Search Results (139)

The combination of computational and experimental methods has become indispensable for optimization and rational enzyme design. Recently, the development of artificial intelligence (AI)-based tools has further streamlined enzyme engineering pipelines, enabling more accurate designs, while reducing the number of variants required for experimental validation. However, due to the intricate complexity of enzymatic systems, significant challenges must be addressed before we take the next step to fully optimize the use of these AI-guided enzyme design methodologies. These challenges include un-curated datasets, the need to consider both the static and dynamic structure of enzymes, and the requirement for effective interdisciplinary collaborations to ensure the integration of computational and experimental approaches. Here, we present recent advances in AI-based computational enzyme design, discussing the main challenges in the field and how a combination with classical physics-based methods could help overcome them. We further explore novel trends that could completely modulate the future of protein design and provide our outlook on the key concepts and future opportunities that will shape the next steps of enzyme design. Full article

Peptide therapeutics offer high target selectivity and low toxicity, but their clinical utility remains constrained by rapid proteolysis in vivo and negligible oral bioavailability. Incorporating non-canonical amino acids (ncAAs) provides a robust molecular engineering framework to overcome these pharmacokinetic bottlenecks. This review analyzes the structural and biophysical design rules of ncAA-mediated peptide stabilization, categorizing them into side-chain steric shielding, backbone conformational constraint, and stereochemical evasion of L-specific proteases. We systematically evaluate the biosynthetic milestones enabling this field, focusing on engineered orthogonal translation systems (tRNA/synthetase pairs, orthogonal ribosomes, quadruplet codons) and metabolic engineering strategies that supply fluorinated and other ncAA precursors de novo. Furthermore, we examine the translation of these technologies into clinical candidates (e.g., modified antimicrobial peptides, antibody–drug conjugates, and PROTACs) and identify scaling, immunogenicity, and computational modeling as key bottlenecks. This review serves as a technical reference for designing next-generation, hyper-stable peptide therapeutics. Full article

Background: Antiretroviral resistance-associated mutations, within the broader context of HIV-1 genetic variability, represent a growing challenge for HIV-1 control, highlighting the need for continuous molecular surveillance and mechanistic understanding of drug resistance. This study aimed to characterize mutations in the pol gene associated with resistance to protease inhibitors and to explore their structural implications. Methods: Viral RNA was extracted from plasma samples of HIV-positive patients, and a 266 bp fragment of the HIV-1 pol gene was amplified by RT-PCR and sequenced using the Sanger method. Sequences showing ≥98% homology were aligned and analyzed using MEGA v11 and the Stanford HIV Drug Resistance Database to identify resistance-associated mutations, while viral subtypes were determined using COMET, jpHMM-HIV, and STAR tools. Amino acid sequences were used for structural modeling with AlphaFold, followed by molecular docking with Nelfinavir using the CB-Dock2 server. Results: Four samples exhibited resistance-associated profiles, including high-level, intermediate, and low-level resistance, with one isolate showing high-level resistance to multiple protease inhibitors. Structural analyses revealed that Nelfinavir preferentially binds to alternative hydrophobic cavities rather than the canonical catalytic site, lacking direct interactions with the Asp25/Asp25′ dyad. Conclusions: These findings suggest a structural mechanism of resistance based on non-canonical ligand binding that may impair effective protease inhibition. Full article

Amyotrophic lateral sclerosis (ALS) remains an intractable motor neuron (MN) disease with a growing patient population and few effective treatments. Here, we review how extracellular phosphoglycerate kinase 1 (ePgk1) improves neurite outgrowth of MNs (NOMN) and axonal growth, both in vitro and in vivo. Our group first elucidated a novel non-canonical function of ePgk1 as a cross-tissue mediator between nerve and muscle tissues. We then discovered that neural membranous Enolase 2 (Eno2) serves as a receptor of ligand ePgk1 and that ePgk1-Eno2 interaction suppresses the Rac1-GTP/p-Pak1-T423/p-P38-T180/pMK2-T334/p-Limk1-S323 axis, reducing p-Cofilin and promoting NOMN and axonal growth, finally suggesting that the 419th aspartic acid residue of Eno2 mediates this interaction. In a crucial preclinical step, we truncated two short 16-amino-acid derivatives from Pgk1, FD-1/-2, each mediating neuroprotection comparable to that of full-length 417-amino-acid Pgk1 in ALS animal models, in terms of improvements of innervated neuromuscular junction, MN cell bodies, motor performance, and endpoint prolongation. In this context, we also discuss the opposite function driven by Eno1-plasminogen interaction and by Eno2-ePgk1 interaction; the latter results in unfavorable for tumorigenesis. Unlike intracellular Pgk1 roles, ePgk1 is an extracellular factor with anti-angiogenic properties, further positioning ePgk1 and its FD-1/-2 as promising protein/peptide drugs for ALS treatment. Full article

Background: Recent work has revealed that protein-coding sequences encode regulatory information influencing mRNA stability and translation through a nascent peptide code. However, the evolutionary origin of this regulatory layer remains unclear. This study aims to determine when peptide-mediated translational control emerged during the evolution of the proteome and genetic code. Methods: Dipeptide-specific effects on mRNA stability and translation were integrated with a phylogenetic timeline of dipeptide emergence derived from dipeptide sequences across proteomes. Each of the 400 canonical dipeptides was assigned an evolutionary age, and experimentally derived regulatory effects were mapped onto this timeline, with associations assessed using rank-based correlation and regression analyses. Results: A weak but statistically significant negative association was observed between dipeptide age and mRNA stability, indicating that more recently evolved dipeptides tend to destabilize transcripts. This trend was stronger at the amino acid level, where later-emerging residues showed greater contributions to reduced mRNA levels. Destabilizing effects were associated with physicochemical properties such as positive charge, side-chain bulkiness, and β-strand propensity. Mapping these effects onto codon space revealed a non-random distribution aligned with the evolutionary and structural organization of the genetic code. Destabilizing effects were also enriched within specific codon exchange groups, indicating that regulatory signals are structured within the degeneracy and mutational neighborhoods of the code. Conclusions: These findings indicate that the nascent peptide code is a late evolutionary innovation linked to amino acid expansion and proteomic complexity, with regulation embedded within both peptide sequences and the degeneracy structure of the standard genetic code. Full article

Threonine aldolases (TAs) are pyridoxal-5-phosphate (PLP)-dependent enzymes that catalyze the reversible aldol condensation between aldehydes and glycine, enabling the asymmetric synthesis of chiral β-hydroxy-α-amino acids. The discovery of new TAs has greatly enriched the enzyme family and spurred investigations into noncanonical reactions beyond conventional aldol condensation, establishing access to structurally diverse amino compounds. Protein engineering has significantly improved the catalytic performance of this enzyme class, particularly addressing the inherent limitation of low C_β-stereoselectivity. Furthermore, the integration of TAs into multi-enzyme cascade systems has broadened their synthetic applicability. This review systematically presents recent progress in biocatalysis with TAs, including the various reaction types, the engineering of TAs for improved catalytic performance, the application of the enzyme in cascade reactions, and future opportunities to broaden the utility of the enzyme class in amino compound synthesis. Full article

Glutamine is the most abundant amino acid in human plasma and tissues and plays essential roles in cellular metabolism, biosynthesis, and redox homeostasis. Beyond these canonical functions, glutamine availability and utilization have emerged as key regulators of multiple cellular stress responses, including the integrated stress response, endoplasmic reticulum stress, metabolic checkpoint signaling, and autophagy. During viral infection, host glutamine metabolism is frequently reprogrammed to meet the energetic and biosynthetic demands of viral replication, thereby inducing or reshaping glutamine-linked stress pathways. Increasing evidence indicates that these stress responses are not merely secondary consequences of infection but actively influence key stages of the viral life cycle, including viral entry, genome replication, protein synthesis, and host antiviral responses. In this review, we summarize current advances in understanding how glutamine metabolism regulates cellular stress responses in the context of both viral and non-viral infections, and how these pathways, in turn, modulate viral pathogenesis and host defense. We discuss the context-dependent roles of glutamine-linked stress signaling in either promoting viral replication or restricting infection, depending on viral species, host cell type, and metabolic conditions. Finally, we highlight emerging concepts and unresolved questions, including the potential of targeting glutamine metabolism and associated stress pathways as host-directed antiviral strategies. A deeper understanding of the interplay between glutamine metabolism, cellular stress responses, and viral infection may provide new insights into disease mechanisms and inform the development of novel therapeutic approaches. Full article

Acidic chitinase (Chia) degrades chitin, a structural polysaccharide in insect exoskeletons, and plays important roles in omnivorous and insectivorous mammals and birds. In birds, gene duplications have generated multiple Chia paralogs with functional divergence, but the molecular basis for this diversification remains unclear. Here, we characterized three chicken Chia paralogs (Chia1–3) and identified distinct pH-dependent enzymatic profiles. Chia1 is enzymatically inactive but was captured by chitin-affinity resin despite lacking a canonical chitin-binding domain, suggesting residual substrate interaction through the catalytic domain or a non-catalytic role. Chia2 exhibits maximal activity at pH 2.0, whereas Chia3 peaks at pH 5.0 and displays broader activity. Exon swapping and site-directed mutagenesis identified residues 104 (Ala in Chia2, Asp in Chia3) and 269 (His vs. Asn) as key contributors to pH-dependent activity differences. Reciprocal substitutions shifted pH profiles accordingly. Structural modeling and computational pKa predictions suggested that D213 and residue 269 may function as a pKa-regulating module influencing catalytic ionization. Comparative sequence analysis revealed lineage-specific conservation of these residues, consistent with adaptive divergence. Our findings show that limited amino acid substitutions can markedly modify pH-dependent enzymatic activity, providing mechanistic insight into how local residue variation contributes to the functional diversification of duplicated genes. Full article

The sustainable synthesis of valuable noncanonical amino acids from renewable raw materials holds significant importance. This research developed a viable chemical–biological coupling process, leveraging the synergistic effect of a solid acid catalyst and the whole cell of E. coli PpLTA to selectively synthesize β-(2-furanyl) serine from corncob. Initially, a novel magnetic solid acid catalyst, Fe₃O₄/C-SO₃H, was successfully fabricated and employed to catalyze the degradation of corncob in a toluene–water biphasic system for furfural production. Under the optimal conditions (catalyst loading of 2.0% w/w and reaction at 170 °C for 20 min), the furfural yield could attain 62.3%. After ten cycles of use, the yield of furfural remained at 44.7% and the retention rate of catalytic activity was 71.7%. Furthermore, the biocompatibility verification results demonstrated that the furfural derived from corncob could be completely transformed by E. coli PpLTA at a concentration of 50 mM, and this furfural system did not generate any by-products that hindered the biotransformation process. This chemical–biological coupling approach offers a technical solution for the efficient production of noncanonical amino acids from biomass resources. Full article

Background: Lyases are used in a wide scope of applications, making them invaluable tools in both industrial biotechnology and molecular biology. Many examples of lyases belong to the extensive family of pyridoxal 5′-phosphate (PLP)-dependent enzymes, which catalyze numerous reactions involved in amino acid metabolism, like tryptophan indole-lyase (Trpase or Tnase), tyrosine phenol-lyase (TPL), and methionine γ-lyase (MGL). Beyond their role in physiological processes, these lyases can also facilitate the synthesis of other biologically active products from non-canonical substrates. Objectives: Up till now there were only two C–S lyases known for the thiosulfinates’ biosynthesis from S-substituted L-cysteine sulfoxides—alliinase and MGL. Our study reveals for the first time that C–C lyases are capable of C–S lyase activity in reactions with S-alkyl, S-allyl and S-benzyl cysteine sulfoxides. Methods: We have compared the kinetic profiles of S-substituted L-cysteine sulfoxide degradation mediated by carbon–sulfur lyase MGL versus carbon–carbon lyases TPL and Trpase. Results: Among other S-alkyl-L-cysteine sulfoxides, petiveriin (S-benzyl-L-cysteine sulfoxide) was proven to be a substrate for all three enzymes. The potential utility of these enzymes in thiosulfinate production was supported by in vitro testing of enzyme-generated thiosulfinates against clinically relevant pathogens such as Candida albicans, Pseudomonas aeruginosa, and Staphylococcus aureus. Conclusions: Both C–S and C–C lyases—MGL, TPL, and Trpase—can be implemented for practical application in thiosulfinate synthesis. Full article

Butidae is a family of teleost fishes with diverse morphological and ecological adaptations, including the marbled goby (Oxyeleotris marmorata), a large species of high economic value in Southeast and East Asia. The previous mitogenomic studies on cultured populations of O. marmorata from non-native habitats have provided limited insights into genetic divergence, structural variation, and evolutionary relationships. Hence, this study presented the complete mitochondrial genome of O. marmorata from its native habitat in Indonesia, providing structural characterization, assessment of genetic diversity, and matrilineal phylogenetic analysis. The circular mitogenome was 16,525 bp, comprising 37 genes and a non-coding control region (CR). The gene organization and strand distribution were conserved among Oxyeleotris species, with 28 genes on the heavy strand and nine on the light strand, and a pronounced A+T compositional bias. The comparative analyses of O. marmorata (from both native and cultured habitats) and Oxyeleotris lineolata mitogenomes revealed minor variations in intergenic spacers, gene overlaps, protein-coding gene (PCGs) lengths, and codon usage patterns. Conversely, the nonsynonymous and synonymous substitution ratios observed in species of the family Butidae and its closest related family (Eleotridae) indicate strong purifying selection in the present dataset. Notably, the ATG was the predominant start codon, whereas the COI gene utilized GTG, and amino acid composition analysis demonstrated high frequencies of arginine, leucine, and serine. Most transfer RNAs retained the canonical cloverleaf secondary structure except for trnS1, which lacked a functional dihydrouridine arm, whereas the CR contained four conserved sequence blocks with variable nucleotide motifs and no detectable tandem repeats. The haplotype analysis of native (Indonesia) and introduced populations (China) highlighted three haplotypes with high diversity (Hd = 1.0000) and substantial nucleotide variation (π = 0.6667). The genetic divergence across 13 PCGs was gene-specific, with COI and ND5 showing the highest variation, while ND4L and ATP8 were highly conserved. The phylogenetic analyses based on concatenated 13 PCGs using both Bayesian Inference and Maximum Likelihood methods revealed that Oxyeleotris forms a monophyletic clade and is closely related to Bostrychus sinensis. In addition, the broader phylogenetic framework inferred the matrilineal relationships within the family Butidae and its closest related family, Eleotridae. This study also recommends expanding analyses to include the mitogenomes of the remaining 17 Oxyeleotris species, together with comprehensive genomic data, to further elucidate their genetic architecture, evolutionary history, and ecological adaptability across diverse aquatic ecosystems. Full article

Rifampicin-resistant tuberculosis (RR-TB) remains a major threat to global TB control, primarily driven by mutations in the rpoB gene of Mycobacterium tuberculosis (Mtb). Most resistance-conferring mutations occur within the 81-base pair RIF resistance determining region (RRDR), particularly at codons S450L, H445Y/D, and D435V, which are strongly associated with high level resistance. However, increasing evidence of low-frequency and disputed variants both within and beyond the RRDR reveals a broader genetic spectrum that contributes to diagnostic uncertainty and variable phenotypic outcomes. This review summarizes current knowledge of high frequency, low frequency, and disputed rpoB mutations and their implications for molecular detection of RIF resistance. Structural analyses show that specific amino acid substitutions alter key hydrogen bonds or create steric hindrance in the RIF-binding pocket, leading to diverse resistance levels. Despite the success of molecular platforms such as Xpert MTB/RIF and line probe assays, their hotspot-based detection limits sensitivity to noncanonical variants. Lowering the minimum inhibitory concentration (MIC) breakpoint and integrating sequencing-based approaches, such as targeted and whole-genome sequencing, can enhance detection accuracy. A combined genomic and phenotypic framework will be essential to close existing diagnostic gaps and advance precision guided management of RIF-resistant and multidrug-resistant tuberculosis. Full article

LD-Transpeptidases (LDTs) are a widely conserved class of peptidoglycan (PG) crosslinking enzymes in bacteria. They are sometimes overlooked as they often act secondary to penicillin binding proteins (PBPs) under standard conditions. However, LDTs are essential in key pathogens such as Clostridioides difficile and are responsible for β-lactam resistance in Mycobacterium tuberculosis and Enterococcus faecium due their low affinity for penicillins and cephalosporins, allowing them to form LD-crosslinks when DD-crosslinking PBPs are inactivated. This role makes LDTs a promising target when developing new treatments for these pathogens. LDTs can perform different enzymatic reactions. Most commonly they reinforce the PG with 3,3-LD-crosslinks or, in a few cases, 1,3-LD-crosslinks, during stationary phase or stress responses. Some LDTs also incorporate endogenous and exogenous non-canonical D-amino acids into the PG. In many Gram-negative bacteria, specialised LDTs tether lipoproteins or outer membrane proteins (OMPs) to the PG to maintain cell envelope integrity; in some cases this regulates virulence factors. Specialised LDTs have also been implied to have roles in polar growth, toxin secretion, and symbiotic colonisation. Recent discoveries include novel subgroups of the major YkuD family and the identification of the VanW family; this has opened new research directions surrounding LDTs. We aim to understand LDTs and their roles to expand our knowledge of PG synthesis and modification and how these enzymes can be targeted for antibiotic treatment. Full article

Background/Objectives: Single-domain antibodies (sdAbs) are derived from camelid heavy-chain antibodies (HCAb). Their small size, high stability, and ease of production, among other properties, makes them highly valuable in biomedical research and therapeutic development. Several sdAb-based molecules are currently progressing through clinical trials, highlighting their translational relevance. As sdAbs originate from HCAb of Camelidae family, they can originate from multiple species including Vicugna pacos, Lama glama, Camelus dromedarius and Camelus bactrianus. Although several reports and databases analyze the structure of sdAbs, comprehensive evaluations on species-dependent structural differences remain scarce. Methods: We assembled MO-IISA, an open-access curated database of sdAbs with known antigen targets by integrating six public resources (iCAN, INDI, SAbDab-nano, sdAb-DB, PLabDab-nano, NbThermo) under harmonized eligibility criteria. Results: The final dataset comprises 2053 sdAbs derived from llamas (Lama glama, n = 1316); alpacas (Vicugna pacos, n = 325), dromedary camels (Camelus dromedarius, n = 377) and Bactrian camels (Camelus bactrianus, n = 35). We quantified region lengths, amino acid frequency, and conservation/entropy across frameworks (FR1–FR4). The average length of all sdAbs was about 124 ± 8 amino acids, with minor interspecies differences. We observed a consistent enrichment of lysines in FR3 (and secondarily FR2) and cysteines primarily in FR1 and FR3, with non-canonical cysteines more frequent in Bactrian and dromedary sdAbs CDRs. CDR2 and, particularly CDR3, contributed most to inter- and intra-species variability, whereas FRs were highly conserved. Conclusions: Species-neutral framework constraints and species-tuned loop adaptations have practical implications for sdAb engineering, species selection, and conjugation strategies. These features are captured in MO-IISA, an open-access database of known-target sdAbs from different species. Full article

To broaden the scope of research on the characteristics and evolutionary relationships within the heat shock protein 70 (Hsp70) family, encompassing its non-canonical members, amino acid sequences of Hsp70-12, Hsp70-13, and Hsp70-14, alongside those of traditional Hsp70, were collected and analyzed. The findings indicate that, during the evolution of metazoans, the various Hsp70 groups diverged from one another. Specifically, Hsp70-12 emerges as the least conserved member, as evidenced by structural alignment data and the Ka/Ks ratio. It not only represents the most distantly related group to traditional Hsp70 but also stands out as the sole alkaline group within the family. In contrast, Hsp70-13 exhibits a close evolutionary relationship with traditional Hsp70, albeit with the notable loss of its C-terminal domain. Hsp70-14 occupies an intermediate position between Hsp70-12 and Hsp70-13. Phylogenetic analysis suggests that these groups diverged prior to the advent of invertebrates. Furthermore, five conserved motifs within the ATP-binding domain of Hsp70, which serve as distinguishing features for Hsp70 groups, were identified. The diverse characters of the non-canonical Hsp70s are probably related to their special cellular location and tissue specificity. Together, the results of this research will help identify and categorize Hsp70s. Further research that aims at identifying additional non-canonical Hsp70 members and elucidating the distinct characteristics and functions of these molecular chaperones will enhance our comprehension of the origin and evolutionary trajectory of the Hsp70 family. Full article