DWV 3C protease uncovers the diverse catalytic triad in insect RNA viruses

Deformed wing virus (DWV) is the most prevalent Iflavirus that is infecting honey bees worldwide. However, the mechanisms of its infection and replication in host cells are poorly understood. In this study, we analyzed the structure and function of DWV 3C protease (3Cpro), which is necessary for the cleavage of the polyprotein to synthesize mature viral proteins. We found that the 3Cpros of DWV and picornaviruses share common enzymatic properties, including sensitivity to the same inhibitors, such as rupintrivir. The predicted structure of DWV 3Cpro by AlphaFold2, the predicted rupintrivir binding domain, and the protease activities of mutant proteins revealed that it has a Cys-His-Asn catalytic triad. Moreover, 3Cpros of other Iflaviruses and Dicistrovirus appear to contain Asn, Ser, Asp, or Glu as the third residue of the catalytic triad, suggesting diversity in insect RNA viruses. Both precursor 3Cpro with RNA-dependent RNA polymerase and mature 3Cpro are present in DWV-infected cells, suggesting that they may have different enzymatic properties and functions. DWV 3Cpro is the first 3Cpro characterized among insect RNA viruses, and our study uncovered both the common and unique characteristics among 3Cpros of Picornavirales. Furthermore, the specific inhibitors of DWV 3Cpro could be used to control DWV infection in honey bees.

Transition Metal Dichalcogenides (TMDC)-Based Nanozymes for Biosensing and Therapeutic Applications

Nanozymes, a type of nanomaterial with enzyme-like properties, are a promising alternative to natural enzymes. In particular, transition metal dichalcogenides (TMDCs, with the general formula MX2, where M represents a transition metal and X is a chalcogen element)-based nanozymes have demonstrated exceptional potential in the healthcare and diagnostic sectors. TMDCs have different enzymatic properties due to their unique nano-architecture, high surface area, and semiconducting properties with tunable band gaps. Furthermore, the compatibility of TMDCs with various chemical or physical modification strategies provide a simple and scalable way to engineer and control their enzymatic activity. Here, we discuss recent advances made with TMDC-based nanozymes for biosensing and therapeutic applications. We also discuss their synthesis strategies, various enzymatic properties, current challenges, and the outlook for future developments in this field.

Associations of clinical features of functional bowel disorders with gut microbiota characteristics in adolescents: A pilot study

Background. Obesity is a multifactorial disease that is one of the important public health problems in the 21st century. Functional bowel disorders in obese children and adolescents are one of the most common pathologies of the digestive tract. The aim: to characterize the bacteriological composition of the colon microbiota in obese adolescents and identify its associations with the clinical manifestations of functional bowel disorders.Materials and methods. The study included 20 adolescents aged 11–17 years. Stool consistency was assessed using the Bristol Stool Scale. The study of biological material was carried out using standard bacteriological methods. To assess the strength of the relationship between the nominal variables, Cramer’s V test was used.Results. It has been shown that in adolescents with obesity and functional bowel disorders, there is a decrease in the number of representatives of bifidoflora, an increase in the microbial density of Escherichia coli with altered enzymatic properties and an excessive growth of potentially pathogenic enterobacteria. In obese adolescents with functional bowel disorders, abdominal pain associated with the act of defecation is associated with the presence of enterobacteria and E. coli with altered enzymatic properties. Rare bowel movements are associated with an increased abundance of Enterococcus spp. and the presence of opportunistic Enterobacteriaceae microorganisms. Frequent bowel movements are associated with the presence of E. coli with altered enzymatic properties and Clostridium spp. In the presence of loose stool, Clostridium spp. is present; unstable stool (alternation of hard and liquid stools in one patient) is associated with a decrease in the number of lactobacilli and E. coli with normal enzymatic properties, the appearance of E. coli with altered enzymatic properties, opportunistic microorganisms of Enterobacteriaceae family and Clostridium spp. Conclusion. The relationship between the clinical symptoms of functional bowel disorders and changes in the composition of the gut microbiota in obese adolescents may be a justification for conducting in-depth studies aimed at finding early predictors of functional bowel diseases associated with obesity in the adult cohort.

Structural and (Pseudo-)Enzymatic Properties of Neuroglobin: Its Possible Role in Neuroprotection

Neuroglobin (Ngb), the third member of the globin family, was discovered in human and murine brains in 2000. This monomeric globin is structurally similar to myoglobin (Mb) and hemoglobin (Hb) α and β subunits, but it hosts a bis-histidyl six-coordinated heme-Fe atom. Therefore, the heme-based reactivity of Ngb is modulated by the dissociation of the distal HisE7-heme-Fe bond, which reflects in turn the redox state of the cell. The high Ngb levels (~100–200 μM) present in the retinal ganglion cell layer and in the optic nerve facilitate the O2 buffer and delivery. In contrast, the very low levels of Ngb (~1 μM) in most tissues and organs support (pseudo-)enzymatic properties including NO/O2 metabolism, peroxynitrite and free radical scavenging, nitrite, hydroxylamine, hydrogen sulfide reduction, and the nitration of aromatic compounds. Here, structural and (pseudo-)enzymatic properties of Ngb, which are at the root of tissue and organ protection, are reviewed, envisaging a possible role in the protection from neuronal degeneration of the retina and the optic nerve.

Highly Enhanced Enzymatic Activity of Mn-Induced Carbon Dots and Their Application as Colorimetric Sensor Probes

Nanomaterial-based enzyme mimetics (nanozymes) have attracted significant interest because of their lower cost and higher stability compared to natural enzymes. In this study, we focused on improving the enzymatic properties of metal induced N-doped carbon dots (N-CDs), which are nanozymes of interest, and their applications for sensory systems. For this purpose, Mn(acetate)2 was introduced during the synthetic step of N-doped carbon dots, and its influence on the enzymatic properties of Mn-induced N-CDs (Mn:N-CDs) was investigated. Their chemical structure was analyzed through infrared spectroscopy and X-ray photoelectron spectrometry; the results suggest that Mn ions lead to the variation in the population of chemical bonding in Mn:N-CDs, whereas these ions were not incorporated into N-CD frameworks. This structural change improved the enzymatic properties of Mn:N-CDs with respect to those of N-CDs when the color change of a 3,3′,5,5′-tetramethylbenzidine/H2O2 solution was examined in the presence of Mn:N-CDs and N-CDs. Based on this enhanced enzymatic property, a simple colorimetric system with Mn:N-CDs was used for the detection of γ-aminobutyric acid, which is an indicator of brain-related disease. Therefore, we believe that Mn:N-CDs will be an excellent enzymatic probe for the colorimetric sensor system.

Genetic fusion of P450 BM3 and formate dehydrogenase towards self-sufficient biocatalysts with enhanced activity

Fusion of multiple enzymes to multifunctional constructs has been recognized as a viable strategy to improve enzymatic properties at various levels such as stability, activity and handling. In this study, the genes coding for cytochrome P450 BM3 from B. megaterium and formate dehydrogenase from Pseudomonas sp. were fused to enable both substrate oxidation catalyzed by P450 BM3 and continuous cofactor regeneration by formate dehydrogenase within one construct. The order of the genes in the fusion as well as the linkers that bridge the enzymes were varied. The resulting constructs were compared to individual enzymes regarding substrate conversion, stability and kinetic parameters to examine whether fusion led to any substantial improvements of enzymatic properties. Most noticeably, an activity increase of up to threefold was observed for the fusion constructs with various substrates which were partly attributed to the increased diflavin reductase activity of the P450 BM3. We suggest that P450 BM3 undergoes conformational changes upon fusion which resulted in altered properties, however, no NADPH channeling was detected for the fusion constructs.