The catalytic activities of cutinase immobilized on zeolite NaY and Candida antarctica lipase B immobilized on an acrylic resin (Novozym 453) were measured in a model transesterification reaction in three imidazolium cation-based ionic liquids (RTILs), supercritical ethane (sc-ethane), sc-CO₂ and n-hexane, at a water activity (a_W) of 0.2 and 0.7. The transesterification activity of cutinase was highest and similar in 1-n-butyl-3-methylimidazolium hexafluorophosphate ([C₄mim][PF₆]), sc-ethane and n-hexane, more than one order of magnitude lower in sc-CO₂, and increased with an increase in a_W. Hydrolysis was not detected in sc-fluids and n-hexane, and was observed in RTILs at a_W 0.7 only. Both initial rates of transesterification and of hydrolysis of Novozym decreased with an increase in a_W. sc-CO₂ did not have a deleterious effect on Novozym activity, which was as high as in sc-ethane and n-hexane. The low reaction rates obtained in this case in RTILs suggested the existence of internal diffusion limitations absent in the cutinase preparation where the enzyme is only adsorbed at the surface of the support. sc-CO₂ did not adversely affect the catalytic activity of cutinase suspended in [C₄mim][PF₆], suggesting a protective effect of the RTIL. In the case of Novozym, a marked increase in the rate of transesterification was obtained in the [C₄mim][PF₆]/sc-CO₂ system, compared to the RTIL alone. This may reflect improved mass transfer of solutes to the pores of the immobilization matrix due to a high concentration of dissolved CO₂ and a reduction in viscosity of the RTIL.

SARS‐CoV‐2 virus has triggered a global pandemic with devastating consequences. The understanding of fundamental aspects of this virus is of extreme importance. In this work, we studied the viral ribonuclease nsp14, one of the most interferon antagonists from SARS‐CoV‐2. Nsp14 is a multifunctional protein with two distinct activities, an N‐terminal 3’‐to‐5’ exoribonuclease (ExoN) and a C‐terminal N7‐methyltransferase (N7‐MTase), both critical for coronaviruses life cycle, indicating nsp14 as a prominent target for the development of antiviral drugs. In coronaviruses, nsp14 ExoN activity is stimulated through the interaction with the nsp10 protein. We have performed a biochemical characterization of nsp14‐nsp10 complex from SARS‐CoV‐2. We confirm the 3’‐5’ exoribonuclease and MTase activities of nsp14 and the critical role of nsp10 in upregulating the nsp14 ExoN activity. Furthermore, we demonstrate that SARS‐CoV‐2 nsp14 N7‐MTase activity is functionally independent of the ExoN activity and nsp10. A model from SARS‐CoV‐2 nsp14‐nsp10 complex allowed mapping key nsp10 residues involved in this interaction. Our results show that a stable interaction between nsp10 and nsp14 is required for the nsp14‐mediated ExoN activity of SARS‐CoV‐2. We studied the role of conserved DEDD catalytic residues of SARS‐CoV‐2 nsp14 ExoN. Our results show that motif I of ExoN domain is essential for the nsp14 function, contrasting to the functionality of these residues in other coronaviruses, which can have important implications regarding the specific pathogenesis of SARS‐CoV‐2. This work unraveled a basis for discovering inhibitors targeting specific amino acids in order to disrupt the assembly of this complex and interfere with coronaviruses replication.

The technological value of nonaqueous enzymology has been recognized for more than thirty years. A detailed understanding of the molecular determinants of enzyme behaviour in nonaqueous media is essential to explore their potential. Computer simulations have provided valuable contributions to this field, having elucidated how the solvent affects the structural and dynamic properties of enzymes, as well as their activity and enantioselectivity. They have also helped to shed light on the effect of hydration and the role of counterions. In this perspective, we describe the major challenges and achievements of molecular simulations of enzymes in different types of nonaqueous solvents, including organic solvents, ionic liquids and supercritical fluids.

The bioenergetics of anaerobic metabolism frequently relies on redox loops performed by membrane complexes with substrate- and quinone-binding sites on opposite sides of the membrane. However, in sulfate respiration (a key process in the biogeochemical sulfur cycle), the substrate- and quinone-binding sites of the QrcABCD complex are periplasmic, and their role in energy conservation has not been elucidated. Here we show that the QrcABCD complex of Desulfovibrio vulgaris is electrogenic, as protons and electrons required for quinone reduction are extracted from opposite sides of the membrane, with a H⁺/e⁻ ratio of 1. Although the complex does not act as a H⁺-pump, QrcD may include a conserved proton channel leading from the N-side to the P-side menaquinone pocket. Our work provides evidence of how energy is conserved during dissimilatory sulfate reduction, and suggests mechanisms behind the functions of related bacterial respiratory complexes in other bioenergetic contexts.