D-Amino acids (D-AAs) are increasingly recognized as valuable precursors for the synthesis of fine chemicals, including pharmaceuticals and pesticides. This study reports the construction of an enzyme cascade system specifically designed for the synthesis of D-AAs from their corresponding racemates and L-isomers. Employing this system, we successfully synthesized seven enantiomerically pure D-AAs at a preparative scale.

Abstract

Enantiomerically pure D-amino acids hold significant potential as precursors for synthesizing various fine chemicals, including peptide-based drugs and other pharmaceuticals. This study focuses on establishing an enzymatic cascade system capable of converting various L-amino acids into their D-isomers. The system integrates four enzymes: ancestral L-amino acid oxidase (AncLAAO-N4), D-amino acid dehydrogenase (DAADH), D-glucose dehydrogenase (GDH), and catalase. AncLAAO-N4 initiates the process by converting L-amino acids to corresponding keto acids, which are then stereo-selectively aminated to D-amino acids by DAADH using NADPH and NH₄Cl. Concurrently, any generated H₂O₂ is decomposed into O₂ and H₂O by catalase, while GDH regenerates NADPH from D-glucose. Optimization of reaction conditions and substrate concentrations enabled the successful synthesis of five D-amino acids, including a D-Phe derivative, three D-Trp derivatives, and D-phenylglycine, all with high enantiopurity (>99 % ee) at a preparative scale (>100 mg). This system demonstrates a versatile approach for producing a diverse array of D-amino acids.

DNA assembly serves as the fundamental technology in the field of synthetic biology. This paper briefly reviews the recent advancements in the assembly of large DNA fragments. We spotlight on in vivo methods using E. coli, B. subtilis, and S. cerevisiae as hosts, and highlight the various applications that can be explored after the assembly of large DNA fragments.

Abstract

Synthetic biology, a newly and rapidly developing interdisciplinary field, has demonstrated increasing potential for extensive applications in the wide areas of biomedicine, biofuels, and novel materials. DNA assembly is a key enabling technology of synthetic biology and a central point for realizing fully synthetic artificial life. While the assembly of small DNA fragments has been successfully commercialized, the assembly of large DNA fragments remains a challenge due to their high molecular weight and susceptibility to breakage. This article provides an overview of the development and current state of DNA assembly technology, with a focus on recent advancements in the assembly of large DNA fragments in Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae. In particular, the methods and challenges associated with the assembly of large DNA fragment in different hosts are highlighted. The advancements in DNA assembly have the potential to facilitate the construction of customized genomes, giving us the ability to modify cellular functions and even create artificial life. It is also contributing to our ability to understand, predict, and manipulate living organisms.

Chromatinized DNA is targeted by proteins and small molecules to regulate chromatin function. For example, anthracycline cancer drugs evict nucleosomes in a mechanism that is still poorly understood. We here developed a flexible method for specific isotope labeling of nucleosomal DNA enabling NMR studies of such nucleosome interactions. We describe the synthesis of segmental one-strand 13C-thymidine labeled 601-DNA, the assignment of the methyl signals, and demonstrate its use to observe site-specific binding events in the nucleosome by aclarubicin, an anthracycline cancer drug that intercalates into the DNA minor grooves. Our results highlight intrinsic conformational heterogeneity in the 601 DNA sequence and show that aclarubicin binds an exposed AT-rich region near the DNA end. Overall, our data point to a model where the drug invades the nucleosome from the terminal ends inward, eventually resulting in histone eviction and nucleosome disruption.

Transcription factors (TFs) play a central role in gene regulation, and their malfunction can result in a plethora of severe diseases. TFs are therefore interesting therapeutic targets, but their involvement in protein-protein interaction networks and the frequent lack of well-defined binding pockets render them challenging targets for classical small molecules. As an alternative, peptide-based scaffolds have proven useful, in particular with an α-helical active conformation. Peptide-based strategies often require extensive structural optimization efforts, which could benefit from a more detailed understanding of the dynamics in inhibitor/protein interactions. In this study, we investigate how truncated stapled α-helical peptides interact with the transcription factor Nuclear Factor-Y (NF-Y). We identified a 13-mer minimal binding core region, for which two crystal structures with an altered C-terminal peptide conformation when bound to NF-Y were obtained. Subsequent molecular dynamics simulations confirmed that the C-terminal part of the stapled peptide is indeed relatively flexible while still showing defined interactions with NF-Y. Our findings highlight the importance of flexibility in the bound state of peptides, which can contribute to overall binding affinity.

Bioluminescence, the mesmerizing natural phenomenon where living organisms produce light through chemical reactions, has long captivated scientists and laypersons alike, offering a rich tapestry of insights into biological function, ecology, evolution as well as the underlying chemistry. This comprehensive review systematically explores the phenomenon of bioluminescence, addressing its historical context, geographic dispersion, and ecological significance with a focus on their chemical mechanisms. We discuss terrestrial bioluminescence in various habitats, including fireflies in Central Europe, luminescent fungi in Brazil's Atlantic rainforest, and glowing species in New Zealand's Waitomo Caves and the Siberian Steppes. The marine section covers deep-sea jellyfish, seasonal bioluminescence in Japan's Toyama Bay, and symbiotic bioluminescent bacteria. Each organism's discovery, ecological function, and distribution are detailed, emphasizing the chemistry behind their luminescence. We conclude with practical experiments in bioluminescence and chemiluminescence for educational purposes. Our goal with this review is to provide a summary of bioluminescence across the diverse ecological contexts, contributing to the broader understanding of this unique biological phenomenon and its chemical mechanisms.

Flavin-dependent N-monooxygenases and cupin/methionyl-tRNA synthetase-like enzymes (hydrazine synthetases) are two key components of bacterial hydrazine biosynthetic pathways. Phylogenetic analysis of these two enzyme families provided a global perspective on the diversity of bacterial hydrazine biosynthesis. A phylogeny-guided enzyme-mining approach led to the identification of new bacterial hydrazines, including those based on 1,3-diaminopropane and putrescine.

Abstract

Cupin/methionyl-tRNA synthetase (MetRS)-like didomain enzymes catalyze nitrogen-nitrogen (N−N) bond formation between Nω-hydroxylamines and amino acids to generate hydrazines, key biosynthetic intermediates of various natural products containing N−N bonds. While the combination of these two building blocks leads to the creation of diverse hydrazine products, the full extent of their structural diversity remains largely unknown. To explore this, we herein conducted phylogeny-guided genome-mining of related hydrazine biosynthetic pathways consisting of two enzymes: flavin-dependent Nω-hydroxylating monooxygenases (NMOs) that produce Nω-hydroxylamine precursors and cupin/MetRS-like enzymes that couple the Nω-hydroxylamines with amino acids via N−N bonds. A phylogenetic analysis identified the largely unexplored sequence spaces of these enzyme families. The biochemical characterization of NMOs demonstrated their capabilities to produce various Nω-hydroxylamines, including those previously not known as precursors of N−N bonds. Furthermore, the characterization of cupin/MetRS-like enzymes identified five new hydrazine products with novel combinations of building blocks, including one containing non-amino acid building blocks: 1,3-diaminopropane and putrescine. This study substantially expanded the variety of N−N bond forming pathways mediated by cupin/MetRS-like enzymes.

We present here a multi-technique analysis, at both supra-fibre and intra-fibre scale, of 3D scaffolds structures made of thermally treated PAN electrospun fibres. Our results show that we can propose biocompatible scaffolds presenting the same topology but with different elastic properties. Therefore, these 3D electrospun PAN fibres scaffolds are a precious tool for the in vitro study of many cell types.

Abstract

Growing cells in a biomimetic environment is critical for tissue engineering as well as for studying the cell biology underlying disease mechanisms. To this aim a range of 3D matrices have been developed, from hydrogels to decellularized matrices. They need to mimic the extracellular matrix to ensure the optimal growth and function of cells. Electrospinning has gained in popularity due to its capacity to individually tune chemistry and mechanical properties and as such influence cell attachment, differentiation or maturation. Polyacrylonitrile (PAN) derived electrospun fibres scaffolds have shown exciting potential due to reports of mechanical tunability and biocompatibility. Building on previous work we fabricate here a range of PAN fibre scaffolds with different concentrations of carbon nanotubes. We characterize them in-depth in respect to their structure, surface chemistry and mechanical properties, using scanning electron microscopy, image processing, ultramicrotomic transmission electron microscopy, x-ray nanotomography, infrared spectroscopy, atomic force microscopy and nanoindentation. Together the data demonstrate this approach to enable finetuning the mechanical properties, while keeping the structure and chemistry unaltered and hence offering ideal properties for comparative studies of the cellular mechanobiology. Finally, we confirm the biocompatibility of the scaffolds using primary rat cardiomyocytes, vascular smooth muscle (A7r5) and myoblast (C2C12) cell lines.

Photoswitchable antibiotic hybrids: Potentials for creating phtoswitchable antibiotic hybrids using norfloxacin/ciproflocacin and azoisoxazole (photoswitch) pharmacophores have been explored. Against Gram-positive pathogens, all hybrids in their trans states exhibited antibacterial activity comparable to the parent drugs. Notably, the potency of the irradiated state (cis isomer) of a hybrid was found to be 2-fold lower than the nonirradiated state (trans isomer).

Abstract

Photopharmacology holds a huge untapped potential to locally treat diseases involving photoswitchable drugs via the elimination of drugs’ off-target effects. The growth of this field has created a pressing demand to develop such light-active drugs. We explored the potential for creating photoswitchable antibiotic hybrids by attaching pharmacophores norfloxacin/ciprofloxacin and azoisoxazole (photoswitch). All compounds exhibited a moderate to a high degree of bidirectional photoisomerization, long thermal cis half-lives, and impressive photoresistance. Gram-negative pathogens were found to be insensitive to these hybrids, while against Gram-positive pathogens, all hybrids in their trans states exhibited antibacterial activity that is comparable to that of the parent drugs. Notably, the toxicity of the irradiated hybrid 6 was found to be 2-fold lower than the nonirradiated trans isomer, indicating that the pre-inactivated cis-enriched drug can be employed for the site-specific treatment of bacterial infection using light, which could potentially eliminate the unwanted exposure of toxic antibiotics to both beneficial and untargeted harmful microbes in our body. Molecular docking revealed different binding affinity of the cis and trans isomers with the topoisomerase IV enzyme, due to their different shapes.

A LigE-type beta-etherase enzyme from lignin-degrading Agrobacterium sp. Is found to convert a phenylcoumaran β-5 lignin dimer into a cis-stilbene, alkene and ketone products, via reaction of glutathione at the α position, the first example of an etherase enzyme attacking the α position of a β-5 unit.

Abstract

A LigE-type beta-etherase enzyme from lignin-degrading Agrobacterium sp. has been identified, which assists degradation of polymeric lignins. Testing against lignin dimer model compounds revealed that it does not catalyse the previously reported reaction of Sphingobium SYK-6 LigE, but instead shows activity for a β-5 phenylcoumaran lignin dimer. The reaction products did not contain glutathione, indicating a catalytic role for reduced glutathione in this enzyme. Three reaction products were identified: the major product was a cis-stilbene arising from C−C fragmentation involving loss of formaldehyde; two minor products were an alkene arising from elimination of glutathione, and an oxidised ketone, proposed to arise from reaction of an intermediate with molecular oxygen. Testing of the recombinant enzyme against a soda lignin revealed the formation of new signals by two-dimensional NMR analysis, whose chemical shifts are consistent with the formation of a stilbene unit in polymeric lignin.

Peptide therapeutics have gained great interest due to their multiple advantages over small molecule and antibody-based drugs. Peptide drugs are easier to synthesize, have the potential for oral bioavailability, and are large enough to target protein-protein interactions that are undruggable by small molecules. However, two major limitations have made it difficult to develop novel peptide therapeutics not derived from natural products, including the metabolic instability of peptides and the difficulty of reaching antibody-like potencies and specificities. Compared to linear and disulfide-monocyclized peptides, multicyclic peptides can provide increased conformational rigidity, enhanced metabolic stability, and higher potency in inhibiting protein-protein interactions. The identification of novel multicyclic peptide binders can be difficult, however, recent advancements in the construction of multicyclic phage libraries have greatly advanced the process of identifying novel multicyclic peptide binders for therapeutically relevant protein targets. This review will describe the current approaches used to create multicyclic peptide libraries, highlighting the novel chemistries developed and the proof-of-concept work done on validating these libraries against different protein targets.