Biosynthetic Gene Cluster of the Glycopeptide Antibiotic Teicoplanin Characterization of Two Glycosyltransferases and the Key Acyltransferase

AbstractThe gene cluster encoding biosynthesis of the clinically important glycopeptide antibiotic teicoplanin has been cloned from Actinoplanes teichomyceticus. Forty-nine putative open reading frames (ORFs) were identified within an 89 kbp genetic locus and assigned roles in teicoplanin biosynthesis, export, resistance, and regulation. Two ORFs, designated orfs 1 and 10*, showed significant homology to known glycosyltransferases. When heterologously expressed in Escherichia coli, these glycosyltransferases were shown to catalyze the transfer of UDP-(N-acetyl)-glucosamine onto, respectively, 3-chloro-β-hydroxytyrosine-6 (3-Cl6βHty) and 4-hydroxyphenylglycine-4 (4Hpg) of the teicoplanin heptapeptide aglycone. The product of another ORF, orf11*, was demonstrated in vitro to transfer n-acetyl-, n-butyryl-, and n-octanoyl-groups from acyl-CoA donors either to a free UDP-aminosugar or to an aminosugar moiety in the teicoplanin pseudoaglycone, thus identifying Orf11* as the key acyltransferase in teicoplanin maturation. These findings should accelerate the combinatorial engineering of new and improved glycopeptide drugs

The Gene Cluster for Fluorometabolite Biosynthesis in Streptomyces cattleya: A Thioesterase Confers Resistance to Fluoroacetyl-Coenzyme A

SummaryA genomic library of Streptomyces cattleya was screened to isolate a gene cluster encoding enzymes responsible for the production of fluorine-containing metabolites. In addition to the previously described fluorinase FlA which catalyzes the formation of 5′-fluoro-5′-deoxyadenosine from S-adenosylmethionine and fluoride, 11 other putative open reading frames have been identified. Three of the proteins encoded by these genes have been characterized. FlB was determined to be the second enzyme in the pathway, catalyzing the phosphorolytic cleavage of 5′-fluoro-5′-deoxyadenosine to produce 5-fluoro-5-deoxy-D-ribose-1-phosphate. The enzyme FlI was found to be an S-adenosylhomocysteine hydrolase, which may act to relieve S-adenosylhomocysteine inhibition of the fluorinase. Finally, flK encodes a thioesterase which catalyzes the selective breakdown of fluoroacetyl-CoA but not acetyl-CoA, suggesting that it provides the producing strain with a mechanism for resistance to fluoroacetate

Combinations of PARP Inhibitors with Temozolomide Drive PARP1 Trapping and Apoptosis in Ewing's Sarcoma.

Ewing's sarcoma is a malignant pediatric bone tumor with a poor prognosis for patients with metastatic or recurrent disease. Ewing's sarcoma cells are acutely hypersensitive to poly (ADP-ribose) polymerase (PARP) inhibition and this is being evaluated in clinical trials, although the mechanism of hypersensitivity has not been directly addressed. PARP inhibitors have efficacy in tumors with BRCA1/2 mutations, which confer deficiency in DNA double-strand break (DSB) repair by homologous recombination (HR). This drives dependence on PARP1/2 due to their function in DNA single-strand break (SSB) repair. PARP inhibitors are also cytotoxic through inhibiting PARP1/2 auto-PARylation, blocking PARP1/2 release from substrate DNA. Here, we show that PARP inhibitor sensitivity in Ewing's sarcoma cells is not through an apparent defect in DNA repair by HR, but through hypersensitivity to trapped PARP1-DNA complexes. This drives accumulation of DNA damage during replication, ultimately leading to apoptosis. We also show that the activity of PARP inhibitors is potentiated by temozolomide in Ewing's sarcoma cells and is associated with enhanced trapping of PARP1-DNA complexes. Furthermore, through mining of large-scale drug sensitivity datasets, we identify a subset of glioma, neuroblastoma and melanoma cell lines as hypersensitive to the combination of temozolomide and PARP inhibition, potentially identifying new avenues for therapeutic intervention. These data provide insights into the anti-cancer activity of PARP inhibitors with implications for the design of treatment for Ewing's sarcoma patients with PARP inhibitors.Research in the M.J.G. laboratory is supported by grants from the Wellcome Trust (086357 and 102696/Z/13/Z; http://www.wellcome.ac.uk/Funding). Research in the S.P.J. laboratory is funded by Cancer Research UK Program Grant C6/A11224 (http://www.cancerresearchuk.org/funding-for-researchers/our-funding-schemes), the European Research Council (http://erc.europa.eu/funding-and-grants)and the European Community Seventh Framework Program grant agreement no. HEALTH-F2-2010-259893 (DDResponse). Core infrastructure funding was provided by Cancer Research UK Grant C6946/A14492 and Wellcome Trust Grant WT092096. S.P.J. receives a salary from the University of Cambridge, supplemented by Cancer Research UK. J.T. was funded by the European Community Seventh Framework Program grant agreement no. HEALTH-F2-2010-259893 (DDResponse). U.M. is supported by a Cancer Research UK Clinician Scientist Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This is the final version of the article. It first appeared from PLOS via http://dx.doi.org/10.1371/journal.pone.014098

Sustainable Hydrogenation of Vinyl Derivatives Using Pd/C Catalysts

The hydrogenation of unsaturated double bonds with molecular hydrogen is an efficient atom-economic approach to the production of a wide range of fine chemicals. In contrast to a number of reducing reagents typically involved in organic synthesis, hydrogenation with H2 is much more sustainable since it does not produce wastes (i.e., reducing reagent residues). However, its full sustainable potential may be achieved only in the case of easily separable catalysts and high reaction selectivity. In this work, various Pd/C catalysts were used for the liquid-phase hydrogenation of O-, S-, and N-vinyl derivatives with molecular hydrogen under mild reaction conditions (room temperature, pressure of 1 MPa). Complete conversion and high hydrogenation selectivity (>99%) were achieved by adjusting the type of Pd/C catalyst. Thus, the proposed procedure can be used as a sustainable method for vinyl group transformation by hydrogenation reactions. The discovery of the stability of active vinyl functional groups conjugated with heteroatoms (O, S, and N) under hydrogenation conditions over Pd/C catalysts opens the way for many useful transformations

Catalysts Derived from Nickel-Containing Layered Double Hydroxides for Aqueous-Phase Furfural Hydrogenation

Changes in the structural and textural properties of NiAl-layered double hydroxides (LDHs) (with 2–4 molar ratios of metals) and state of nickel that occur in different steps of the synthesis of nickel catalysts were studied using XRD, thermal analysis, TPR, low-temperature nitrogen adsorption, XANES, EXAFS, and electron microscopy methods. Relations between nickel content, catalyst reduction conditions, state of nickel, and its catalytic properties were revealed. It was shown that the use of NiAl LDH as the catalyst precursor even at a high content of active metal allows for the obtaining of the dispersed particles of supported nickel that are active in the aqueous-phase hydrogenation of furfural. The catalyst activity and conversion of furfural were found to increase with elevation of the catalyst reduction temperature and the corresponding growth of the fraction of reduced nickel. However, a lower reduction temperature (500 °C) makes it possible to form smaller nickel particles with the size of 4–6 nm, and a high Ni content (Ni:Al = 4) can be used to obtain the active Ni@NiAlOx catalyst. Under mild reaction conditions (90 °C, 2.0 MPa), the furfural conversion reached 93%, and furfuryl alcohol was formed with the selectivity of 70%. Under more severe reaction conditions (150 °C, 3.0 MPa), complete conversion of furfural was achieved, and cyclopentanol and tetrahydrofurfuryl alcohol were the main hydrogenation products

Synthesis of CuAl-, CoAl-, and CuCoAl-Catalysts from Layered Hydroxides for Furfural Hydrogenation

Catalysts based on CuAl-, CoAl-, and CuCoAl-layered hydroxides with M2+/Al = 2 and Cu/Co = 1 molar ratio were obtained. The effect of amount of cobalt on the structural properties, morphology, surface cations distribution, oxide phase formation, thermal stability of the samples and reduction of metals from them was studied. The effect of reaction conditions (temperature, time, pressure, solvent) and conditions of preliminary treatment of catalysts on their catalytic properties in furfural hydrogenation was established. High selectivity to furfuryl alcohol was observed for all the samples irrespective of pretreatment and reaction conditions. The synergetic effect in furfural hydrogenation between Co and Cu in the CuCoAlOx catalysts was revealed when ethanol is used as a solvent

EWSCs are sensitive to PARP1 trapping.

<p><b>(A)</b> Relative viability of mock-transfected and PARP1 siRNA-transfected ES8 cells treated with vehicle or olaparib. Asterisks indicate <i>student’s paired t-test P</i> value **P<0.01, ns = not significant. <b>(B)</b> PARP1 expression in cells transfected with a scrambled control or a titration of PARP1_1 siRNA and their relative viability following treatment with vehicle or olaparib. <b>(C)</b> IC50 values of parental ES8 and PARPi-resistant OLAR5 cells to five different PARPi and the fold difference between them. <b>(D)</b> Western blot of PARP1 expression in ES8 and OLAR5 cells. Viability values are the mean of technical triplicates and representative of 3 independent experiments.</p

DNA DSB repair by HR is functional in EWSCs.

<p><b>(A)</b> Western blot of ES8 cells treated with olaparib for the times indicated. Markers are grouped as part of ATM or ATR signaling. Tubulin served as a loading control. (<b>B)</b> Western blot of ES8 cells treated with camptothecin and harvested at various time points following drug washout. GAPDH served as a loading control. <b>(C)</b> Percentage of EdU-positive and EdU-negative ES8 cells with >5 nuclear RAD51 foci following 6-hour treatment with vehicle or olaparib (ola). <b>(D)</b> Olaparib log GI₅₀ (μM) of cell lines mock-transfected or transfected with CtIP or BRCA1 siRNA as indicated.</p

Temozolomide enhances PARP inhibitor sensitivity in multiple tumour types.

<p>List of cell lines screened against a combination of olaparib and temozolomide. Whether enhancement of PARP inhibitor sensitivity with temozolomide is observed (✔) or not (✖) is indicated.</p