ChemBioChem最新文献

Transketolases (TKs) are thiamine diphosphate (ThDP)-dependent enzymes that catalyze the transfer of two-carbon units in a stereoselective manner, making them valuable biocatalysts for sustainable processes. Most known TKs are about 650 amino acids long; however, a second type found in Archaea and many Bacteria consists of two proteins, each of about 300 amino acids. Exploring the unique features and differences of split TKs may help in assessing their potential use in biocatalysis and for uncovering new reactivities. Additionally, it could provide valuable information on how their structure relates to their function, especially compared to full-length TKs. In this study, we significantly expanded the known repertoire of split TKs approximately 14-fold to the best of our knowledge, by identifying and providing accessions of nearly 500 putative split-TK subunit pairs. Moreover, we doubled the number of experimentally produced and tested split TKs by cloning, purifying, and testing ten candidates retrieved from genomes and in-house metagenomes. Interestingly, pQR2809 and pQR2812, derived from hyperthermophilic organisms, showed enhanced thermostability compared to other TK examples in the literature, maintaining partial activity after heating at 90 °C or 100 °C for 1 hour, respectively.

Copper depletion is being billed as a viable approach for cancer treatment. Vittorio and co-workers successfully demonstrated that Cuprior, an FDA-approved drug that binds with copper, effectively enhances anti-GD2 immunotherapy and improves the responses for neuroblastoma patients. These findings highlight the important role of copper chelation in modulating the tumor microenvironment. This study presented a novel approach to potentiate immunotherapies for neuroblastoma patients, warranting further investigations into copper depletion as a potential adjuvant therapy for other tumors.

O-GlcNAcylation is an important biological process in regulating the function of many nucleocytoplasmic proteins in cells.  Enhancement of O-GlcNAcylation was associated with cancer development and progression.  Here, we demonstrated the involvement of O-GlcNAcylation in melanoma metastasis.  Using the data from GEO database, we found that O-GlcNAcylation and its related enzymes, including glutamine fructose-6-phosphate amidotransferase (GFAT), O-GlcNAc transferase (OGT), and O-GlcNAcase (OGA); were elevated in metastatic melanoma compared with primary tumors and normal tissues.  Functional analyses in melanoma cell lines--MNT-1, SK-MEL-28, and A-375 showed that suppression of O-GlcNAcylation by siRNA against OGT significantly reduces the migration and invasion abilities of the cells.  Phosphorylation of Akt and NFkB was drastically suppressed after knockdown of OGT, suggesting the role of O-GlcNAcylation in regulating the Akt-NFkB signaling pathway.  In addition, we found that the NFkB target genes, such as ZEB-2 and MCT-1, were significantly upregulated in metastatic tumors compared with primary tumors.  MCT-1 expression in melanoma tissues was also correlated with O-GlcNAcylation level.  Taken together, we have demonstrated in this study the possible role of O-GlcNAcylation in controlling melanoma metastasis via upregulating MCT-1 expression through activation of Akt-NFkB signaling pathway.

N-glycosides exhibit diverse biological and pharmacological activities, making their efficient synthesis crucial for both biological research and drug development. Traditional acid-promoted N-glycosylation methods, which rely on the formation of oxocarbenium intermediates, often face significant challenges. These methods are water-sensitive and typically require neighboring group participation to achieve high selectivity. Furthermore, they depend on acid activation, rendering them incompatible with alkyl amine. Additionally, low-nucleophilicity amides often need to be converted into their TMS-derivatives to enhance reactivity, limiting the direct use of such substrates. In contrast, radical-based strategies have emerged as a promising alternative, addressing many of these limitations and leading to notable advances in N-glycosylation. This review explores the unique properties of N-glycosides, the inherent challenges of traditional N-glycosylation techniques, and the transformative advantages offered by radical-based approaches. Specifically, it highlights recent advancements in radical-mediated N-glycosylation, including photoredox radical strategies, radical/ionic hybrid approaches, and metallophotoredox catalysis, accompanied by a detailed discussion of the underlying mechanisms. Finally, the ongoing challenges and potential future directions of N-glycoside synthesis using radical strategies are presented.

Bacillibactin (BB) is a microbial siderophore produced by Bacillus species. BB is biosynthesized from 2,3-dihydroxybenzoic acid (2,3-DHB), Gly, and L-Thr by nonribosomal peptide synthetase (NRPS) enzymes DhbE, DhbB, and DhbF. The biosynthetic gene cluster (dhb) is also conserved in some strains of thermophilic genera, Geobacillus, Anoxybacillus and Parageobacillus. However, the production of BB from these thermophilic bacteria has not been characterized. Here, we report in vivo and in vitro characterization of BB biosynthesis in Parageobacillus sp. KH3-4 which grows at 65°C. We confirmed BB production in this thermophilic bacterium and the gene cluster active. In vitro enzymatic analysis revealed that 4'-phosphopantetheinyltransferase (PPTase) encoded in the same gene cluster is responsible for the post-translational maturation of carrier proteins. DhbE and DhbF showed substrate preference to 2,3-DHB and Gly and L-Thr, respectively, consistent with the chemical structure of BB. With the purified enzymes, we successfully reconstituted the NRPS assembly line in vitro. In addition, using chemically synthesized acyl-N-acetylcysteamine substrate analogues, BB analogues possessing methylbenzoyl groups instead of 2,3-DHB were detected. This study provides a new insight into secondary metabolism in thermophiles, and it expands the temperature limitation of NRPS enzymes.

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.

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.

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.