Unveiling a CAAX Protease-Like Protein Involved in Didemnin Drug Maturation and Secretion.

The assembly line biosynthesis of the powerful anticancer-antiviral didemnin cyclic peptides is proposed to follow a prodrug release mechanism in Tristella bacteria. This strategy commences with the formation of N-terminal prodrug scaffolds and culminates in their cleavage during the cellular export of the mature products. In this study, a comprehensive exploration of the genetic and biochemical aspects of the enzymes responsible for both the assembly and cleavage of the acylated peptide prodrug scaffolds is provided. This process involves the assembly of N-acyl-polyglutamine moieties orchestrated by the nonribosomal peptide synthetase DidA and the cleavage of these components at the post-assembly stage by DidK, a transmembrane CAAX hydrolase homolog. The findings not only shed light on the complex prodrug mechanism that underlies the synthesis and secretion of didemnin compounds but also offer novel insights into the expanded role of CAAX hydrolases in microbes. Furthermore, this knowledge can be leveraged for the strategic design of genome mining approaches aimed at discovering new bioactive natural products that employ similar prodrug biochemical strategies.

Conserved Enzymatic Cascade for Bacterial Azoxy Biosynthesis.

Azoxy compounds exhibit a wide array of biological activities and possess distinctive chemical properties. Although there has been considerable interest in the biosynthetic mechanisms of azoxy metabolites, the enzymatic basis responsible for azoxy bond formation has remained largely enigmatic. In this study, we unveil the enzyme cascade that constructs the azoxy bond in valanimycin biosynthesis. Our research demonstrates that a pair of metalloenzymes, comprising a membrane-bound hydrazine synthase and a nonheme diiron azoxy synthase, collaborate to convert an unstable pathway intermediate to an azoxy product through a hydrazine-azo-azoxy pathway. Additionally, by characterizing homologues of this enzyme pair from other azoxy metabolite pathways, we propose that this two-enzyme cascade could represent a conserved enzymatic strategy for azoxy bond formation in bacteria. These findings provide significant mechanistic insights into biological N-N bond formation and should facilitate the targeted isolation of bioactive azoxy compounds through genome mining.

Enzymatic Photodecarboxylation on Secondary and Tertiary Carboxylic Acids.

Photoenzymatic decarboxylation of bulky secondary and tertiary carboxylic acids catalyzed by engineered Chlorella variabilis fatty acid photodecarboxylase (CvFAP) is reported. Rational design and directed evolution of wild-type CvFAP are used to improve the reactivity and expand potential applications. Moreover, engineered CvFAP can catalyze light-driven kinetic resolution of α-substituted carboxylic acid. Our work sheds light on the production of chiral building blocks and bioactive molecules from bulky carboxylic acids via the photoenzymatic way.

Computer simulation of the structural properties of fatty-acid modified PAMAM dendrimers at pH 5 and 7.

In this work, we performed coarse-grained molecular dynamics (CGMD) simulations of G3, G4, and G5 polyamidoamine (PAMAM) dendrimers grafting with fatty acid (FTA) chains. The FTA chains of varying length and grafting densities (50% and 100% of surface terminals) correspond to pH 7 and 5, respectively. Our findings suggested that the structural properties of dendrimers were determined by dendrimer generation, polymerization degrees, and pH. With one exception, the size of the FTA grafting dendrimer shrank after fatty acid attachment. Because of the protonation of the dendrimer's interior amines at low pH, the FTA chains are distributed at the dendrimer's surface group. At pH 7, the FTA chains that have aggregated in the interior of the dendrimer cause chain crowding. Our research provided references on drug encapsulation and the lower toxicity of these hydrophobically modified nanoparticles.

Molecular Insights into the Improved Bioactivity of Interferon Conjugates Attached to a Helical Polyglutamate.

Attaching polymers, especially polyethylene glycol (PEG), to protein drugs has emerged as a successful strategy to prolong circulation time in the bloodstream. The hypothesis is that the flexible chain wobbles on the protein's surface, thus resisting potential nonspecific adsorption. Such a theoretical framework may be challenged when a helical polyglutamate is used to conjugate with target proteins. In this study, we investigated the structure-activity relationships of polyglutamate-interferon conjugates P(EG3Glu)-IFN using molecular simulations. Our results show that the local crowding effect induced by oligoethylene glycols (i.e., EG3) is the primary driving force for helix formation in P(EG3Glu), and its helicity can be effectively increased by reducing the free volume of the two termini. Furthermore, it was found that the steric hindrance induced by IFN is not conductive to the helicity of P(EG3Glu) but contributes to its dominant orientation relative to interferon. The orientation of IFN relative to the helical P(EG3Glu) can help to protect the protein drug from neutralizing antibodies while maintaining its bioactivity. These findings suggest that the helical structure and its orientation are critical factors to consider when updating the theoretical framework for protein-polymer conjugates.

Phenoxyl mediators improve enzymatic degradation of organic pollutants: Effect and mechanism.

A mediation strategy can effectively overcome the low reaction activity of enzymes with nonspecific substrates. In this study, we demonstrated how phenol compounds can mitigate the substrate limitation of HRP in catalytic degradation of various organic pollutants. In a classical HRP/H2O2 system, phenol and natural phenolic compounds (4-HBA & pHBA), exhibited up to over 100-fold enhancement in eliminating organic dyes and persistent antibiotics while the loading is only 2-5 wt%. A combination of molecular modelling, docking and frontier orbital energy analysis was employed to elucidate the catalytic performance and mechanism. We revealed that (1) generating phenoxyl radicals required the proximity of mediators to the HRP active centre, and (2) the subsequent efficient radical transfer to pollutants was determined by the large energy gap between the SOMO energy of phenoxyl radicals and the HOMO energy of phenols. When considering phenols as pollutants, we showed a synergistic effect on catalytic degradation of phenols, dyes, and tetracycline with a removal efficiency of 71-92 %. Overall, this work not only demonstrates that phenoxyl mediators can overcome the lower efficiency and substrate-specificity limitations of the HRP/H2O2 system but also revealed their structure-mediation relationship, implying great potential in the biodegradation of diverse pollutants and their mixtures.

Sequence Effect of Peptide-Based Materials on Delivering Interferon-α (IFN-α): A Molecular Dynamic Perspective.

Peptide-based biomaterials exhibit great potentials in developing drug delivery platforms due to their biocompatibility and biodegradability beyond poly(ethylene glycol). How different amino acids in peptides used for delivery play their roles is still unclear at the microscopic level. This work compared the assembly behaviors of a series of peptides around interferon-α (IFN-α). Through all-atom molecular simulations, the sequence effect of peptides on delivering interferon-α was quantitively characterized. The hydrophobic elastin-like peptide (VPGAG)n preferred to self-aggregate into dense clusters, rather than encapsulate IFN-α. The hydrophilic zwitterionic peptides with repeating unit "KE" tended to phase-separate from IFN-α in the mixture. In contrast, peptides with a hybrid sequence, i.e., (VPKEG)n, exhibited the highest contact preference, and the formed protective shell endowed IFN-α with better thermal stability and stealth property and achieved a subtle balance between protecting IFN-α and subsequent releasing. Further energy decomposition analysis revealed that the positively charged Lys contributed most to the binding affinity while the negatively charged Glu contributed most to the hydrophilic property of peptide-based materials. In summary, this article reveals why peptides composed of repeating hydrophobic and charged residues could be a potential choice for delivering therapeutic proteins in the form of solution.

Molecular simulation of zwitterionic polypeptides on protecting glucagon-like peptide-1 (GLP-1).

Owing to their anti-fouling properties, zwitterionic polypeptides demonstrate great advantage on protecting protein drugs. When conjugated to glucagon-like peptide-1 (GLP-1), a drug for type-II diabetes, zwitterionic polypeptides confer better pharmacokinetics than uncharged counterparts. However, its microscopic mechanism is still unclear due to the complicated conformational space. To address this challenge, this work explored the interaction modes of GLP-1 with the unconnected repeat units, instead of the full-length polypeptides. The three repeat units are two zwitterionic pentapeptides VPKEG and VPREG, and one uncharged control VPGAG. Our molecular simulations revealed that the helical conformation of GLP-1 was stabilized by adding 40 polypeptides. Both VPGAG and VPREG formed dense packing shells around GLP-1, but the driving forces were hydrophobic and electrostatic interactions, respectively. In contrast, the packing shell composed of VPKEG was most loose, while could still stabilize GLP-1. The moderate electrostatic interactions endowed VPKEG an anti-fouling property, thereby avoiding non-specific interaction with other amino acids. The strong electrostatic interactions exerted by arginine promoted atomic contacts between VPREG and other residues, making it as "hydrophobic" as VPGAG. In summary, the combination of hydrophobic and moderate electrostatic interactions in VPKEG brings about a subtle balance between stabilizing GLP-1 and avoiding non-specific interaction.

Unfolding of a diblock chain and its anomalous diffusion induced by active particles.

We study the structural and dynamical behavior of an A-B diblock chain in the bath of active Brownian particles (ABPs) by Brownian dynamics simulations in two dimensions. We are interested in the situation that the effective interaction between the A segments is attractive, while that between the B segments is repulsive. Therefore, in thermal (nonactive) equilibrium, the A block "folds" into a compact globule, while the B block is in the expanded coil state. Interestingly, we find that the A block could "unfold" sequentially like unknitting a sweater, driven by the surrounding ABPs when the propelling strength on them is beyond a certain value. This threshold value decreases and then levels off as the length of the B block increases. We also find a simple power-law relation between the unfolding time of the A block and the self-propelling strength and an exponential relation between the unfolding time and the length of the B block. Finally, we probe the translational and rotational diffusion of the chain and find that both of them show "super-diffusivity" in a large time window, especially when the self-propelling strength is small and the A block is in the folded state. Such super-diffusivity is due to the strong asymmetric distribution of ABPs around the chain. Our work provides new insights into the behavior of a polymer chain in the environment of active objects.

Molecular dynamics simulation of G-actin interacting with PAMAM dendrimers.

Understanding the interactions of dendrimers as drug/gene delivery vectors with proteins is important for functional optimization. Here, atomistic molecular dynamics simulations are employed to study the interactions between six positively-charged polyamidoamine dendrimers of the second generation (G2 PAMAM) and G-actin. We find that the structure of G-actin is relatively stable after dendrimers' binding. PAMAM dendrimers also do not significantly change the secondary structure of G-actin. Furthermore, we find the formation of dendrimer-actin complex is mainly driven by electrostatic interactions. Moreover, we suggest the secondary structure change of local domains of G-actin is probably responsible to the inhibition of actin polymerization.

Beating of grafted chains induced by active Brownian particles.

We study the interplay between active Brownian particles (ABPs) and a "hairy" surface in two-dimensional geometry. We find that the increase of propelling force leads to and enhances inhomogeneous accumulation of ABPs inside the brush region. Oscillation of chain bundles (beating like cilia) is found in company with the formation and disassembly of a dynamic cluster of ABPs at large propelling forces. Meanwhile chains are stretched and pushed down due to the effective shear force by ABPs. The decrease of the average brush thickness with propelling force reflects the growth of the beating amplitude of chain bundles. Furthermore, the beating phenomenon is investigated in a simple single-chain system. We find that the chain swings regularly with a major oscillatory period, which increases with chain length and decreases with the increase of propelling force. We build a theory to describe the phenomenon and the predictions on the relationship between the period and amplitude for various chain lengths, and propelling forces agree very well with simulation data.

Polymer-Nucleic Acid Interactions.

Gene therapy is an important therapeutic strategy in the treatment of a wide range of genetic disorders. Polymers forming stable complexes with nucleic acids (NAs) are non-viral gene carriers. The self-assembly of polymers and nucleic acids is typically a complex process that involves many types of interaction at different scales. Electrostatic interaction, hydrophobic interaction, and hydrogen bonds are three important and prevalent interactions in the polymer/nucleic acid system. Electrostatic interactions and hydrogen bonds are the main driving forces for the condensation of nucleic acids, while hydrophobic interactions play a significant role in the cellular uptake and endosomal escape of polymer-nucleic acid complexes. To design high-efficiency polymer candidates for the DNA and siRNA delivery, it is necessary to have a detailed understanding of the interactions between them in solution. In this chapter, we survey the roles of the three important interactions between polymers and nucleic acids during the formation of polyplexes and summarize recent understandings of the linear polyelectrolyte-NA interactions and dendrimer-NA interactions. We also review recent progress optimizing the gene delivery system by tuning these interactions.

Structural characteristics of hydrated protons in the conductive channels: effects of confinement and fluorination studied by molecular dynamics simulation.

The relationship between the proton conductive channel and the hydrated proton structure is of significant importance for understanding the deformed hydrogen bonding network of the confined protons which matches the nanochannel. In general, the structure of hydrated protons in the nanochannel of the proton exchange membrane is affected by several factors. To investigate the independent effect of each factor, it is necessary to eliminate the interference of other factors. In this paper, a one-dimensional carbon nanotube decorated with fluorine was built to investigate the independent effects of nanoscale confinement and fluorination on the structural properties of hydrated protons in the nanochannel using classical molecular dynamics simulation. In order to characterize the structure of hydrated protons confined in the channel, the hydrogen bonding interaction between water and the hydrated protons has been studied according to suitable hydrogen bond criteria. The hydrogen bond criteria were proposed based on the radial distribution function, angle distribution and pair-potential energy distribution. It was found that fluorination leads to an ordered hydrogen bonding structure of the hydrated protons near the channel surface, and confinement weakens the formation of the bifurcated hydrogen bonds in the radial direction. Besides, fluorination lowers the free energy barrier of hydronium along the nanochannel, but slightly increases the barrier for water. This leads to disintegration of the sequential hydrogen bond network in the fluorinated CNTs with small size. In the fluorinated CNTs with large diameter, the lower degree of confinement produces a spiral-like sequential hydrogen bond network with few bifurcated hydrogen bonds in the central region. This structure might promote unidirectional proton transfer along the channel without random movement. This study provides the cooperative effect of confinement dimension and fluorination on the structure and hydrogen bonding of the slightly acidic water in the nanoscale channel.