Enzymes are the cornerstone of biocatalysis, biosynthesis and synthetic biology. However, their applicability is often limited by low substrate selectivity. A prime example is the bifunctional linalool/nerolidol synthase (LNS) that can use both geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) to produce linalool and nerolidol, respectively. This bifunctionality can lead to undesired byproducts in synthetic biology applications. To enhance enzyme specificity and create monofunctional linalool synthases, we modified amino acids in the loop between helices C and D of four bifunctional mushroom LNSs. Through these modifications, we successfully shifted the substrate preference of two LNSs (ApLNS from Agrocybe pediades and HsLNS from Hypholoma sublateritium) from FPP towards GPP. Although complete monofunctionality was not achieved, we significantly increased linalool yield by 13 times while minimizing nerolidol production to 1% of the wildtype HsLNS. Docking simulations revealed a substantial reduction in the FPP binding score compared to that of the wildtype. Molecular dynamics simulations suggested that Tyr300 in the apo HsLNS mutant has a greater tendency to adopt an inward orientation. Together with Met77, the inward-facing Tyr300 creates a steric barrier that prevents the longer FPP molecule from entering the substrate binding pocket, thereby greatly reducing its activity towards FPP. This study demonstrates the potential of enzyme engineering to design substrate-specific terpene synthases, which is a challenging task and few successful examples are available. The insights gained can inform future enzyme design efforts, including the development of artificial intelligence models.
Low efficiency and high surface runoff of 2,4-dichlorophenoxyacetic acid (2,4-D) from agricultural field threaten crop yield severely. Layered double hydroxides (LDH) have shown promising adsorption properties for 2,4-D. However, the comparison of two environmentally friendly LDHs (i.e. Mg/Al-LDH vs Mg/Fe-LDH) on adsorption of 2,4-D and corresponding intrinsic mechanisms are still unclear, and the studies on the leaching control of 2,4-D by LDHs in soil environment are particularly limited. In this study, Mg/Al-LDH and Mg/Fe-LDH were selected to conduct their adsorption kinetics experiment for 2,4-D combined with the characterization technology. The results showed that the adsorption capacity of Mg/Al-LDH and Mg/Fe-LDH for 2,4-D was up to 242 mg kg−1 and 64 mg kg−1, respectively, which were negatively correlated with pH. Adsorption mechanisms of both Mg/Al-LDH and Mg/Fe-LDH for 2,4-D are dominated by chemical adsorption, including electrostatic attraction and inner sphere complexation, but no interlayer adsorption mechanism. Mg/Al-LDH contains smaller metal ion radius, which provides greater surface charge density, resulting in greater electrostatic attraction and inner sphere complexation to 2,4-D compared to Mg/Fe-LDH. The greater adsorption capacity of Mg/Al-LDH for 2,4-D was driven by the higher adsorption energy (E ads) and lower electron density, as corroborated by density functional theory (DFT) calculation. The soil column experiment further verified that Mg/Al-LDH could control the loss of 2,4-D more effectively, and the leaching amount could be significantly reduced by 61.7%, while the effect of Mg/Fe-LDH was only 24.2%. This study provides theoretical guidance for screening more potential LDH types to solve the leaching loss of 2,4-D from soil and improve its effectiveness in agricultural production.
The co-circulation of influenza and SARS-CoV-2 has led to co-infection events, primarily affecting children and older adults, who are at higher risk for severe disease. Although co-infection prevalence is relatively low, it is associated with worse outcomes compared to mono-infections. Previous studies have shown that the outcomes of co-infection depend on multiple factors, including viral interference, virus-host interaction and host response. Children and the elderly exhibit distinct patterns of antiviral response, which involve airway epithelium, mucociliary clearance, innate and adaptive immune cells, and inflammatory mediators. This review explores the pathogeneses of SARS-CoV-2 and influenza co-infection, focusing on the antiviral responses in children and the elderly. By comparing immature immunity in children and immune senescence in older adults, we aim to provide insights for the clinical management of severe co-infection cases.
Antibiotic treatment is crucial for controlling bacterial infections, but it is greatly hindered by the global prevalence of multidrug-resistant (MDR) bacteria. Although traditional Chinese medicine (TCM) monomers have shown high efficacy against MDR infections, the inactivation of bacteria induced by TCM is often incomplete and leads to infection relapse. The synergistic combination of TCM and antibiotics emerges as a promising strategy to mitigate the limitations inherent in both treatment modalities when independently administered. This review begins with a succinct exploration of the molecular mechanisms such as the antibiotic resistance, which informs the antibiotic discovery efforts. We subsequently provide an overview of the therapeutic effects of TCM/antibiotic combinations that have been developed. Finally, the factors that affect the therapeutic outcomes of these combinations and their underlying molecular mechanisms are systematically summarized. This overview offers insights into alternative strategies to treat clinical infections associated with MDR bacteria and the development of novel TCM/antibiotic combination therapies, with the goal of guiding their appropriate usage and further development.