Significance: Major pathogenic enterobacteria and protozoan parasites from the phylum Euglenozoa, such as trypanosomatids, are endowed with glutathione (GSH)-spermidine (Sp) derivatives that play important roles in signaling and metal and thiol-redox homeostasis. For some Euglenozoa lineages, the GSH-Sp conjugates represent the main redox cosubstrates around which entire new redox systems have evolved. Several proteins underwent molecular adaptations to synthesize and utilize the new polyamine-based thiols. Recent Advances: The genomes of closely related organisms have recently been sequenced, which allows mining and analysis of gene sequences that belong to these peculiar redox systems. Similarly, the three-dimensional structures of several of these proteins have been solved, which allows for comparison with their counterparts in classical redox systems that rely on GSH/glutaredoxin and thioredoxin. Critical Issues: The evolutionary and structural aspects related to the emergence and use of GSH-Sp conjugates in Euglenozoa are reviewed focusing on unique structural specializations that proteins developed to use N¹,N⁸-bisglutathionylspermidine (trypanothione) as redox cosubstrate. An updated overview on the biochemical and biological significance of the major enzymatic activities is also provided. Future Directions: A thiol-redox system strictly dependent on trypanothione is a feature unique to trypanosomatids. The physicochemical properties of the polyamine-GSH conjugates were a major driving force for structural adaptation of proteins that use these thiols as ligand and redox cofactor. In fact, the structural differences of indispensable components of this system can be exploited toward selective drug development. Future research should clarify whether additional cellular processes are regulated by the trypanothione system. Antioxid. Redox Signal. 00, 000–000.

The SARS-CoV-2 main protease (M^pro) has a pivotal role in mediating viral genome replication and transcription of the coronavirus, making it a promising target for drugs against the COVID-19 pandemic. Here, a crystal structure is presented in which M^pro adopts an inactive state that has never been observed before, called new-inactive. It is shown that the oxyanion loop, which is involved in substrate recognition and enzymatic activity, adopts a new catalytically incompetent conformation and that many of the key interactions of the active conformation of the enzyme around the active site are lost. Solvation/desolvation energetic contributions play an important role in the transition from the inactive to the active state, with Phe140 moving from an exposed to a buried environment and Asn142 moving from a buried environment to an exposed environment. In new-inactive M^pro a new cavity is present near the S2′ subsite, and the N-terminal and C-terminal tails, as well as the dimeric interface, are perturbed, with partial destabilization of the dimeric assembly. This novel conformation is relevant both for comprehension of the mechanism of action of M^pro within the catalytic cycle and for the successful structure-based drug design of antiviral drugs.

Trypanosomes are flagellated protozoan parasites (kinetoplastids) that have a unique redox metabolism based on the small dithiol trypanothione (T(SH)₂). Although GSH may still play a biological role in trypanosomatid parasites beyond being a building block of T(SH)₂, most of its functions are replaced by T(SH)₂ in these organisms. Consequently, trypanosomes have several enzymes adapted to using T(SH)₂ instead of GSH, including the glutaredoxins (Grxs). However, the mechanistic basis of Grx specificity for T(SH)₂ is unknown. Here, we combined fast-kinetic and biophysical approaches, including NMR, MS, and fluorescent tagging, to study the redox function of Grx1, the only cytosolic redox-active Grx in trypanosomes. We observed that Grx1 reduces glutathione-containing disulfides (including oxidized trypanothione) in very fast reactions (k > 5 × 10⁵ M^-1 s^-1). We also noted that disulfides without a GSH are much slower oxidants, suggesting a strongly selective binding of the GSH molecule. Not surprisingly, oxidized Grx1 was also reduced very fast by T(SH)2 (4.8 × 10⁶ M^-1 s^-1); however, GSH-mediated reduction was extremely slow (39 M^-1 s^-1). This kinetic selectivity in the reduction step of the catalytic cycle suggests that Grx1 uses preferentially a dithiol mechanism, forming a disulfide on the active site during the oxidative half of the catalytic cycle and then being rapidly reduced by T(SH)₂ in the reductive half. Thus, the reduction of glutathionylated substrates avoids GSSG accumulation in an organism lacking glutathione reductase. These findings suggest that Grx1 has played an important adaptive role during the rewiring of the thiol-redox metabolism of kinetoplastids.

Many enzymes work as homodimers with two distant catalytic sites, but the reason for this choice is often not clear. For the main protease M^pro of SARS-CoV-2, dimerization is essential for function and plays a regulatory role during the coronaviral replication process. Here, to analyze a possible allosteric mechanism, we use X-ray crystallography, native mass spectrometry, isothermal titration calorimetry, and activity assays to study the interaction of M^pro with three peptide substrates. Crystal structures show how the plasticity of M^pro is exploited to face differences in the sequences of the natural substrates. Importantly, unlike in the free form, the M^pro dimer in complex with these peptides is asymmetric and the structures of the substrates nsp5/6 and nsp14/15 bound to a single subunit show allosteric communications between active sites. We identified arginines 4 and 298 as key elements in the transition from symmetric to asymmetric dimers. Kinetic data allowed the identification of positive cooperativity based on the increase in the processing efficiency (kinetic allostery) and not on the better binding of the substrates (thermodynamic allostery). At the physiological level, this allosteric behavior may be justified by the need to regulate the processing of viral polyproteins in time and space.