ChemBioChem最新文献

Enzyme functional analysis is a multifaceted process that can be used for various purposes, such as screening for specific activities, as well as developing, optimising, and validating processes or final products. Functional analysis methods are crucial for assessing enzyme performance and catalytic properties. Laccase, a well-known blue multi-copper oxidase, holds immense potential in diverse industries such as pharmaceuticals, paper and pulp, food and beverages, textiles, and biorefineries due to its clean oxidation process and versatility in handling a wide range of substrates. Despite its prominence, the use of laccase encounters challenges in selecting appropriate functional analysis substrates and methods. This review delves into the substrates utilised in qualitative and quantitative techniques for laccase activity analysis. Although laccase catalyses mono-electron oxidation of aromatic hydroxyl, amine, and thiol compounds efficiently, using molecular oxygen as an electron acceptor, the review identifies limitations in the specificity of the commonly employed substrates, concerns regarding the stability of certain compounds and highlights potential strategies.

There is no doubt that breakthroughs in the enzyme-mediated formation of the oxetane ring in paclitaxel biosynthesis constitute significant milestones in the biosynthesis of complex natural products. In this review, we summarize the understanding of the biosynthesis of the oxetane ring of paclitaxel from different viewpoints. Generally, it covers five aspects, (1) a different understanding of the mechanistic formation of the oxetane ring on the basis of sound chemical reasoning, (2) a reasonable speculation of the biosynthetic pathways and suitable surrogate substrates for oxetane ring formation based on the natural and chemical logical analysis, (3) Taxus genome-enabled enzymes identification, (4) the discovery of different enzymes that mediate oxetane ring formation, and (5) a mechanistic investigation involving the use of isotopic labelling experiments and quantum chemical calculations. This review provides a detailed overview of the history of the studies on the oxetane ring formation in paclitaxel biosynthesis, which may be useful for a better understanding this process in combined view of nature, chemistry and biology logics, also for efficient heterologous reconstruction of the paclitaxel biosynthetic pathway in the future.

Antimicrobial peptides (AMPs) are recognized as one of the most ancient components of innate immunity, playing a pivotal role as the first line of host defense systems. These evolutionarily conserved molecules have been identified in various organisms, from prokaryotes to humans. AMPs establish a delicate balanced relationship between host and microbes, by simultaneously regulating the biological activities of pathogens and commensal microbes. Given the escalating global concern over antibiotic resistance, there is an urgent need to explore alternative strategies to combat challenging infectious diseases. AMPs have emerged as promising candidates employed in clinical practice due to their sustainable bactericidal properties. Witnessed by deep understanding of AMPs actions toward host and bacteria, the potential applications of AMPs extend far beyond infection control. Emerging developments harnessed natural capabilities of AMPs to optimize their roles in modulating host signaling, treating diverse diseases, advancing biosensing and bioimaging technologies. In this Concept paper, we provide a comprehensive overview of the diversity and properties of AMPs. Additionally, we elaborate on the mechanisms underlying AMP activity and bacterial responses counteracting AMP's functions. Most importantly, we discuss potential biomedical applications of AMPs and offer perspectives on their future development.

As one of the essential components of reactive oxygen species (ROS), peroxynitrite (ONOO-) plays an indispensable role in redox homeostasis and signal transduction processes, and its deviant levels are associated with numerous clinical diseases. Therefore, accurate and rapid detection of intracellular ONOO- levels is crucial for revealing its role in physiological and pathological processes. Herein, we constructed a ratiometric fluorescent probe to detect ONOO- levels in biological systems. The probe represents fast reaction rate (within 15 min), outstanding selectivity, good sensitivity (LOD = 13.32 nM) and stability to ONOO-, and it was successfully used for visualizing endogenous ONOO- in living cells. More importantly, it has also been used for tracking the changes of intracellular ONOO- during drug-induced hepatotoxicity with ratiometric fluorescence.

Access to synthetic oligonucleotides is crucial for applications in diagnostics, therapeutics, synthetic biology, and nanotechnology. Traditional solid phase synthesis is limited by sequence length and complexities, low yields, high costs and poor sustainability. Similarly, polymerase-based approaches such as in vitro transcription and primer extension reactions do not permit any control on the positioning of modifications and display poor substrate tolerance. In response, biocatalytic and chemoenzymatic strategies have emerged as promising alternatives, offering selective and efficient pathways for oligonucleotide synthesis. These methods leverage the precision and efficiency of enzymes to construct oligonucleotides with high fidelity. Recent advancements have focused on optimized systems and/or engineered enzymes enabling the incorporation of chemically modified nucleotides. Biocatalytic approaches, particularly those using DNA/RNA polymerases provide advantages in milder reaction conditions and enhanced sustainability. Chemoenzymatic methods, combining chemical synthesis and enzymes, have proven to be effective in overcoming limitations of traditional solid phase synthesis. This review summarizes recent developments in biocatalytic and chemoenzymatic strategies to construct oligonucleotides, highlighting innovations in enzyme engineering, substrate and reaction condition optimization for various applications. We address crucial details of the methods, their advantages, and limitations as well as important insights for future research directions in oligonucleotide production.

Peptides and proteins are important functional biomolecules both inside and outside of living organisms. The ability to prepare various types of functionalized peptides and proteins is essential for understanding fundamental biological processes, such as protein folding and post-translational modifications (PTMs), and for developing new therapeutics for many diseases, such as cancers and neurodegenerative diseases. The o-aminoaniline moiety was first proposed for activation to a thioester precursor and used for native chemical ligation to prepare large peptides and proteins. In the past decade, the function of o-aminoaniline has been greatly expanded to facilitate the preparation of homogeneously modified peptide and protein samples, where the modifications can include cyclization, C-terminus diversification, etc. Many o-aminoaniline derivatives have also been developed to overcome the inherent limitations of previous versions. In this review, we attempt to summarize the recent developments of different o-aminoaniline derivatives, focusing on their application to the preparation of functional peptide and protein molecules.

Polyphosphate kinases (PPK) play crucial roles in various biological processes, including energy storage and stress responses, through their interaction with inorganic polyphosphate (polyP) and the intracellular nucleotide pool. Members of the PPK family 2 (PPK2s) catalyse polyP-consuming phosphorylation of nucleotides. In this study, we characterised two PPK2 enzymes from Bacillus cereus (BcPPK2) and Lysinibacillus fusiformis (LfPPK2) to investigate their substrate specificity and potential for selective nucleotide synthesis. Both enzymes exhibited a broad substrate scope, selectively converting over 85 % of pyrimidine nucleoside monophosphates (NMPs) to nucleoside diphosphates (NDPs), while nucleoside triphosphate (NTP) formation was observed only with purine NMPs. Preparative enzymatic synthesis of cytidine diphosphate (CDP) was applied to achieve an yield of 49 %. Finally, structural analysis of five crystal structures of BcPPK2 and LfPPK2 provided insights into their active sites and substrate interactions. This study highlights PPK2-II enzymes as promising biocatalysts for the efficient and selective synthesis of pyrimidine NDPs.

The Rous sarcoma virus (RSV) is an onco-retrovirus that infects avian species such as the chicken (Gallus gallus). RSV is the first oncovirus to be described, and the oncogenic activity of this virus is related to the expression of a tyrosine kinase that induces carcinogenic transformation. Interestingly, we have noted that the RSV genome contains various potential G4-forming sequences. Among these, two sequences in the GAG and POL genes show high G4-forming potential. Additionally, the SRC oncogene also harbours a putative G4 forming sequence. In this study, we have characterised the G4 formation and topology in these three loci in the RSV-DNA. We have found that these sequences form dynamic G4 structures in physiological conditions, and such dynamicity may be associated with their cellular functions. Further, we have also established that these G4s are recognised by G4 interacting small-molecule ligands and the G4-stabilising protein nucleolin. The binding of these ligands induces structural shifts in the G4s, leading to changes in structure and stability. Thus, the RSV-DNA G4s may be further studied as targets to control its infection and oncogenic effects.

Bacterial infections, particularly those caused by drug-resistant bacteria, represent a pressing global health challenge. During the interaction between pathogen infection and host defense, bacterial infections initiate the host's immune response, which involves the activation of proteases that play a critical role in antibacterial defense. Granzyme B (GzmB), a key immune-related biomarker associated with cytotoxic T lymphocytes (CTLs), plays a pivotal role in this process. Therefore, detecting the activity of GzmB is crucial for understanding the host immune response to bacterial infections and for developing therapeutic strategies to overcome bacterial virulence. In this study, we designed and synthesized three granzyme B-activated near-infrared molecular probes. Among them, the probe HCy-F demonstrates in situ imaging capability, enabling precise quantification of GzmB activity. This development offers a valuable tool for monitoring immune responses and optimizing immunotherapy approaches for combating drug-resistant pathogens.

Vitamin D receptor (VDR) plays a critical role in regulating multiple biological processes, including bone metabolism and cell differentiation, by mediating transcriptional activation in response to ligand binding. We have constructed an environmentally fluorescent probe 2 for VDR to facilitate real-time observation of its ligand-dependent conformational changes in living cells. This probe 2 was synthesized by introducing a dansyl fluorophore via an ethynyl group at the C11 position of 1α,25(OH)₂D₃. Probe 2 exhibited strong environmentally responsive fluorescence, showing increased intensity and a blue shift of the peak wavelength upon binding to VDR due to the increased hydrophobicity of the environment.