Evolutionary conservation of the structure and function of meiotic Rec114-Mei4 and Mer2 complexes.

Meiosis-specific Rec114-Mei4 and Mer2 complexes are thought to enable Spo11-mediated DNA double-strand break (DSB) formation through a mechanism that involves DNA-dependent condensation. However, the structure, molecular properties, and evolutionary conservation of Rec114-Mei4 and Mer2 are unclear. Here, we present AlphaFold models of Rec114-Mei4 and Mer2 complexes supported by nuclear magnetic resonance (NMR) spectroscopy, small-angle X-ray scattering (SAXS), and mutagenesis. We show that dimers composed of the Rec114 C terminus form α-helical chains that cup an N-terminal Mei4 α helix, and that Mer2 forms a parallel homotetrameric coiled coil. Both Rec114-Mei4 and Mer2 bind preferentially to branched DNA substrates, indicative of multivalent protein-DNA interactions. Indeed, the Rec114-Mei4 interaction domain contains two DNA-binding sites that point in opposite directions and drive condensation. The Mer2 coiled-coil domain bridges coaligned DNA duplexes, likely through extensive electrostatic interactions along the length of the coiled coil. Finally, we show that the structures of Rec114-Mei4 and Mer2 are conserved across eukaryotes, while DNA-binding properties vary significantly. This work provides insights into the mechanism whereby Rec114-Mei4 and Mer2 complexes promote the assembly of the meiotic DSB machinery and suggests a model in which Mer2 condensation is the essential driver of assembly, with the DNA-binding activity of Rec114-Mei4 playing a supportive role.

NMR-guided directed evolution.

Directed evolution is a powerful tool for improving existing properties and imparting completely new functionalities to proteins1-4. Nonetheless, its potential in even small proteins is inherently limited by the astronomical number of possible amino acid sequences. Sampling the complete sequence space of a 100-residue protein would require testing of 20100 combinations, which is beyond any existing experimental approach. In practice, selective modification of relatively few residues is sufficient for efficient improvement, functional enhancement and repurposing of existing proteins5. Moreover, computational methods have been developed to predict the locations and, in certain cases, identities of potentially productive mutations6-9. Importantly, all current approaches for prediction of hot spots and productive mutations rely heavily on structural information and/or bioinformatics, which is not always available for proteins of interest. Moreover, they offer a limited ability to identify beneficial mutations far from the active site, even though such changes may markedly improve the catalytic properties of an enzyme10. Machine learning methods have recently showed promise in predicting productive mutations11, but they frequently require large, high-quality training datasets, which are difficult to obtain in directed evolution experiments. Here we show that mutagenic hot spots in enzymes can be identified using NMR spectroscopy. In a proof-of-concept study, we converted myoglobin, a non-enzymatic oxygen storage protein, into a highly efficient Kemp eliminase using only three mutations. The observed levels of catalytic efficiency exceed those of proteins designed using current approaches and are similar with those of natural enzymes for the reactions that they are evolved to catalyse. Given the simplicity of this experimental approach, which requires no a priori structural or bioinformatic knowledge, we expect it to be widely applicable and to enable the full potential of directed enzyme evolution.

Beyond the VSG layer: Exploring the role of intrinsic disorder in the invariant surface glycoproteins of African trypanosomes.

In the bloodstream of mammalian hosts, African trypanosomes face the challenge of protecting their invariant surface receptors from immune detection. This crucial role is fulfilled by a dense, glycosylated protein layer composed of variant surface glycoproteins (VSGs), which undergo antigenic variation and provide a physical barrier that shields the underlying invariant surface glycoproteins (ISGs). The protective shield's limited permeability comes at the cost of restricted access to the extracellular host environment, raising questions regarding the specific function of the ISG repertoire. In this study, we employ an integrative structural biology approach to show that intrinsically disordered membrane-proximal regions are a common feature of members of the ISG super-family, conferring the ability to switch between compact and elongated conformers. While the folded, membrane-distal ectodomain is buried within the VSG layer for compact conformers, their elongated counterparts would enable the extension beyond it. This dynamic behavior enables ISGs to maintain a low immunogenic footprint while still allowing them to engage with the host environment when necessary. Our findings add further evidence to a dynamic molecular organization of trypanosome surface antigens wherein intrinsic disorder underpins the characteristics of a highly flexible ISG proteome to circumvent the constraints imposed by the VSG coat.

Structural insights into the interaction between adenovirus C5 hexon and human lactoferrin.

Adenovirus (AdV) infection of the respiratory epithelium is common but poorly understood. Human AdV species C types, such as HAdV-C5, utilize the Coxsackie-adenovirus receptor (CAR) for attachment and subsequently integrins for entry. CAR and integrins are however located deep within the tight junctions in the mucosa where they would not be easily accessible. Recently, a model for CAR-independent AdV entry was proposed. In this model, human lactoferrin (hLF), an innate immune protein, aids the viral uptake into epithelial cells by mediating interactions between the major capsid protein, hexon, and yet unknown host cellular receptor(s). However, a detailed understanding of the molecular interactions driving this mechanism is lacking. Here, we present a new cryo-EM structure of HAdV-5C hexon at high resolution alongside a hybrid structure of HAdV-5C hexon complexed with human lactoferrin (hLF). These structures reveal the molecular determinants of the interaction between hLF and HAdV-C5 hexon. hLF engages hexon primarily via its N-terminal lactoferricin (Lfcin) region, interacting with hexon's hypervariable region 1 (HVR-1). Mutational analyses pinpoint critical Lfcin contacts and also identify additional regions within hLF that critically contribute to hexon binding. Our study sheds more light on the intricate mechanism by which HAdV-C5 utilizes soluble hLF/Lfcin for cellular entry. These findings hold promise for advancing gene therapy applications and inform vaccine development. IMPORTANCE: Our study delves into the structural aspects of adenovirus (AdV) infections, specifically HAdV-C5 in the respiratory epithelium. It uncovers the molecular details of a novel pathway where human lactoferrin (hLF) interacts with the major capsid protein, hexon, facilitating viral entry, and bypassing traditional receptors such as CAR and integrins. The study's cryo-EM structures reveal how hLF engages hexon, primarily through its N-terminal lactoferricin (Lfcin) region and hexon's hypervariable region 1 (HVR-1). Mutational analyses identify critical Lfcin contacts and other regions within hLF vital for hexon binding. This structural insight sheds light on HAdV-C5's mechanism of utilizing soluble hLF/Lfcin for cellular entry, holding promise for gene therapy and vaccine development advancements in adenovirus research.

Enantioselective, BroÌ·nsted Acid-Catalyzed Anti-selective Hydroamination of Alkenes.

Chiral pyrrolidines are common structural motives in natural products as well as active pharmaceutical ingredients, explaining the need for methods for their enantioselective synthesis. While several, often metal-catalyzed, methods for their preparation do exist, the enantioselective synthesis of pyrrolidines containing quaternary stereocenters remains challenging. Herein, we report a BroÌ·nsted acid-catalyzed intramolecular hydroamination that provides such pyrrolidines from simple starting materials in high yield and enantioselectivity. Key to an efficient reaction was the use of an electron-deficient protective group on nitrogen, the common nosyl-protecting group, to avoid deactivation of the BroÌ·nsted acid by deprotonation. The reaction proceeds as a stereospecific anti-addition indicating a concerted reaction. Furthermore, kinetic studies show Michaelis-Menten behavior, suggesting the formation of a precomplex similar to those observed in enzymatic catalysis.

Amyloid Typing in Cardiac Amyloidosis Using Western Blotting.

BACKGROUND: Cardiac amyloidosis (CA) is characterized by the extracellular deposition of misfolded protein in the heart. Precise identification of the amyloid type is often challenging, but critical, since the treatment and prognosis depend on the disease form and the type of deposited amyloid. Coexistence of clinical conditions such as old age, monoclonal gammopathy, chronic inflammation, or peripheral neuropathy in a patient with cardiomyopathy creates a differential diagnosis between the major types of CA: amyloidosis light chains (AL), amyloidosis transthyretin (ATTR) and amyloidosis A (AA). OBJECTIVES: To demonstrate the utility of the Western blotting (WB)-based amyloid typing method in patients diagnosed with cardiac amyloidosis where the type of amyloid was not obvious based on the clinical context. METHODS: Congo red positive endomyocardial biopsy specimens were studied in patients where the type of amyloid was uncertain. Amyloid proteins were extracted and identified by WB. Mass spectrometry (MS) of the electrophoretically resolved protein-in-gel bands was used for confirmation of WB data. RESULTS: WB analysis allowed differentiation between AL, AA, and ATTR in cardiac biopsies based on specific immunoreactivity of the electrophoretically separated proteins and their characteristic molecular weight. The obtained results were confirmed by MS. CONCLUSIONS: WB-based amyloid typing method is cheaper and more readily available than the complex and expensive gold standard techniques such as MS analysis or immunoelectron microscopy. Notably, it is more sensitive and specific than the commonly used immunohistochemical techniques and may provide an accessible diagnostic service to patients with amyloidosis in Israel.

General Strategy for Incorporation of Functional Group Handles into Covalent Organic Frameworks via the Ugi Reaction.

The library of imine-linked covalent organic frameworks (COFs) has grown significantly over the last two decades, featuring a variety of morphologies, pore sizes, and applications. An array of synthetic methods has been developed to expand the scope of the COF functionalities; however, most of these methods were designed to introduce functional scaffolds tailored to a specific application. Having a general approach to diversify COFs via late-stage incorporation of functional group handles would greatly facilitate the transformation of these materials into platforms for a variety of useful applications. Herein, we report a general strategy to introduce functional group handles in COFs via the Ugi multicomponent reaction. To demonstrate the versatility of this approach, we have synthesized two COFs with hexagonal and kagome morphologies. We then introduced azide, alkyne, and vinyl functional groups, which could be readily utilized for a variety of post-synthetic modifications. This facile approach enables the functionalization of any COFs containing imine linkages.

Safety and Efficacy of Neoadjuvant Chemoimmunotherapy in Gastric Cancer Patients with a PD-L1 Positive Status: A Case Report.

INTRODUCTION: The landscape of gastric cancer treatment has changed owing to the widespread use of immune checkpoint inhibitors. Autophagy, involved in regulating the immune system, is a potential trigger of immunity in tumors. This study aims to find molecular-based evidence for the effectiveness of FLOT chemotherapy with immune checkpoint inhibitors in gastric cancer patients. MATERIALS AND METHODS: Three patients with advanced gastric cancer received FLOT neoadjuvant chemotherapy with immunotherapy and surgery. IHC was used to determine the PD-L1 status. Real-time PCR was used to analyze expression patterns of transcriptional growth factors, AKT/mTOR signaling components, PD-1, PD-L1, PD-L2 and LC3B. The LC3B content was measured via Western blotting analysis. RESULTS: The combination of FLOT neoadjuvant chemotherapy and immunotherapy was found to be efficient in patients with a PD-L1-positive status. Gastric tumors with a PD-L1-positive status exhibited autophagy activation and decreased PD-1 expression. CONCLUSIONS: FLOT chemotherapy combined with immune checkpoint inhibitors showed high efficacy in gastric cancer patients with a positive PD-L1 status. Autophagy was involved in activating the tumor immunity. Further research is needed to clarify the mechanism of effective anticancer treatment.

Acute Kidney Injury and Hair-Straightening Products: A Case Series.

RATIONALE & OBJECTIVE: Keratin-based hair-straightening treatment is a popular hair-styling method. The majority of keratin-based hair-straightening products in Israel contain glycolic acid derivatives, which are considered safe when used topically. Systemic absorption of these products is possible, and anecdotal reports have described kidney toxicity associated with their use. We report a series of cases of severe acute kidney injury (AKI) following use of hair-straightening treatment in Israel during the past several years. STUDY DESIGN: Case series. SETTING & PARTICIPANTS: We retrospectively identified 26 patients from 14 medical centers in Israel who experienced severe AKI and reported prior treatment with hair-straightening products in 2019-2022. FINDINGS: The 26 patients described had a median age of 28.5 (range, 14-58) years and experienced severe AKI following a hair-straightening procedure. The most common symptoms at presentation were nausea, vomiting, and abdominal pain. Scalp rash was noted in 10 (38%) patients. Two patients experienced a recurrent episode of AKI following a repeat hair-straightening treatment. Seven patients underwent kidney biopsies, which demonstrated intratubular calcium oxalate deposition in 6 and microcalcification in tubular cells in 1. In all biopsies, signs of acute tubular injury were present, and an interstitial infiltrate was noted in 4 cases. Three patients required temporary dialysis. LIMITATIONS: Retrospective uncontrolled study, small number of kidney biopsies. CONCLUSIONS: This series describes cases of AKI with prior exposure to hair-straightening treatments. Acute oxalate nephropathy was the dominant finding on kidney biopsies, which may be related to absorption of glycolic acid derivatives and their metabolism to oxalate. This case series suggests a potential underrecognized cause of AKI in the young healthy population. Further studies are needed to confirm this association and to assess the extent of this phenomenon as well as its pathogenesis.

Metallic water: Transient state under ultrafast electronic excitation.

The modern means of controlled irradiation by femtosecond lasers or swift heavy ion beams can transiently produce such energy densities in samples that reach collective electronic excitation levels of the warm dense matter state, where the potential energy of interaction of the particles is comparable to their kinetic energies (temperatures of a few eV). Such massive electronic excitation severely alters the interatomic potentials, producing unusual nonequilibrium states of matter and different chemistry. We employ density functional theory and tight binding molecular dynamics formalisms to study the response of bulk water to ultrafast excitation of its electrons. After a certain threshold electronic temperature, the water becomes electronically conducting via the collapse of its bandgap. At high doses, it is accompanied by nonthermal acceleration of ions to a temperature of a few thousand Kelvins within sub-100 fs timescales. We identify the interplay of this nonthermal mechanism with the electron-ion coupling, enhancing the electron-to-ions energy transfer. Various chemically active fragments are formed from the disintegrating water molecules, depending on the deposited dose.

Novel anti-repression mechanism of H-NS proteins by a phage protein.

H-NS family proteins, bacterial xenogeneic silencers, play central roles in genome organization and in the regulation of foreign genes. It is thought that gene repression is directly dependent on the DNA binding modes of H-NS family proteins. These proteins form lateral protofilaments along DNA. Under specific environmental conditions they switch to bridging two DNA duplexes. This switching is a direct effect of environmental conditions on electrostatic interactions between the oppositely charged DNA binding and N-terminal domains of H-NS proteins. The Pseudomonas lytic phage LUZ24 encodes the protein gp4, which modulates the DNA binding and function of the H-NS family protein MvaT of Pseudomonas aeruginosa. However, the mechanism by which gp4 affects MvaT activity remains elusive. In this study, we show that gp4 specifically interferes with the formation and stability of the bridged MvaT-DNA complex. Structural investigations suggest that gp4 acts as an 'electrostatic zipper' between the oppositely charged domains of MvaT protomers, and stabilizes a structure resembling their 'half-open' conformation, resulting in relief of gene silencing and adverse effects on P. aeruginosa growth. The ability to control H-NS conformation and thereby its impact on global gene regulation and growth might open new avenues to fight Pseudomonas multidrug resistance.

Isochores and Heat Capacity of Liquid Water in Terms of the Ion-Molecular Model.

Thermodynamics of liquid water in terms of a non-standard approach-the ion-molecular model-is considered. Water is represented as a dense gas of neutral H2O molecules and single charged H3O+ and OH- ions. The molecules and ions perform thermal collisional motion and interconvert due to ion exchange. The energy-rich process-vibrations of an ion in a hydration shell of molecular dipoles-well known to spectroscopists with its dielectric response at 180 cm-1 (5 THz), is suggested to be key for water dynamics. Taking into account this ion-molecular oscillator, we compose an equation of state of liquid water to obtain analytical expressions for the isochores and heat capacity.

Functional Activity of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes on a Mouse Renal Subcapsular Xenograft Model.

In the treatment of coronary heart disease, the most promising approach for replacing lost contractile elements involves obtaining cardiomyocytes through cardiac differentiation of pluripotent cells. The objective of this study is to develop a technology for creating a functional layer of cardiomyocytes derived from iPSCs, capable of generating rhythmic activity and synchronous contractions. To expedite the maturation of cardiomyocytes, a renal subcapsular transplantation model was employed in SCID mice. Following explantation, the formation of the cardiomyocyte contractile apparatus was assessed using fluorescence and electron microscopy, while the cytoplasmic oscillation of calcium ions was evaluated through visualization using the fluorescent calcium binding dye Fluo-8. The results demonstrate that transplanted human iPSC-derived cardiomyocyte cell layers, placed under the fibrous capsules of SCID mouse kidneys (for up to 6 weeks), initiate the development of an organized contractile apparatus and retain functional activity along with the ability to generate calcium ion oscillations even after removal from the body.

The Prognostic Value of Anti-PLA2R Antibodies Levels in Primary Membranous Nephropathy.

Anti-PLA2R antibodies (Ab) are a diagnostic and prognostic biomarker in primary membranous nephropathy (PMN). We assessed the relationship between the levels of anti-PLA2R Ab at diagnosis and different variables related to disease activity and prognosis in a western population of PMN patients. Forty-one patients with positive anti-PLA2R Ab from three nephrology departments in Israel were enrolled. Clinical and laboratory data were collected at diagnosis and after one year of follow-up, including serum anti-PLA2R Ab levels (ELISA) and glomerular PLA2R deposits on biopsy. Univariable statistical analysis and permutation-based ANOVA and ANCOVA tests were performed. The median [(interquartile range (IQR)) age of the patients was 63 [50-71], with 28 (68%) males. At the time of diagnosis, 38 (93%) of the patients had nephrotic range proteinuria, and 19 (46%) had heavy proteinuria (≥8 gr/24 h). The median [IQR] level of anti-PLA2R at diagnosis was 78 [35-183] RU/mL. Anti-PLA2R levels at diagnosis were correlated with 24 h proteinuria, hypoalbuminemia and remission after one year (p = 0.017, p = 0.003 and p = 0.034, respectively). The correlations for 24 h proteinuria and hypoalbuminemia remained significant after adjustment for immunosuppressive treatment (p = 0.003 and p = 0.034, respectively). Higher levels of anti-PLA2R Ab at diagnosis in patients with active PMN from a western population are associated with higher proteinuria, lower serum albumin and remission one year after the diagnosis. This finding supports the prognostic value of anti-PLA2R Ab levels and their possible use in stratifying PMN patients.

Dexamethasone Inhibits Heparan Sulfate Biosynthetic System and Decreases Heparan Sulfate Content in Orthotopic Glioblastoma Tumors in Mice.

Glioblastoma (GB) is an aggressive cancer with a high probability of recurrence, despite active chemoradiotherapy with temozolomide (TMZ) and dexamethasone (DXM). These systemic drugs affect the glycosylated components of brain tissue involved in GB development; however, their effects on heparan sulfate (HS) remain unknown. Here, we used an animal model of GB relapse in which SCID mice first received TMZ and/or DXM (simulating postoperative treatment) with a subsequent inoculation of U87 human GB cells. Control, peritumor and U87 xenograft tissues were investigated for HS content, HS biosynthetic system and glucocorticoid receptor (GR, Nr3c1). In normal and peritumor brain tissues, TMZ/DXM administration decreased HS content (5-6-fold) but did not affect HS biosynthetic system or GR expression. However, the xenograft GB tumors grown in the pre-treated animals demonstrated a number of molecular changes, despite the fact that they were not directly exposed to TMZ/DXM. The tumors from DXM pre-treated animals possessed decreased HS content (1.5-2-fold), the inhibition of HS biosynthetic system mainly due to the -3-3.5-fold down-regulation of N-deacetylase/N-sulfotransferases (Ndst1 and Ndst2) and sulfatase 2 (Sulf2) expression and a tendency toward a decreased expression of the GRalpha but not the GRbeta isoform. The GRalpha expression levels in tumors from DXM or TMZ pre-treated mice were positively correlated with the expression of a number of HS biosynthesis-involved genes (Ext1/2, Ndst1/2, Glce, Hs2st1, Hs6st1/2), unlike tumors that have grown in intact SCID mice. The obtained data show that DXM affects HS content in mouse brain tissues, and GB xenografts grown in DXM pre-treated animals demonstrate attenuated HS biosynthesis and decreased HS content.

Azetidomonamide and Diazetidomonapyridone Metabolites Control Biofilm Formation and Pigment Synthesis in Pseudomonas aeruginosa.

Synthesis of azetidine-derived natural products by the opportunistic pathogen Pseudomonas aeruginosa is controlled by quorum sensing, a process involving the production and sensing of diffusible signal molecules that is decisive for virulence regulation. In this study, we engineered P. aeruginosa for the titratable expression of the biosynthetic aze gene cluster, which allowed the purification and identification of two new products, azetidomonamide C and diazetidomonapyridone. Diazetidomonapyridone was shown to have a highly unusual structure with two azetidine rings and an open-chain diimide moiety. Expression of aze genes strongly increased biofilm formation and production of phenazine and alkyl quinolone virulence factors. Further physiological studies revealed that all effects were mainly mediated by azetidomonamide A and diazetidomonapyridone, whereas azetidomonamides B and C had little or no phenotypic impact. The P450 monooxygenase AzeF which catalyzes a challenging, stereoselective hydroxylation of the azetidine ring converting azetidomonamide C into azetidomonamide A is therefore crucial for biological activity. Based on our findings, we propose this group of metabolites to constitute a new class of diffusible regulatory molecules with community-related effects in P. aeruginosa.

Structural basis for osmotic regulation of the DNA binding properties of H-NS proteins.

H-NS proteins act as osmotic sensors translating changes in osmolarity into altered DNA binding properties, thus, regulating enterobacterial genome organization and genes transcription. The molecular mechanism underlying the switching process and its conservation among H-NS family members remains elusive. Here, we focus on the H-NS family protein MvaT from Pseudomonas aeruginosa and demonstrate experimentally that its protomer exists in two different conformations, corresponding to two different functional states. In the half-opened state (dominant at low salt) the protein forms filaments along DNA, in the fully opened state (dominant at high salt) the protein bridges DNA. This switching is a direct effect of ionic strength on electrostatic interactions between the oppositely charged DNA binding and N-terminal domains of MvaT. The asymmetric charge distribution and intramolecular interactions are conserved among the H-NS family of proteins. Therefore, our study establishes a general paradigm for the molecular mechanistic basis of the osmosensitivity of H-NS proteins.

Comparative genetic variability of pink salmon from different parts of their range: native Pacific, artificially introduced White Sea and naturally invasive Atlantic Scottish rivers.

Trans-oceanic movement, stocking and subsequent establishment of Pacific pink salmon (Oncorhynchus gorbuscha) into the Atlantic White Sea area have resulted in their spreading further across the northern Atlantic, with spawning being reported in a number of regions within this area. Such expansions of non-native species bring potential risks to the ecosystems in question. It has not yet been established if the spawning events of pink salmon observed are the result of self-sustaining populations in these areas, or are because of repeated invasions of strayers from the White Sea stocks. In 2017 pink salmon were observed in a number of Scottish rivers in historically large numbers. This study set out to examine genetic variation in these fish and compare this to fish in Pacific founder regions and the White Sea translocated populations. A total of 286 samples from Scotland, the Atlantic White Sea, the Pacific Okhotsk region and Northern Pacific Bering Sea were screened using a 1018 bp sequenced region of the Cytochrome b mtDNA gene and 205 of these samples for 13 microsatellites. Significant bottleneck and founder effects were observed in the White Sea stocks in both mitochondrial and nuclear DNA, including loss of diversity and changes in haplotype and allele proportions. Scottish fish were indistinguishable from White Sea populations and as such it was not possible to determine if the fish were strayers from this region or returning fish from previous spawning events in Scotland. Therefore, although the fish caught in Scotland have their origins in the White Sea population, it may not be easy to determine whether self-sustaining populations have, or are becoming, established in the UK using genetic analysis and other techniques may need to be used.

Redox Reactions of Biologically Active Molecules upon Cold Atmospheric Pressure Plasma Treatment of Aqueous Solutions.

Cold atmospheric pressure plasma (CAPP) is widely used in medicine for the treatment of diseases and disinfection of bio-tissues due to its antibacterial, antiviral, and antifungal properties. In agriculture, CAPP accelerates the imbibition and germination of seeds and significantly increases plant productivity. Plasma is also used to fix molecular nitrogen. CAPP can produce reactive oxygen and nitrogen species (RONS). Plasma treatment of bio-tissue can lead to numerous side effects such as lipid peroxidation, genotoxic problems, and DNA damage. The mechanisms of occurring side effects when treating various organisms with cold plasma are unknown since RONS, UV-Vis light, and multicomponent biological tissues are simultaneously involved in a heterogeneous environment. Here, we found that CAPP can induce in vitro oxidation of the most common water-soluble redox compounds in living cells such as NADH, NADPH, and vitamin C at interfaces between air, CAPP, and water. CAPP is not capable of reducing NAD+ and 1,4-benzoquinone, despite the presence of free electrons in CAPP. Prolonged plasma treatment of aqueous solutions of vitamin C, 1,4-hydroquinone, and 1,4-benzoquinone respectively, leads to their decomposition. Studies of the mechanisms in plasma-induced processes can help to prevent side effects in medicine, agriculture, and food disinfection.

Methionine sulfoxide reductase B from Corynebacterium diphtheriae catalyzes sulfoxide reduction via an intramolecular disulfide cascade.

Corynebacterium diphtheriae is a human pathogen that causes diphtheria. In response to immune system-induced oxidative stress, C. diphtheriae expresses antioxidant enzymes, among which are methionine sulfoxide reductase (Msr) enzymes, which are critical for bacterial survival in the face of oxidative stress. Although some aspects of the catalytic mechanism of the Msr enzymes have been reported, several details still await full elucidation. Here, we solved the solution structure of C. diphtheriae MsrB (Cd-MsrB) and unraveled its catalytic and oxidation-protection mechanisms. Cd-MsrB catalyzes methionine sulfoxide reduction involving three redox-active cysteines. Using NMR heteronuclear single-quantum coherence spectra, kinetics, biochemical assays, and MS analyses, we show that the conserved nucleophilic residue Cys-122 is S-sulfenylated after substrate reduction, which is then resolved by a conserved cysteine, Cys-66, or by the nonconserved residue Cys-127. We noted that the overall structural changes during the disulfide cascade expose the Cys-122-Cys-66 disulfide to recycling through thioredoxin. In the presence of hydrogen peroxide, Cd-MsrB formed reversible intra- and intermolecular disulfides without losing its Cys-coordinated Zn2+, and only the nonconserved Cys-127 reacted with the low-molecular-weight (LMW) thiol mycothiol, protecting it from overoxidation. In summary, our structure-function analyses reveal critical details of the Cd-MsrB catalytic mechanism, including a major structural rearrangement that primes the Cys-122-Cys-66 disulfide for thioredoxin reduction and a reversible protection against excessive oxidation of the catalytic cysteines in Cd-MsrB through intra- and intermolecular disulfide formation and S-mycothiolation.