Reaction mechanism of the farnesyl pyrophosphate C-methyltransferase towards the biosynthesis of pre-sodorifen pyrophosphate by Serratia plymuthica 4Rx13.

Classical terpenoid biosynthesis involves the cyclization of the linear prenyl pyrophosphate precursors geranyl-, farnesyl-, or geranylgeranyl pyrophosphate (GPP, FPP, GGPP) and their isomers, to produce a huge number of natural compounds. Recently, it was shown for the first time that the biosynthesis of the unique homo-sesquiterpene sodorifen by Serratia plymuthica 4Rx13 involves a methylated and cyclized intermediate as the substrate of the sodorifen synthase. To further support the proposed biosynthetic pathway, we now identified the cyclic prenyl pyrophosphate intermediate pre-sodorifen pyrophosphate (PSPP). Its absolute configuration (6R,7S,9S) was determined by comparison of calculated and experimental CD-spectra of its hydrolysis product and matches with those predicted by semi-empirical quantum calculations of the reaction mechanism. In silico modeling of the reaction mechanism of the FPP C-methyltransferase (FPPMT) revealed a SN2 mechanism for the methyl transfer followed by a cyclization cascade. The cyclization of FPP to PSPP is guided by a catalytic dyad of H191 and Y39 and involves an unprecedented cyclopropyl intermediate. W46, W306, F56, and L239 form the hydrophobic binding pocket and E42 and H45 complex a magnesium cation that interacts with the diphosphate moiety of FPP. Six additional amino acids turned out to be essential for product formation and the importance of these amino acids was subsequently confirmed by site-directed mutagenesis. Our results reveal the reaction mechanism involved in methyltransferase-catalyzed cyclization and demonstrate that this coupling of C-methylation and cyclization of FPP by the FPPMT represents an alternative route of terpene biosynthesis that could increase the terpenoid diversity and structural space.

TFAM-dependent and independent dynamics of mtDNA levels in C2C12 myoblasts caused by redox stress.

TFAM is an essential protein factor for the initiation of transcription of the mtDNA. It also functions as a packaging factor, which stabilizes the mtDNA pool. To investigate the regulatory role of TFAM for regeneration and proliferation of the mtDNA pool, we exposed the muscle cell line C2C12 to a severe redox stress (H2O2) or to a moderate redox stress (GSH depletion), determined the dynamics of the mtDNA levels and correlated this with the TFAM protein levels. H2O2 caused a concentration-dependent loss of mtDNA molecules. The mtDNA levels recovered slowly within 3 days after H2O2 stress. The TFAM protein was less degraded than the mtDNA indicating an accumulation of TFAM protein per mtDNA after H2O2 stress. Overexpression of TFAM did not protect against the mtDNA loss after H2O2 stress but shortened the recovery time. GSH depletion led to a proliferation of the mtDNA pool. Although the mtDNA levels increased the TFAM protein levels were unaffected by the GSH depletion. We conclude that the accumulation of the TFAM protein after H2O2 stress contributes to the regeneration of the mtDNA pool but that other mechanisms, independent from the TFAM protein amount have to be postulated to explain the proliferation of the mtDNA pool after GSH depletion.

Life imaging of peroxynitrite in rat microglial and astroglial cells: Role of superoxide and antioxidants.

Free radicals, such as superoxide and nitric oxide, are known to play a role in a number of inflammatory and degenerative brain diseases, in which resident microglia upregulate the inducible nitric oxide synthase (iNOS) and thus produce large amounts of nitric oxide. Simultaneously, microglia generate superoxide mainly via NADPH-oxidase, which reacts at a diffusion-limited rate with nitric oxide to form the powerful oxidant peroxynitrite. We used mixed astroglial/microglial cultures to study the effects of iNOS induction by lipopolysaccharide and interferon-gamma on free radical formation. Using the fluorogenic compound 2,7-dihydrodichlorofluorescein diacetate, we monitored cellular peroxynitrite formation by confocal laser microscopy. Peroxynitrite formation in continuously nitric oxide-producing microglial cells was rather limited. However, activation of the superoxide-generating enzyme NADPH-oxidase dramatically increased DCF fluorescence within a few minutes. We conclude that superoxide is the limiting factor for peroxynitrite formation. Since the formation and oxidant activity of peroxynitrite depends strongly on the availability of cellular antioxidants, we investigated the capacity of several compounds to influence peroxynitrite formation. Among the substances under investigation in this study, glutathione and the synthetic compound ebselen had a major effect on preventing peroxynitrite formation, whereas ascorbate failed to decrease peroxynitrite levels.