Search Results (95)

The shikimate pathway is the fundamental metabolic route for aromatic amino acid biosynthesis in bacteria, plants, and fungi, but is absent in mammals. This review explores how multi-enzyme synergy and allosteric regulation coordinate metabolic flux through this pathway by focusing on three key enzymes: 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase, chorismate mutase, and tryptophan synthase. We examine the structural diversity and distribution of these enzymes across evolutionary domains, highlighting conserved catalytic mechanisms alongside species-specific regulatory adaptations. The review covers directed evolution strategies that have transformed naturally regulated enzymes into standalone biocatalysts with enhanced activity and expanded substrate scope, enabling synthesis of non-canonical amino acids and complex organic molecules. Industrial applications demonstrate the pathway’s potential for sustainable production of pharmaceuticals, polymer precursors, and specialty chemicals through engineered microbial platforms. Additionally, we discuss the therapeutic potential of inhibitors targeting pathogenic organisms, particularly their mechanisms of action and antimicrobial efficacy. This comprehensive review establishes the shikimate pathway as a paradigmatic system where understanding allosteric networks enables the rational design of biocatalytic platforms, providing blueprints for biotechnological innovation and demonstrating how evolutionary constraints can be overcome through protein engineering to create superior industrial biocatalysts. Full article

Molecular modelling is a vital tool in the discovery and characterisation of bioactive peptides, providing insights into their structural properties and interactions with biological targets. Many models predicting bioactive peptide function or structure rely on their intrinsic properties, including the influence of amino acid composition, sequence, and chain length, which impact stability, folding, aggregation, and target interaction. Homology modelling predicts peptide structures based on known templates. Peptide–protein interactions can be explored using molecular docking techniques, but there are challenges related to the inherent flexibility of peptides, which can be addressed by more computationally intensive approaches that consider their movement over time, called molecular dynamics (MD). Virtual screening of many peptides, usually against a single target, enables rapid identification of potential bioactive peptides from large libraries, typically using docking approaches. The integration of artificial intelligence (AI) has transformed peptide discovery by leveraging large amounts of data. AlphaFold is a general protein structure prediction tool based on deep learning that has greatly improved the predictions of peptide conformations and interactions, in addition to providing estimates of model accuracy at each residue which greatly guide interpretation. Peptide function and structure prediction are being further enhanced using Protein Language Models (PLMs), which are large deep-learning-derived statistical models that learn computer representations useful to identify fundamental patterns of proteins. Recent methodological developments are discussed in the context of canonical peptides, as well as those with modifications and cyclisations. In designing potential peptide therapeutics, the main outstanding challenge for these methods is the incorporation of diverse non-canonical amino acids and cyclisations. Full article

3,4-Dihydroxy-L-phenylalanine (DOPA) is a promising noncanonical amino acid (ncAA) that introduces novel catechol chemical features into proteins, expanding their functional potential. However, the most common approach to incorporating ncAAs into proteins relies on stop codon suppression, which is often limited by the competition of endogenous translational termination machinery. Here, we employed a special in vitro protein expression system that facilitates the efficiency of DOPA incorporation into proteins by removing essential Class I peptide release factors through targeted degradation. In the absence of both RF1 and RF2, we successfully demonstrated DOPA incorporation at all three stop codons (TAG, TAA, and TGA). By optimizing the concentration of engineered DOPA-specific aminoacyl-tRNA synthetase (DOPARS), DOPA, and DNA template, we achieved a synthesis yield of 2.24 µg of sfGFP with 100% DOPA incorporation in a 20 μL reaction system. DOPARS exhibited a dissociation constant (Kd) of 11.7 μM for DOPA but showed no detectable binding to its native counterpart, tyrosine. Additionally, DOPA was successfully incorporated into a reverse transcriptase, which interfered with its activity. This system demonstrates a fast and efficient approach for precise DOPA incorporation into proteins, paving the way for advanced protein engineering applications. Full article

Background: In biotechnology, B. subtilis is established for heterologous protein production. In addition, the species provides a variety of bioactive metabolites, including the non-ribosomally produced surfactin lipopeptide. However, to control the formation of the target product-forming enzyme, different expression systems could be introduced, including the principle of genetic code expansion by the incorporation of externally supplied non-canonical amino acids. Methods: Integration of an amber stop codon into the srfA operon and additional chromosomal integration of an aminoacyl-tRNA synthetase/tRNA mutant pair from Methanococcus jannaschii enabled site-directed incorporation of the non-canonical amino acid O-methyl-L-tyrosine (OMeY). In different fed-batch bioreactor approaches, OMeY-associated surfactin production was quantified by high-performance thin-layer chromatography (HPTLC). Physiological adaptations of the B. subtilis production strain were analyzed by mass spectrometric proteomics. Results: Using a surfactin-forming B. subtilis production strain, which enables high cell density fermentation processes, the principle of genetic code expansion was introduced. Accordingly, the biosynthesis of the surfactin-forming non-ribosomal peptide synthetase (NRPS) was linked to the addition of the non-canonical amino acid OMeY. In OMeY-associated fed-batch bioreactor fermentation processes, a maximum surfactin titre of 10.8 g/L was achieved. In addition, the effect of surfactin induction was investigated by mass spectrometric proteome analyses. Among other things, adaptations in the B. subtilis motility towards a more sessile state and increased abundances of surfactin precursor-producing enzymes were detected. Conclusions: The principle of genetic code expansion enabled a precise control of the surfactin bioproduction as a representative of bioactive secondary metabolites in B. subtilis. This allowed the establishment of inducer-associated regulation at the post-transcriptional level with simultaneous use of the native promoter system. In this way, inductor-dependent control of the production of the target metabolite-forming enzyme could be achieved. Full article

In addition to the 20 canonical amino acids encoded in the genetic code, there are two non-canonical ones: selenocysteine and pyrrolysine. The discovery of pyrrolysine synthetases (PylRSs) was a key event in the field of genetic code expansion research. The importance of this discovery is mainly due to the fact that the translation systems involving PylRS, pyrrolysine tRNA (tRNA^Pyl) and pyrrolysine are orthogonal to the endogenous translation systems of organisms that do not use this amino acid in protein synthesis. In addition, pyrrolysine synthetases belonging to different groups are also mutually orthogonal. This orthogonality is based on the structural features of PylRS and tRNA^Pyl, which include identical elements, such as a condensed core, certain base pairs and the structural motifs of tRNA^Pyl. This suggests that targeted structural changes in these molecules enable changes in their specificity for the amino acid and the codon. Such modifications were successfully used to obtain different aaRS/tRNA pairs that allow the incorporation of unnatural amino acids into peptides. This review presents the results of recent studies related to the correlation between the structure and activity of PylRS and tRNA^Pyl and the use of pyrrolysine synthetases to extend the genetic code. Full article

Background: An estimated 10–15% of all genetic diseases are attributable to variants in noncanonical splice sites, auxiliary splice sites and deep-intronic variants. Most of these unstudied variants are classified as variants of uncertain significance (VUS), which are not clinically actionable. This study investigated two novel splice-altering variants, CHM NM_000390.4:c.941-11T>G and CACNA1F NM_005183.4:c.2576+4_2576+5del implicated in choroideremia and cone dystrophy (COD), respectively, resulting in significant visual loss. Methods: Next-generation sequencing was employed to identify the candidate variants in CHM and CACNA1F, which were confirmed using Sanger sequencing. Cascade analysis was undertaken when additional family members were available. Functional analysis was conducted by cloning genomic regions of interest into gateway expression vectors, creating variant and wildtype midigenes, which were subsequently transfected into HEK293 cells. RNA was harvested and amplified by RT-PCR to investigate the splicing profile for each variant compared to the wildtype. Novel variants were reclassified according to ACMG/AMP and ClinGen SVI guidelines. Results: Midigene functional analysis confirmed that both variants disrupted splicing. The CHM NM_000390.4:c.941-11T>G variant caused exon 8 skipping, leading to a frameshift and the CACNA1F NM_005183.4:c.2576+4_2576+5del variant caused a multimodal splice defect leading to an in-frame insertion of seven amino acids and a frameshift. With this evidence, the former was upgraded to likely pathogenic and the latter to a hot VUS. Conclusions: This study adds to the mutational spectrum of splicing defects implicated in retinal degenerations by identifying and characterising two novel variants in CHM and CACNA1F. Our results highlight the importance of conducting functional analysis to investigate the consequences of intronic splice-altering variants and the significance of reclassifying VUS to confirm a genetic diagnosis. Full article

The bioavailability, release, and stability of pharmaceuticals under physicochemical conditions is the major cause of drug candidates failing during their clinical trials. Therefore, extensive efforts have been invested in the development of novel drug delivery systems that are able to transport drugs to a desired site and improve bioavailability. Hydrogels, and peptide hydrogels in particular, have been extensively investigated due to their excellent biocompatibility and biodegradability properties. However, peptide hydrogels often have weak mechanical strength, which limits their therapeutic efficacy. Therefore, a number of methods for improving their rheological properties have been established. This review will cover the broad area of drug delivery, focusing on the recent developments in this research field. We will discuss the variety of different types of nanocarrier drug delivery systems and then, more specifically, the significance and perspectives of peptide-based hydrogels. In particular, the interplay of intermolecular forces that govern the self-assembly of peptide hydrogels, progress made in understanding the distinct morphologies of hydrogels, and applications of non-canonical amino acids in hydrogel design will be discussed in more detail. Full article

The physicochemical properties of amino acid residues from the AAindex database are widely used as predictors in building models for predicting both protein structures and properties. It should be noted, however, that the AAindex database contains data only for the 20 canonical amino acids. Non-canonical amino acids, while less common, are not rare; the Protein Data Bank includes proteins with more than 1000 distinct non-canonical amino acids. In this study, we propose a method to evaluate the physicochemical properties from the AAindex database for non-canonical amino acids and assess the prediction quality. We implemented our method as a bioinformatics tool and estimated the physicochemical properties of non-canonical amino acids from the PDB with the chemical composition presentation using SMILES encoding obtained from the PDBechem databank. The bioinformatics tool and resulting database of the estimated properties are freely available on the author’s website and available for download via GitHub. Full article

Amine transaminases (ATA) are critical players in producing non-canonical amino acids, essential building blocks in pharmaceuticals and fine chemicals. Significant progress has been made in discovering and engineering enzymes in this field, enhancing their use in organic synthesis. However, challenges such as co-factor regeneration, substrate, and product inhibition remain significant limitations to widespread industrial enzyme application. (Chemo-)enzymatic cascades offer efficient and environmentally friendly pathways for synthesizing amino acids, reducing the need for multiple synthesis steps and saving the purification of intermediates. This review focuses specifically on the synthesis of non-canonical amino acids, emphasizing the use of enzymatic and chemoenzymatic cascades involving ATA. Full article

The synthetic peptide of lumican C-terminal 13 amino acids with the cysteine replaced by an alanine, hereafter referred to as lumikine (LumC13_C-A: YEALRVANEVTLN), binds to TGFβ type I receptor/activin-like kinase5 (TBR1/ALK5) in the activated TGFβ receptor complex to promote corneal epithelial wound healing. The present study aimed to identify the minimum essential amino acid epitope necessary to exert the effects of lumikine via ALK5 and to determine the role of the Y (tyrosine) residue for promoting corneal epithelium wound healing. This study also aimed to determine the signaling pathway(s) triggered by lumican–ALK5 binding. For such, adult Lum knockout (Lum^−/−) mice (~8–12 weeks old) were subjected to corneal epithelium debridement using an Agerbrush^®. The injured eyes were treated with 10 µL eye drops containing 0.3 µM synthetic peptides designed based on the C-terminal region of lumican for 5–6 h. To unveil the downstream signaling pathways involved, inhibitors of the Alk5 and EGFR signaling pathways were co-administered or not. Corneas isolated from the experimental mice were subjected to whole-mount staining and imaged under a ZEISS Observer to determine the distance of epithelium migration. The expression of EGFR ligands was determined following a scratch assay with HTCE (human telomerase-immortalized cornea epithelial cells) in the presence or not of lumikine. Results indicated that shorter LumC-terminal peptides containing EVTLN and substitution of Y with F in lumikine abolishes its capability to promote epithelium migration indicating that Y and EVTLN are essential but insufficient for Lum activity. Lumikine activity is blocked by inhibitors of Alk5, EGFR, and MAPK signaling pathways, while EGF activity is only suppressed by EGFR and MAPK inhibitors. qRT-PCR of scratched HTCE cells cultures treated with lumikine showed upregulated expression of several EGFR ligands including epiregulin (EREG). Treatment with anti-EREG antibodies abolished the effects of lumikine in corneal epithelium debridement healing. The observations suggest that Lum/lumikine binds Alk5 and promotes the noncanonical Smad-independent TGFβ/TBRs signaling pathways during the healing of corneal epithelium debridement. Full article

Peptide-based antibiotics (PBAs), including antimicrobial peptides (AMPs) and their synthetic mimics, have received significant interest due to their diverse and unique bioactivities. The integration of high-throughput sequencing and bioinformatics tools has dramatically enhanced the discovery of enzymes, allowing researchers to identify specific genes and metabolic pathways responsible for producing novel PBAs more precisely. Cell-free systems (CFSs) that allow precise control over transcription and translation in vitro are being adapted, which accelerate the identification, characterization, selection, and production of novel PBAs. Furthermore, these platforms offer an ideal solution for overcoming the limitations of small-molecule antibiotics, which often lack efficacy against a broad spectrum of pathogens and contribute to the development of antibiotic resistance. In this review, we highlight recent examples of how CFSs streamline these processes while expanding our ability to access new antimicrobial agents that are effective against antibiotic-resistant infections. Full article

In the fullerene cone HIV-1 capsid, the central channels of the hexameric and pentameric capsomers each contain a ring of arginine (Arg18) residues that perform essential roles in capsid assembly and function. In both the hexamer and pentamer, the Arg18 rings coordinate inositol hexakisphosphate, an assembly and stability factor for the capsid. Previously, it was shown that amino-acid substitutions of Arg18 can promote pentamer incorporation into capsid-like particles (CLPs) that spontaneously assemble in vitro under high-salt conditions. Here, we show that these Arg18 mutant CLPs contain a non-canonical pentamer conformation and distinct lattice characteristics that do not follow the fullerene geometry of retroviral capsids. The Arg18 mutant pentamers resemble the hexamer in intra-oligomeric contacts and form a unique tetramer-of-pentamers that allows for incorporation of an octahedral vertex with a cross-shaped opening in the hexagonal capsid lattice. Our findings highlight an unexpected degree of structural plasticity in HIV-1 capsid assembly. Full article

Despite being frequently encountered, the effect of oxidative or reductive stress on the intracellular metabolism and the response of the intracellular metabolome of yeasts is severely understudied. Non-conventional yeasts are attracting increasing attention due to their large substrate portfolio of non-canonical pathways as well as their production and secretion of proteins. To understand the effects of both stresses on yeast, the conventional model yeast S. cerevisiae and the non-conventional model yeast P. pastoris were perturbed with 5 mM of hydrogen peroxide for oxidative stress and 20 mM of dithiothreitol for reductive stress in well-defined chemostat cultures at a steady state, and fermentation profiles, intracellular amino acid levels, and intracellular glutathione levels were measured. Although stable profiles of extracellular metabolites were observed, significant changes were measured in intracellular amino acid levels within the first five minutes. Collectively, the amino acids ranged from 0.5 to 400 µmol/gDW, with the most significant increase upon the induction of oxidative stress being seen in cysteine (up to 90%) for S. cerevisiae and in aspartate (up to 80%) for P. pastoris. Upon the induction of reductive stress, asparagine nearly halves in S. cerevisiae, while tryptophan decreases by 60% in P. pastoris. By inspecting the time traces of each amino acid, possible mechanisms of pathway kinetics are speculated. This work furthers our understanding of the response of metabolism to oxidative stress in two model yeasts. Full article

GCN1 is recognized as a factor that is essential for the activation of GCN2, which is a sensor of amino acid starvation. This function is evolutionarily conserved from yeast to higher eukaryotes. However, recent studies have revealed non-canonical functions of GCN1 that are independent of GCN2, such as its participation in cell proliferation, apoptosis, and the immune response, beyond the borders of species. Although it is known that GCN1 and GCN2 interact with ribosomes to accomplish amino acid starvation sensing, recent studies have reported that GCN1 binds to disomes (i.e., ribosomes that collide each other), thereby regulating both the co-translational quality control and stress response. We propose that GCN1 regulates ribosome-mediated signaling by dynamically changing its partners among RWD domain-possessing proteins via unknown mechanisms. We recently demonstrated that GCN1 is essential for cell proliferation and whole-body energy regulation in mice. However, the manner in which ribosome-initiated signaling via GCN1 is related to various physiological functions warrants clarification. GCN1-mediated mechanisms and its interaction with other quality control and stress response signals should be important for proteostasis during aging and neurodegenerative diseases, and may be targeted for drug development. Full article

Introducing 3-aminotyrosine (aY), a noncanonical amino acid (ncAA), into green fluorescent protein (GFP)-like chromophores shows promise for achieving red-shifted fluorescence. However, inconsistent results, including undesired green fluorescent species, hinder the effectiveness of this approach. In this study, we optimized expression conditions for an aY-derived cpGFP (aY-cpGFP). Key factors like rich culture media and oxygen restriction pre- and post-induction enabled high-yield, high-purity production of the red-shifted protein. We also engineered two variants of aY-cpGFP with enhanced brightness by mutating a few amino acid residues surrounding the chromophore. We further investigated the sensitivity of the aY-derived protein to metal ions, reactive oxygen species (ROS), and reactive nitrogen species (RNS). Incorporating aY into cpGFP had minimal impact on metal ion reactivity but increased the response to RNS. Expanding on these findings, we examined aY-cpGFP expression in mammalian cells and found that reductants in the culture media significantly increased the red-emitting product. Our study indicates that optimizing expression conditions to promote a reduced cellular state proved effective in producing the desired red-emitting product in both E. coli and mammalian cells, while targeted mutagenesis-based protein engineering can further enhance brightness and increase method robustness. Full article