérium est donc incorporé dans tous les cofacteurs de réduction et serait transféré sur les unités isopréniques dérivées de l IPP (C-2) ou du DMAPP (C-4). Les positions du deutérium ont été déterminées dans les triterpénoïdes de série hopane par RMN 13C en analysant les déplacements vers les champs forts des signaux du 13C. L introduction de deutérium en position C-2 a été confirmée, la présence de deutérium en position C-4 et d une activité IPP isomérase chez Zymomonas mobilis a pu être mise en évidence.The enzymesoftheMEP pathwaypresentinmanymicroorganismsbutabsentin animals,are potential targetsfordevelopingnew drugsagainst diseases causedby pathogenic bacteria or parasites.Inthisobjective, thedevelopmentof potential inhibitorsof thispathway hasbeen considered.Thedeoxyxylulosephosphatesynthase (DXS),the firstenzyme of theMEP pathway, was chosenas a targetandanaloguesof its substrate,pyruvate,weresynthesized andtheirbiochemical characteristicshave beenestablished.signals of 13C.Inaddition, toimprove the understanding of thebiosynthesisofisoprenoids, experimentswere performedonZymomonasmobilistodeterminetheoriginof thehydrogen atomsof isopentenyldiphosphate(IPP)anddimethylallyldiphosphate(DMAPP) from the MEP.Weare particularly interested instudyinginvivotheprotonationofallylic cationic intermediate inthereactioncatalyzedby thelastenzymeoftheMEP pathway(LytB). The proton donoristhereducing cofactor(FADH2)usedbothas an electronsdonorand protons donor.Tohighlightthesigningof thesereduction steps, themetabolism of Zymomonasmobilisis usedbecauseitallowstheformationofapoolofdeuterated NADPH by incubating[1-2H]glucose. Deuteriumisincorporated inallreducing cofactors NADP2H and thusFAD2H2andwould betransferredtotheisoprene units derivedfromIPP orDMAPP. The positions of deuterium were determined in hopanoids by 13C NMR by analyzing the shifted signals of 13C.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Nouveau test d'activité des NAD+glycohydrolases (activité catalytique des enzymes cristallisées)

STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

New Application of <i>cyclo</i>Saligenyl Prodrugs Approach for the Delivery of Fosfoxacin Derivatives in Mycobacteria

In this work, we implemented for the first time the cycloSaligenyl prodrug strategy to increase the bioavailability of fosmidomycin phosphate analogs in bacteria. Here, we report the synthesis of 34 cycloSaligenyl prodrugs of fosfoxacin and its derivatives. Among them, fifteen double prodrugs efficiently prevented the growth of the non-pathogenic, fast-growing Mycobacterium smegmatis

A Cytosolic Arabidopsis d-Xylulose Kinase Catalyzes the Phosphorylation of 1-Deoxy-d-Xylulose into a Precursor of the Plastidial Isoprenoid Pathway

Plants are able to integrate exogenous 1-deoxy-d-xylulose (DX) into the 2C-methyl-d-erythritol 4-phosphate pathway, implicated in the biosynthesis of plastidial isoprenoids. Thus, the carbohydrate needs to be phosphorylated into 1-deoxy-d-xylulose 5-phosphate and translocated into plastids, or vice versa. An enzyme capable of phosphorylating DX was partially purified from a cell-free Arabidopsis (Arabidopsis thaliana) protein extract. It was identified by mass spectrometry as a cytosolic protein bearing d-xylulose kinase (XK) signatures, already suggesting that DX is phosphorylated within the cytosol prior to translocation into the plastids. The corresponding cDNA was isolated and enzymatic properties of a recombinant protein were determined. In Arabidopsis, xylulose kinases are encoded by a small gene family, in which only two genes are putatively annotated. The additional gene is coding for a protein targeted to plastids, as was proved by colocalization experiments using green fluorescent protein fusion constructs. Functional complementation assays in an Escherichia coli strain deleted in xk revealed that the cytosolic enzyme could exclusively phosphorylate xylulose in vivo, not the enzyme that is targeted to plastids. xk activities could not be detected in chloroplast protein extracts or in proteins isolated from its ancestral relative Synechocystis sp. PCC 6803. The gene encoding the plastidic protein annotated as “xylulose kinase” might in fact yield an enzyme having different phosphorylation specificities. The biochemical characterization and complementation experiments with DX of specific Arabidopsis knockout mutants seedlings treated with oxo-clomazone, an inhibitor of 1-deoxy-d-xylulose 5-phosphate synthase, further confirmed that the cytosolic protein is responsible for the phosphorylation of DX in planta

Striatal neurones have a specific ability to respond to phasic dopamine release

International audienceThe cAMP/protein kinase A (PKA) signalling cascade is ubiquitous, and each step in this cascade involves enzymes that are expressed in multiple isoforms. We investigated the effects of this diversity on the integration of the pathway in the target cell by comparing prefrontal cortical neurones with striatal neurones which express a very specific set of signalling proteins. The prefrontal cortex and striatum both receive dopaminergic inputs and we analysed the dynamics of the cAMP/PKA signal triggered by dopamine D-1 receptors in these two brain structures. Biosensor imaging in mouse brain slice preparations showed profound differences in the D-1 response between pyramidal cortical neurones and striatal medium spiny neurones: the cAMP/PKA response was much stronger, faster and longer lasting in striatal neurones than in pyramidal cortical neurones. We identified three molecular determinants underlying these differences: different activities of phosphodiesterases, particularly those of type 4, which strongly damp the cAMP signal in the cortex but not in the striatum; stronger adenylyl cyclase activity in the striatum, generating responses with a faster onset than in the cortex; and DARPP-32, a phosphatase inhibitor which prolongs PKA action in the striatum. Striatal neurones were also highly responsive in terms of gene expression since a single sub-second dopamine stimulation is sufficient to trigger c-Fos expression in the striatum, but not in the cortex. Our data show how specific molecular elements of the cAMP/PKA signalling cascade selectively enable the principal striatal neurones to respond to brief dopamine stimuli, a critical process in incentive learning