20 research outputs found

    Etude de deux enzymes paralogues Mra Y et WECA impliquées dans la biosynthèse de la paroi bactérienne, membres d une famille de protéines membranaires Polyprenyl-phosphate n-acetyl-hexosamine 1-phosphate transferases

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    L enzyme mray catalyse la premiere etape membranaire de la biosynthese du peptidoglycane. Elle est essentielle à la viabilite de la bacterie, ce qui fait de cette enzyme une cible privilegiee pour la recherche de nouveaux antibiotiques. La topologie membranaire de mray a ete determinee. Elle est composee de 10 segments transmembranaires, 4 sequences periplasmiques, et 5 sequences cytoplasmiques contenant de nombreux residus invariants. Une partie de ce travail de these a concerne la cartographie du site actif de la translocase mray et l etude de son mecanisme catalytique. Pour cela, dix-neuf proteines mutantes de mray de bacillus subtilis ont ete produites par mutagenese dirigee des residus invariants. Les plasmides portant ces mutations ont ete testes par complementation fonctionnelle in vivo. Quatorze proteines mutantes, qui n assurent pas la complementation d une souche mutante mray thermosensible, ont alors ete purifiees a homogeneite et leur analyse enzymatique a ete effectuee. Les etudes d activite a differents ph suggerent l implication du residu d98 dans la deprotonation de l undecaprenyl-phosphate (c55-p) pendant la catalyse. L activite enzymatique des proteines mutantes a differentes concentrations de mg2+ a ete determinee. Les resultats suggerent l implication des residus d174, d177 et h45 dans la fixation du mg2+. L etude du mecanisme catalytique de l enzyme mray a ete entreprise. Les resultats de nos experiences supportent l hypothese selon laquelle la formation du lipide i s effectuerait selon un mecanisme catalytique en une etape, a savoir l attaque directe d un oxyanion du phosphate de l undecaprenyl-phosphate sur le phosphate ß du substrat nucleotidique. Un autre volet de ce travail a ete consacre a l etude de la transferase membranaire weca, paralogue de mray et membre de la meme famille d enzymes, les polyprenyl-phosphate n-acetyl-hexosamine 1-phosphate transferases. Pour la premiere fois, nous avons reussi la surproduction, l extraction des membranes et la purification a homogeneite de cette enzyme. Sa caracterisation biochimique a egalement ete effectuee. Une partie importante de cette these concerne l etude et la recherche de nouveaux antibiotiques. Sur la base de la structure de la liposidomycine, un inhibiteur naturel de mray, de nouveaux composes ont ete synthetises. Les effets de ces derniers sur l activite enzymatique de mray ont ete analyses. Une inhibition interessante a ete observee avec un de ces composes avec une ic50 de 0,58 mm. La derniere partie de cette these concerne l etude de l interaction entre les intermediaires lipidiques de la biosynthese du peptidoglycane et un nouveau lantibiotique produit par bacillus claucii, baptise claucine. Ce travail a ete realise par des approches de biophysique. Nous avons montre que les lipides i et ii sont des cibles de ce nouveau lantibiotique. En revanche, l undecaprenyl-phosphate, l udp-murnac-pentapeptide ou son analogue pyrophospho-murnac-pentapeptide n interagissent pas avec la claucine.The mray transferase is an integral membrane protein that catalyzes an essential step of peptidoglycan biosynthesis, namely the transfer of the phospho-n-acetylmuramoyl pentapeptide motif onto the undecaprenyl phosphate carrier lipid. It belongs to a large superfamily of eukaryotic and prokaryotic prenyl sugar transferases. Nineteen polar residues located in the five cytoplasmic segments of mray appeared as invariants in the sequences of mray orthologues. A certain number of these invariant residues were found to be conserved in the whole superfamily. To assess the importance of these residues in the catalytic process, site-directed mutagenesis was performed using the bacillus subtilis mray as a model. Fourteen residues were shown to be important for mray activity by an in vivo functional complementation assay using a constructed conditional mray mutant strain. The corresponding mutant proteins were purified and biochemically characterized. None of these mutations did significantly affect the binding of both the nucleotidic and lipidic substrates, but the kcat was drastically reduced in almost all cases. The important residues for activity appeared to be distributed in all the cytoplasmic segments, indicating that these five regions contribute to the structure of the catalytic site. Our data show that the d98 residue which is invariant in the whole superfamily should be involved in the deprotonation of the lipid substrate during the catalytic process. The effects of magnesium (required for mray activity) on the activity of the wild-type and mutant proteins were tested. Moreover, our results suggest the involvement of h45, d174 and d177 residues in the binding of the metal ion. We also studied the catalytic mechanism of mray. Based on our present results, we here propose a reaction proceeding via a single displacement mechanism. In this one-step mechanism, the oxyanion of the undecaprenyl-phosphate attacks the b phosphate of the nucleotidic substrate leading to the formation of lipid i and the liberation of ump. During this thesis, we were also interested in studying the weca transferase, an integral membrane protein, paralogue of mray. In this thesis and for the first time, the weca protein was overproduced and purified to near homogeneity in milligram quantities. Its kinetic parameters and biochemical characterization were determined. Another aspect of our work concerned the research of new antibacterial drugs. Indeed, mray, an essential integral membrane protein is a promising potential target to be exploited for new antibacterial discovery. Based on the structure of the liposidomycin, a natural inhibitor of mray, new compounds were synthesized. Their effects on the enzymatic activity of mray were analyzed. In the case of one compound, an important inhibition was observed with an ic50 of 0.58 mm. In the last part of this manuscript we investigated the specificity of interaction of a new type a lantibiotic, termed claucin, isolated from bacillus claucii with the lipid precursors of the peptidoglycan biosynthesis. Our data show that these lipid intermediates are the targets of this new lantibiotic. However, no interaction was observed when undecaprenyl-phosphate, udp-murnac-pentapetide and its analogue the pyrophosphate-murnac-pentapeptide were tested.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

    Purification and Characterization of the Bacterial UDP-GlcNAc:Undecaprenyl-Phosphate GlcNAc-1-Phosphate Transferase WecA▿

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    To date, the structural and functional characterization of proteins belonging to the polyprenyl-phosphate N-acetylhexosamine-1-phosphate transferase superfamily has been relentlessly held back by problems encountered with their overexpression and purification. In the present work and for the first time, the integral membrane protein WecA that catalyzes the transfer of the GlcNAc-1-phosphate moiety from UDP-GlcNAc onto the carrier lipid undecaprenyl phosphate, yielding undecaprenyl-pyrophosphoryl-GlcNAc, the lipid intermediate involved in the synthesis of various bacterial cell envelope components, was overproduced and purified to near homogeneity in milligram quantities. An enzymatic assay was developed, and the kinetic parameters of WecA as well as the effects of pH, salts, cations, detergents, and temperature on the enzyme activity were determined. A minimal length of 35 carbons was required for the lipid substrate, and tunicamycin was shown to inhibit the enzyme at submicromolar concentrations

    Antioxidant and anticancer activities of chamomile (Matricaria recutita L.)

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    Abstract Objectives The present study aimed at determining the antioxidant activity, total phenols and flavonoids and to evaluate the antiproliferative activity of ethanolic extract of Matricaria recutita L. (chamomile). The antioxidant activities were measured using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay. The total phenolic content was measured by the Folin–Ciocalteu assay. The flavonoid content was determined using the aluminum chloride method. The MTT assay was used to estimate the antiproliferative activities against human hepatoma (HepG2) cancer cell line. We assessed the mode of action of the extract as a cancer preventive agent and reported its ability to regulate tumor angiogenesis by down regulating in a dose dependent manner the expression of some proteins involved in the process. Results The percentage inhibition of DPPH scavenging activity was dose-dependent ranging between (94.8% ± 0.03) at 1.50 mg/mL and (84.2% ± 0.86) at 0.15 mg/mL. It showed high polyphenols (21.4 ± 0.327 mg GAE/g) and high flavonoids content (157.9 ± 2.22 mg QE/g). Effect of extract was investigated against HepG2 cells. A dose-dependent reduction in cell viability was recorded in cells treated with the extract. The IC50 was ~ 300 µg/mL. It significantly inhibited the level of important prerequisite angiogenesis markers both in HepG2 cells and ex vivo

    Catalytic mechanism of MraY and WecA, two paralogues of the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase superfamily.

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    International audienceThe MraY transferase catalyzes the first membrane step of bacterial cell wall peptidoglycan biosynthesis, namely the transfer of the N-acetylmuramoyl-pentapeptide moiety of the cytoplasmic precursor UDP-MurNAc-pentapeptide to the membrane transporter undecaprenyl phosphate (C55P), yielding C55-PP-MurNAc-pentapeptide (lipid I). A paralogue of MraY, WecA, catalyzes the transfer of the phospho-GlcNAc moiety of UDP-N-acetylglucosamine onto the same lipid carrier, leading to the formation of C55-PP-GlcNAc that is essential for the synthesis of various bacterial cell envelope components. These two enzymes are members of the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase superfamily, which are essential for bacterial envelope biogenesis. Despite the availability of detailed biochemical information on the MraY enzyme, and the recently published crystal structure of MraY of Aquifex aeolicus, the molecular basis for its catalysis remains poorly understood. This knowledge can contribute to the design of potential inhibitors. Here, we report a detailed catalytic study of the Bacillus subtilis MraY and Thermotoga maritima WecA transferases. Both forward and reverse exchange reactions required the presence of the second substrate, C55P and uridine monophosphate (UMP), respectively. Both enzymes did not display any pyrophosphatase activity on the nucleotide substrate. Moreover, we showed that the nucleotide substrate UDP-MurNAc-pentapeptide, as well as the nucleotide product UMP, can bind to MraY in the absence of lipid ligands. Therefore, our data are in favour of a single displacement mechanism. During this "one-step" mechanism, the oxyanion of the polyprenyl-phosphate attacks the β-phosphate of the nucleotide substrate, leading to the formation of lipid product and the liberation of UMP. The involvement of an invariant aspartyl residue in the deprotonation of the lipid substrate is discussed

    Characteristics and in-hospital outcomes of patients with acute coronary syndromes and heart failure in the United Arab Emirates

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    <p>Abstract</p> <p>Background</p> <p>Heart failure (HF) is a serious complication of acute coronary syndromes (ACS), and is associated with high in-hospital mortality and poor long-term survival. The aims of this study were to describe the clinical characteristics, management and in-hospital outcomes of coronary syndrome (ACS) patients with HF in the United Arab Emirates.</p> <p>Findings</p> <p>The study was selected from the Gulf Registry of Acute Coronary Events (Gulf RACE), a prospective multi-national, multicenter registry of patients hospitalized with ACS in six Middle East countries. The present analysis was focused on participants admitted to various hospitals in the UAE with a diagnosis of ACS in 2007 and were analyzed in terms of HF (Killip class II/III and IV) on admission. Of 1691 patients (mean age: 52.6 ± 11.7 years; 210 Females, 1481 Males) with ACS, 356 (21%) had an admission diagnosis of HF (Killip class II/III and IV). HF patients were less frequently males (19.2% vs. 34.3%; P <it><</it> 0.001). HF was more frequently associated with hypertension (64.3% vs. 43.9%; P < 0.001), hyperlipidemia (49.4% vs. 31.8%; P < 0.001) and diabetes mellitus (DM) (51.1% vs. 36.2%; P < 0.001). HF was significantly associated with in-hospital mortality (OR = 11.821; 95% CI: 5.385-25.948; P < 0.001). In multivariate logistic regression, age, hyperlipidemia, heart rate and DM were associated with higher in-hospital HF.</p> <p>Conclusions</p> <p>HF is observed in about 1 in 5 patients with ACS in the UAE and is associated with a significant increase in in-hospital mortality and other adverse outcomes.</p

    Prevalence of Undiagnosed Diabetes and Quality of Care in Diabetic Patients Followed at Primary and Tertiary Clinics in Abu Dhabi, United Arab Emirates

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    AIMS: To investigate the prevalence of undiagnosed type 2 diabetes (T2D) at primary health care (PHC) clinics, and to assess the quality of care of diabetic patients followed at a tertiary hospital diabetes center in Abu Dhabi, United Arab Emirates (UAE). METHODS: Between May 2009 and October 2010, adult patients attending two PHC clinics, and adult diabetic patients attending the diabetes center, were invited to participate in the study. After overnight fast, participants returned for interview and laboratory tests. Undiagnosed T2D was defined by FPG ≥ 7.0 mmol/l or HbA1c ≥ 6.5%. Quality of care was assessed by reported care practices and achievement of internationally recognized targets. RESULTS: Out of 239 patients at PHC clinics without history of T2D, 14.6% had undiagnosed T2D, and 31% had increased risk of diabetes (FPG 5.6-7.0 mmol/l or HbA1c 5.7-6.5%). The independent predictors of undiagnosed T2D were age (adjusted OR per year 1.07, 95% CI 1.04-1.11, p < 0.001) and BMI ≥ 25 (adjusted OR 4.2, 95% CI 0.91-19.7, p = 0.033). Amongst all 275 diagnosed T2D patients, including those attending PHC clinics and those followed at the diabetes center, it was found that 40.1% followed dietary recommendations, 12% reported visiting a diabetes educator, 28.2% walked for exercise, and 13.5% attained recognized targets of HbA1c < 7%, blood pressure < 130/80 mmHg, and LDL cholesterol < 2.6 mmol/l. CONCLUSIONS: Almost half of the adult patients attending PHC clinics had undiagnosed T2D, or increased diabetes risk. Care practices, and achievement of treatment targets, were suboptimal

    Characteristics, Management, and In-Hospital Outcomes of Diabetic Patients with Acute Coronary Syndrome in the United Arab Emirates

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    We describe the baseline characteristics, management, and in-hospital outcomes of patients in the United Arab Emirates (UAE) with DM admitted with an acute coronary syndrome (ACS) and assess the influence of DM on in-hospital mortality. Data was analyzed from 1697 patients admitted to various hospitals in the UAE with a diagnosis of ACS in 2007 as part of the 1st Gulf RACE (Registry of Acute Coronary Events). Of 1697 patients enrolled, 668 (39.4%) were diabetics. Compared to patients without DM, diabetic patients were more likely to have a past history of coronary artery disease (49.1% versus 30.1%, P<0.001), hypertension (67.2% versus 36%, P<0.001), and prior revascularization (21% versus 11.4%, P<0.001). They experienced more in-hospital recurrent ischemia (8.5% versus 5.1%; P=0.004) and heart failure (20% versus 10%; P<0.001). The mortality rate was 2.7% for diabetics and 1.6% for nondiabetics (P=0.105). After age adjustment, in-hospital mortality increased by 3.5% per year of age (P=0.016). This mortality was significantly higher in females than in males (P=0.04). ACS patients with DM have different clinical characteristics and appear to have poorer outcomes
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