Metal-dependent formate dehydrogenases (FDHs) catalyze, under mild conditions, the reversible reduction of CO2 to formate, a versatile C1 feedstock that can contribute to a carbon-neutral economy. Metal-dependent FDHs are the most widespread selenoproteins found in bacteria, and around 44% of them include selenocysteine (Sec) as a ligand to the Mo/W active site. In the sulfate-reducer Nitratidesulfovibrio vulgaris Hildenborough, the main FDH responsible for CO2 reduction is the W/Sec-dependent FdhAB, which is among the most active CO2 reductases reported so far. In contrast to most metal-dependent FDHs, this enzyme is relatively O2-tolerant and can be purified aerobically. In this work, we evaluated the role of Sec in the catalytic and stability properties of the W/Sec-FdhAB. For that, a Sec-to-Cys variant (U192C) was created, its catalytic and spectroscopic properties were characterized, and its crystal structure was determined. Sec substitution by Cys strongly affects activity, decreases the KM for formate, and increases susceptibility to O2. While Sec-to-Cys replacement induces only weak changes of the WV EPR signals, using 77Se-labeled enzyme, we could show that Sec undoubtedly coordinates the W metal in the WV redox state. The crystal structure of U192C confirmed previous findings on the redox switch mechanism of activation and protection of FdhAB, while revealing a putative catalytic intermediate of FdhAB with Arg441 orienting a CO2 substrate analog (probably SO2) in the active site. Overall, the results indicate that Sec plays a critical role in the high activity displayed by W/Sec-FdhAB, and that it may also be involved in or modulate the proton transfer to and from the active site.Metal-dependent formate dehydrogenases (FDHs) catalyze, under mild conditions, the reversible reduction of CO2 to formate, a versatile C1 feedstock that can contribute to a carbon-neutral economy. Metal-dependent FDHs are the most widespread selenoproteins found in bacteria, and around 44% of them include selenocysteine (Sec) as a ligand to the Mo/W active site. In the sulfate-reducer Nitratidesulfovibrio vulgaris Hildenborough, the main FDH responsible for CO2 reduction is the W/Sec-dependent FdhAB, which is among the most active CO2 reductases reported so far. In contrast to most metal-dependent FDHs, this enzyme is relatively O2-tolerant and can be purified aerobically. In this work, we evaluated the role of Sec in the catalytic and stability properties of the W/Sec-FdhAB. For that, a Sec-to-Cys variant (U192C) was created, its catalytic and spectroscopic properties were characterized, and its crystal structure was determined. Sec substitution by Cys strongly affects activity, decreases the KM for formate, and increases susceptibility to O2. While Sec-to-Cys replacement induces only weak changes of the WV EPR signals, using 77Se-labeled enzyme, we could show that Sec undoubtedly coordinates the W metal in the WV redox state. The crystal structure of U192C confirmed previous findings on the redox switch mechanism of activation and protection of FdhAB, while revealing a putative catalytic intermediate of FdhAB with Arg441 orienting a CO2 substrate analog (probably SO2) in the active site. Overall, the results indicate that Sec plays a critical role in the high activity displayed by W/Sec-FdhAB, and that it may also be involved in or modulate the proton transfer to and from the active site.

Summary The emergence of bacterial resistance to different antibiotics in clinical use, together with the knowledge on the mechanisms by which bacteria resist the action of aminoglycosides, have contributed to the renewed interest in these molecules as potential antimicrobials. Here, we give an overview on natural and semisynthetic aminoglycosides and their structural features and modes of action, focusing on the structural insight underlying resistance mechanisms. Developments on carbohydrate chemistry and microarray technology are highlighted as powerful approaches toward generation of new aminoglycosides and for screening their interactions with RNAs and proteins. The link between antibiotic uptake and the human gut microbiome is also addressed, focusing on gut microbiome function and composition, antibiotic-induced alterations in host health, and antibiotic resistance. In addition, strategies to modulate human microbiome responses to antibiotics are discussed as novel approaches for aminoglycoside usage and for the effectiveness of antibiotic therapy.

AbstractThe current chromatographic approaches used in protein purification are not keeping pace with the increasing biopharmaceutical market demand. With the upstream improvements, the bottleneck shifted towards the downstream process. New approaches rely in Anything But Chromatography methodologies and revisiting former techniques with a bioprocess perspective. Protein crystallization and precipitation methods are already implemented in the downstream process of diverse therapeutic biological macromolecules, overcoming the current chromatographic bottlenecks. Promising work is being developed in order to implement crystallization and precipitation in the purification pipeline of high value therapeutic molecules. This review focuses in the role of these two methodologies in current industrial purification processes, and highlights their potential implementation in the purification pipeline of high value therapeutic molecules, overcoming chromatographic holdups.