Stereoselective Ring-Opening (Co)polymerization of β-Butyrolactone and ε-Decalactone Using an Yttrium Bis(phenolate) Catalytic System

An effective route for ring-opening copolymerization of β-butyrolactone (BBL) with ε-decalactone (ε-DL) is reported. Microstructures of the block copolymers characterized by 13C NMR spectroscopy revealed syndiotactic-enriched poly(3-hydroxybutyrate) (PHB) blocks. Several di- and triblock copolymers (PDL-b-PHB and PDL-b-PHB-b-PDL, respectively) were successfully synthesized by sequential addition of the monomers using (salan)Y(III) complexes as catalysts. The results from MALDI-ToF mass spectrometry confirmed the presence of the copolymers. Moreover, thermal properties of the block copolymers were also investigated and showed that the microphase separation of PDL-b-PHB copolymers into PHB- and PDL-rich domains has an impact on the glass transition temperatures of both blocks

A New Hydroxylated Nonaprenylhydroquinone from the Mediterranean Marine Sponge Sarcotragus spinosulus

Chemical investigation of the Mediterranean sponge Sarcotragus spinosulus led to the isolation of a new hydroxylated nonaprenylhydroquinone, along with two known metabolites, hepta- and octaprenylhydroquinones. The structure of the new metabolite was assigned by extensive 1D and 2D NMR analyses and MS studies. The antileukemic effect of the three compounds towards the chronic myelogenous leukemia (CML) cells line K562 was also evaluated

Insights into the hyperthermostability and unusual region-specificity of archaeal Pyrococcus abyssi tRNA m1A57/58 methyltransferase

The S-adenosyl-l-methionine dependent methylation of adenine 58 in the T-loop of tRNAs is essential for cell growth in yeast or for adaptation to high temperatures in thermophilic organisms. In contrast to bacterial and eukaryotic tRNA m1A58 methyltransferases that are site-specific, the homologous archaeal enzyme from Pyrococcus abyssi catalyzes the formation of m1A also at the adjacent position 57, m1A57 being a precursor of 1-methylinosine. We report here the crystal structure of P. abyssi tRNA m1A57/58 methyltransferase (PabTrmI), in complex with S-adenosyl-l-methionine or S-adenosyl-l-homocysteine in three different space groups. The fold of the monomer and the tetrameric architecture are similar to those of the bacterial enzymes. However, the inter-monomer contacts exhibit unique features. In particular, four disulfide bonds contribute to the hyperthermostability of the archaeal enzyme since their mutation lowers the melting temperature by 16.5°C. His78 in conserved motif X, which is present only in TrmIs from the Thermococcocales order, lies near the active site and displays two alternative conformations. Mutagenesis indicates His78 is important for catalytic efficiency of PabTrmI. When A59 is absent in tRNAAsp, only A57 is modified. Identification of the methylated positions in tRNAAsp by mass spectrometry confirms that PabTrmI methylates the first adenine of an AA sequence

MALDI-TOF MS Profiling of Annonaceous Acetogenins in Annona muricata Products for Human Consumption

Annonaceous acetogenins are proposed as environmental neurotoxicants consumed through medicinal and alimentary habits and responsible for atypical parkinsonian syndromes observed in tropical areas. Potential sources of exposure still have to be determined, as, to date, only a few batches of products for human consumption were searched for these compounds. To assess the presence of acetogenins, we propose a fast, sensitive and accurate method of screening, using MALDI-TOF MS, with minimal sample preparation. Development of the technique is discussed. Its application to leaves of herbal tea, pulp and bottled nectar of Annona muricata is presented

MALDI-TOF MS Profiling of Annonaceous Acetogenins in Annona muricata Products for Human Consumption

Annonaceous acetogenins are proposed as environmental neurotoxicants consumed through medicinal and alimentary habits and responsible for atypical parkinsonian syndromes observed in tropical areas. Potential sources of exposure still have to be determined, as, to date, only a few batches of products for human consumption were searched for these compounds. To assess the presence of acetogenins, we propose a fast, sensitive and accurate method of screening, using MALDI-TOF MS, with minimal sample preparation. Development of the technique is discussed. Its application to leaves of herbal tea, pulp and bottled nectar of Annona muricata is presented

Structural and functional insights into Archaeoglobus fulgidusArchaeoglobus\ fulgidus m2^2G10_{10} tRNA methyltransferase Trm11 and its Trm112 activator

International audiencetRNAs play a central role during the translation process and are heavily post-transcriptionally modified to ensure optimal and faithful mRNA decoding. These epitranscriptomics marks are added by largely conserved proteins and defects in the function of some of these enzymes are responsible for neurodevelopmental disorders and cancers. Here, we focus on the Trm11 enzyme, which forms N$^2$-methylguanosine (m$^2$G) at position 10 of several tRNAs in both archaea and eukaryotes. While eukaryotic Trm11 enzyme is only active as a complex with Trm112, an allosteric activator of methyltransferases modifying factors (RNAs and proteins) involved in mRNA translation, former studies have shown that some archaeal Trm11 proteins are active on their own. As these studies were performed on Trm11 enzymes originating from archaeal organisms lacking TRM112 gene, we have characterized Trm11 (AfTrm11) from the $Archaeoglobus\ fulgidus$ archaeon, which genome encodes for a Trm112 protein (AfTrm112). We show that AfTrm11 interacts directly with AfTrm112 similarly to eukaryotic enzymes and that although AfTrm11 is active as a single protein, its enzymatic activity is strongly enhanced by AfTrm112. We finally describe the first crystal structures of the AfTrm11-Trm112 complex and of Trm11, alone or bound to the methyltransferase inhibitor sinefungin

A Catalytic Intermediate and Several Flavin Redox States Stabilized by Folate-Dependent tRNA Methyltransferase from Bacillus subtilis

International audienceThe flavoprotein TrmFO catalyzes the C5 methylation of uridine 54 in the TΨC loop of tRNAs using 5,10-methylenetetrahydrofolate (CH2THF) as a methylene donor and FAD as a reducing agent. Here, we report biochemical and spectroscopic studies that unravel the remarkable capability of Bacillus subtilis TrmFO to stabilize, in the presence of oxygen, several flavin-reduced forms, including an FADH• radical, and a catalytic intermediate endowed with methylating activity. The FADH• radical was characterized by high-field electron paramagnetic resonance and electron nuclear double-resonance spectroscopies. Interestingly, the enzyme exhibited tRNA methylation activity in the absence of both an added carbon donor and an external reducing agent, indicating that a reaction intermediate, containing presumably CH2THF and FAD hydroquinone, is present in the freshly purified enzyme. Isolation by acid treatment, under anaerobic conditions, of noncovalently bound molecules, followed by mass spectrometry analysis, confirmed the presence in TrmFO of nonmodified FAD. Addition of formaldehyde to the purified enzyme protects the reduced flavins from decay by probably preventing degradation of CH2THF. The absence of air-stable reduced FAD species during anaerobic titration of oxidized TrmFO, performed in the absence or presence of added CH2THF, argues against their thermodynamic stabilization but rather implicates their kinetic trapping by the enzyme. Altogether, the unexpected isolation of a stable catalytic intermediate suggests that the flavin-binding pocket of TrmFO is a highly insulated environment, diverting the reduced FAD present in this intermediate from uncoupled reactions

Profiling of modified nucleosides from ribonucleic acid digestion by supercritical fluid chromatography coupled to high resolution mass spectrometry

International audienceA method based on supercritical fluid chromatography coupled to high resolution mass spectrometry for the profiling of canonical and modified nucleosides was optimized, and compared to classical reverse-phase liquid chromatography in terms of separation, number of detected modified nucleosides and sensitivity. Limits of detection and quantification were measured using statistical method and quantifications of twelve nucleosides of a tRNA digest from E. coli are in good agreement with previously reported data. Results highlight the complementarity of both separation techniques to cover the largest view of nucleoside modifications for forthcoming epigenetic studies