Cell-Free Synthesis of Natural Compounds from Genomic DNA of Biosynthetic Gene Clusters.

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 because of the lack of specific functions or substrates in the host cell, the cell's sensitivity in vital physiological processes to the heterologous components, or constrained mass transfer across the membrane. In this study, 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. Specifically, we synthesize indigoidine and rhabdopeptides with the in vitro produced multidomain nonribosomal peptide synthetases BpsA and KJ12ABC from the organisms Streptomyces lavendulae and Xenorhabdus KJ12.1, respectively. These in vitro produced proteins are analyzed in yield, post-translational modification and in their ability to synthesize the natural compounds, and compared to recombinantly produced proteins. Our study highlights cell-free PURE system as suitable setting for the characterization of biosynthetic gene clusters that can potentially be harnessed for the rapid engineering of biosynthetic pathways.

Charge translocation by mitochondrial NADH:ubiquinone oxidoreductase (complex I) from Yarrowia lipolytica measured on solid-supported membranes.

The charge translocation by purified reconstituted mitochondrial complex I from the obligate aerobic yeast Yarrowia lipolytica was investigated after adsorption of proteoliposomes to solid-supported membranes. In presence of n-decylubiquinone (DBQ), pulses of NADH provided by rapid solution exchange induced charge transfer reflecting steady-state pumping activity of the reconstituted enzyme. The signal amplitude increased with time, indicating 'deactive→active' transition of the Yarrowia complex I. Furthermore, an increase of the membrane-conductivity after addition of 5-(N-ethyl-N-isopropyl)amiloride (EIPA) was detected which questiones the use of EIPA as an inhibitor of the Na+/H+-antiporter-like subunits of complex I. This investigation shows that electrical measurements on solid-supported membranes are a suitable method to analyze transport events and 'active/deactive' transition of mitochondrial complex I.

Manganese ions enhance mitochondrial H2O2 emission from Krebs cycle oxidoreductases by inducing permeability transition.

Manganese-induced toxicity has been linked to mitochondrial dysfunction and an increased generation of reactive oxygen species (ROS). We could recently show in mechanistic studies that Mn2+ ions induce hydrogen peroxide (H2O2) production from the ubiquinone binding site of mitochondrial complex II (IIQ) and generally enhance H2O2 formation by accelerating the rate of superoxide dismutation. The present study with intact mitochondria reveals that manganese additionally enhances H2O2 emission by inducing mitochondrial permeability transition (mPT). In mitochondria fed by NADH-generating substrates, the combination of Mn2+ and different respiratory chain inhibitors led to a dynamically increasing H2O2emission which was sensitive to the mPT inhibitor cyclosporine A (CsA) as well as Ru-360, an inhibitor of the mitochondrial calcium uniporter (MCU). Under these conditions, flavin-containing enzymes of the mitochondrial matrix, e.g. the mitochondrial 2-oxoglutaratedehydrogenase (OGDH), were major sources of ROS. With succinate as substrate, Mn2+ stimulated ROS production mainly at complex II, whereby the applied succinate concentration had a marked effect on the tendency for mPT. Also Ca2+ increased the rate of H2O2 emission by mPT, while no direct effect on ROS-production of complex II was observed. The present study reveals a complex scenario through which manganese affects mitochondrial H2O2 emission: stimulating its production from distinct sites (e.g. site IIQ), accelerating superoxide dismutation and enhancing the emission via mPT which also leads to the loss of soluble components of the mitochondrial antioxidant systems and favors the ROS production from flavin-containing oxidoreductases of the Krebs cycle.

Q-site inhibitor induced ROS production of mitochondrial complex II is attenuated by TCA cycle dicarboxylates.

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.