The impact of complex II (succinate:ubiquinone oxidoreductase) on the mitochondrial production of reactive oxygen species (ROS) has been underestimated for a long time. However, recent studies with intact mitochondria revealed that complex II can be a significant source of ROS. Using submitochondrial particles from bovine heart mitochondria as a system that allows the precise setting of substrate concentrations we could show that mammalian complex II produces ROS at subsaturating succinate concentrations in the presence of Q-site inhibitors like atpenin A5 or when a further downstream block of the respiratory chain occurred. Upon inhibition of the ubiquinone reductase activity, complex II produced about 75% hydrogen peroxide and 25% superoxide. ROS generation was attenuated by all dicarboxylates that are known to bind competitively to the substrate binding site of complex II, suggesting that the oxygen radicals are mainly generated by the unoccupied flavin site. Importantly, the ROS production induced by the Q-site inhibitor atpenin A5 was largely unaffected by the redox state of the Q pool and the activity of other respiratory chain complexes. Hence, complex II has to be considered as an independent source of mitochondrial ROS in physiology and pathophysiology.
A variety of chemicals can be produced in a living host cell via optimized and engineered biosynthetic pathways. Despite the successes, pathway engineering remains demanding and partly impossible owing to the lack of specific functions or substrates in the host cell, its sensitivity in vital physiological processes to the heterologous components, or constrained mass transfer across the membrane. In this study, we demonstrate that cellfree systems can be useful in driving the characterization and engineering of biosynthetic pathways. We show that complex multidomain proteins involved in natural compound biosynthesis can be produced from encoding DNA in vitro in a minimal complex PURE system to directly run multistep reactions. We prove the concept of this approach on the direct synthesis of indigoidine and rhabdopeptides with the in vitro produced multidomain megasynthases BpsA and KJ12ABC. The in vitro produced proteins are analyzed in detail, i.e., in yield, quality, post-translational modification and specific activity, and compared to recombinantly produced proteins. Our study highlights cell-free PURE systems as suitable setting for the rapid engineering of biosynthetic pathways.
18A variety of chemicals can be produced in a living host cell via optimized and engineered 19 biosynthetic pathways. Despite the successes, pathway engineering remains demanding 20 and partly impossible owing to the lack of specific functions or substrates in the host 21 cell, its sensitivity in vital physiological processes to the heterologous components, or 22 constrained mass transfer across the membrane. In this study, we demonstrate that cell-23 free systems can be useful in driving the characterization and engineering of 24 biosynthetic pathways. We show that complex multidomain proteins involved in natural 25 compound biosynthesis can be produced from encoding DNA in vitro in a minimal 26 complex PURE system to directly run multistep reactions. We prove the concept of this 27 approach on the direct synthesis of indigoidine and rhabdopeptides with the in vitro 28 produced multidomain megasynthases BpsA and KJ12ABC. The in vitro produced 29 proteins are analyzed in detail, i.e., in yield, quality, post-translational modification and 30 specific activity, and compared to recombinantly produced proteins. Our study 31 highlights cell-free PURE systems as suitable setting for the rapid engineering of 32 biosynthetic pathways. 33 34 Keywords: cell-free protein synthesis, PURE system, natural products, biosynthetic 35 pathways, non-ribosomal peptide synthetase, polyketide synthase
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