Structure and Function of a Class III Metal-Independent Lanthipeptide Synthetase.

In bacteria, Ser/Thr protein kinase-like sequences are found as part of large multidomain polypeptides that biosynthesize lanthipeptides, a class of natural products distinguished by the presence of thioether cross-links. The kinase domain phosphorylates Ser or Thr residues in the peptide substrates. Subsequent ß-elimination by a lyase domain yields electrophilic dehydroamino acids, which can undergo cyclase domain-catalyzed cyclization to yield conformationally restricted, bioactive compounds. Here, we reconstitute the biosynthetic pathway for a class III lanthipeptide from Bacillus thuringiensis NRRL B-23139, including characterization of a two-component protease for leader peptide excision. We also describe the first crystal structures of a class III lanthipeptide synthetase, consisting of the lyase, kinase, and cyclase domains, in various states including complexes with its leader peptide and nucleotide. The structure shows interactions between all three domains that result in an active conformation of the kinase domain. Biochemical analysis demonstrates that the three domains undergo movement upon binding of the leader peptide to establish interdomain allosteric interactions that stabilize this active form. These studies inform on the regulatory mechanism of substrate recognition and provide a framework for engineering of variants of biotechnological interest.

Mutational fitness landscape of human influenza H3N2 neuraminidase.

Influenza neuraminidase (NA) has received increasing attention as an effective vaccine target. However, its mutational tolerance is not well characterized. Here, the fitness effects of >6,000 mutations in human H3N2 NA are probed using deep mutational scanning. Our result shows that while its antigenic regions have high mutational tolerance, there are solvent-exposed regions with low mutational tolerance. We also find that protein stability is a major determinant of NA mutational fitness. The deep mutational scanning result correlates well with mutational fitness inferred from natural sequences using a protein language model, substantiating the relevance of our findings to the natural evolution of circulating strains. Additional analysis further suggests that human H3N2 NA is far from running out of mutations despite already evolving for >50 years. Overall, this study advances our understanding of the evolutionary potential of NA and the underlying biophysical constraints, which in turn provide insights into NA-based vaccine design.

Prevalence and mechanisms of evolutionary contingency in human influenza H3N2 neuraminidase.

Neuraminidase (NA) of human influenza H3N2 virus has evolved rapidly and been accumulating mutations for more than half-century. However, biophysical constraints that govern the evolutionary trajectories of NA remain largely elusive. Here, we show that among 70 natural mutations that are present in the NA of a recent human H3N2 strain, >10% are deleterious for an ancestral strain. By mapping the permissive mutations using combinatorial mutagenesis and next-generation sequencing, an extensive epistatic network is revealed. Biophysical and structural analyses further demonstrate that certain epistatic interactions can be explained by non-additive stability effect, which in turn modulates membrane trafficking and enzymatic activity of NA. Additionally, our results suggest that other biophysical mechanisms also contribute to epistasis in NA evolution. Overall, these findings not only provide mechanistic insights into the evolution of human influenza NA and elucidate its sequence-structure-function relationship, but also have important implications for the development of next-generation influenza vaccines.

Wastewater-leachate treatment by microalgae: Biomass, carbohydrate and lipid production.

Increases in wastewater discharges and the generation of municipal solid wastes have resulted in deleterious effects on the environment, causing eutrophication and pollution of water bodies. It is therefore necessary to investigate sustainable bioremediation alternatives. Wastewater treatment using consortia of microalgae-bacteria is an attractive alternative because it allows the removal and recycling of nutrients, with the additional advantage of biomass production and its subsequent conversion into valuable by-products. The present study aims to integrate wastewater and landfill leachate treatment with the production of microalgal biomass, considering not only its valorization in terms of lipid and carbohydrate content but also the effect of nutrient limitation on biomass formation. The effect of treating a mixture of raw wastewater with different leachate ratios (0%, 7%, 10% and 15%) was investigated using a microalgae-bacteria consortium. Two microalgae (Desmodesmus spp. and Scenedesmus obliquus) were used. Nutrient removal, biomass concentration, carbohydrate, lipid and Fatty Acid Methyl Ester (FAMEs) content and morphological changes were evaluated. Removals of 82% of NH4+ and 43% of orthophosphate from a wastewater-leachate mixture (containing 167 mg/L NH4+ and 23 mg/L PO43-) were achieved. The highest final yield was obtained using Desmodesmus spp. (1.95 ±â€¯0.3 g/L). The microalgae were observed to accumulate high lipid (20%) and carbohydrate (41%) contents under nutrient limiting conditions. The concentration of Polyunsaturated Fatty Acids (PUFAs) also increased. Morphological changes including the disintegration of coenobia were observed. By using a mixture of wastewater-leachate it is possible to remove nutrients, since microalgae tolerate high ammonia concentrations, and simultaneously increase the algal biomass concentration containing precursors to allow biofuel production.

Fullerenes in Biology and Medicine.

Fullerenes and related carbon based derivatives have shown a growing relevance in biology and medicine, mainly due to the unique electronic and structural properties that make them excellent candidates for multiple functionalization. This review focuses on the most recent developments of fullerene derivatives for different biological applications.

Characterization of New Cationic N,N-Dimethyl[70]fulleropyrrolidinium Iodide Derivatives as Potent HIV-1 Maturation Inhibitors.

HIV-1 maturation can be impaired by altering protease (PR) activity, the structure of the Gag-Pol substrate, or the molecular interactions of viral structural proteins. Here we report the synthesis and characterization of new cationic N,N-dimethyl[70]fulleropyrrolidinium iodide derivatives that inhibit more than 99% of HIV-1 infectivity at low micromolar concentrations. Analysis of the HIV-1 life cycle indicated that these compounds inhibit viral maturation by impairing Gag and Gag-Pol processing. Importantly, fullerene derivatives 2a-c did not inhibit in vitro PR activity and strongly interacted with HIV immature capsid protein in pull-down experiments. Furthermore, these compounds potently blocked infectivity of viruses harboring mutant PR that are resistant to multiple PR inhibitors or mutant Gag proteins that confer resistance to the maturation inhibitor Bevirimat. Collectively, our studies indicate fullerene derivatives 2a-c as potent and novel HIV-1 maturation inhibitors.