Biochemical Properties Research Articles

Biochemical features and biotechnological potential of a proteolytic extract from a psychrophilic Antarctic bacterium.

Proteases are hydrolases that act on peptide bonds, releasing amino acids and/or oligopeptides, and are involved in essential functions in all organisms. They represent an important segment of the global enzyme market, with applications in the food, leather, detergent, and pharmaceutical industries. Depending on their industrial use, proteases should exhibit high activity under extreme conditions, such as low temperatures, e.g. cold-active protease may have potential uses in the detergent industry. Cold-active enzymes show high catalytic constants (kcat) at low temperatures and thermolability, allowing their inactivation at moderate temperatures. This work aimed to characterize an extracellular proteolytic extract produced by an Antarctic isolate identified as Flavobacterium sp. strain AU13, and to evaluate its biotechnological potential as a detergent additive. By mass spectrometry analysis, we identified a major 50kDa protease, with high identity with an epralysin from Pseudomonas fluorescens Pf0-1, an alkaline extracellular metalloprotease belonging to the serralysin subfamily. The AU13 proteolytic extract showed metalloprotease activity and, maximal activity over a wide pH range (pH 5 to 8); it also showed maximal activity at 40°C, suggesting that this extracellular protease is a cold-active enzyme. The AU13 proteolytic extract demonstrated stable and compatible activity with surfactants and oxidants, making it a promising additive for commercial laundry detergents. Its ability to function effectively in cold-water washing conditions offers a significant advantage over conventional enzymes, potentially improving energy efficiency in industrial processes. The biochemical properties and performance of the AU13 proteolytic extract in the presence of laundry detergents, suggest that AU13 produces an extracellular protease with a biotechnological potential.

Feeding broilers with the addition of an Ilex paraguariensis coproduct affects zootechnical performance and meat quality responses.

During the harvest ofIlex paraguariensis, approximately 2-5 tons per hectare of thick stems are left on the soil surface. The outer portion of these stems, referred to as the coproduct, constitutes 30% of the total residue mass. Although this coproduct has been partially characterized in terms of its phytochemical profile, its technological applications remain unexplored. The objective of this research is to evaluate whether broilers fed with feed incorporating I. paraguariensis harvest prunings will exhibit improved zootechnical performance as well as enhanced biochemical and impacts on meat quality. One-day-old Cobb 500 broilers (n = 300) were raised for 42days in the broiler shed at the experimental farm in housed in 2.0 m2 pens with a 10cm layer of poultry litter and equipped with tubular feeders and nipple drinkers. They were arranged in a completely randomized design, comprising four treatments with five replicates of 15 birds each. The treatments consisted of the following diets: a basal diet (0%; the control), feed with 1% coproduct (1% treatment), feed with 2% coproduct (2% treatment), and feed with 3% coproduct (3% treatment). Broilers were assessed for zootechnical performance, intestinal morphometry, and serum biochemical properties. Additionally, meat quality was evaluated, including centesimal composition, chlorogenic acid content, antioxidant activity, metal concentration, and fatty acid profile. Chlorogenic acid was not detected in the meat of broiler chickens. The inclusion of the coproduct impacted both zootechnical performance and meat quality, with a linear effect proportional to the concentration of the additive used in the diet; that is, the worst performance was seen in chickens that consumed 3% of the co-product. It reduced feed consumption and weight gain, lowered cholesterol and triglyceride levels in broiler blood, but increased polyunsaturated fatty acids in the meat, one effect verified for the two largest inclusions (2 and 3% of the co-product). In the intestine, greater villus height and levels of reactive oxygen species were observed in the highest dose of the additive, a group of birds in which greater activity of the enzymes creatine kinase and pyruvate kinase was also observed. In general, none of the doses tested proved to be effective in enhancing productive performance; in addition, it did not increase the concentration of chlorogenic acid in the meat, which would be our hypothesis of having a nutraceutical food.

Factor XIII: Driving (Cross-)Links in Hemostasis, Thrombosis, and Disease.

Blood clots are complex structures composed of blood cells and proteins held together by the structural framework provided by an insoluble fibrin network. Factor (F)XIII is a protransglutaminase essential for stabilizing the fibrin network. Activated FXIII(a) introduces novel covalent crosslinks within and between fibrin and other plasma and cellular proteins, and thereby promotes fibrin biochemical and mechanical integrity. These irreversible modifications are also major determinants of clot composition and functional properties. As such, FXIII has central roles in hemostasis and wound healing, thrombosis, and many proinflammatory diseases associated with coagulation activation. FXIII's biochemical properties are as interesting as its biology is unusual, giving rise to unique and still undefined mechanisms. Here we review features underlying FXIII biology, biochemical function, biophysical impact, and (patho)physiologic implications in hemostasis, thrombosis, and disease.

A complex interplay between histone variants and DNA methylation.

Nucleosomes, the chromatin building blocks, play an important role in controlling DNA and chromatin accessibility. Nucleosome remodeling and the incorporation of distinct histone variants confer unique structural and biochemical properties, influencing the targeting of multiple epigenetic pathways, particularly DNA methylation. This stable epigenetic mark suppresses transposable element expression in plants and mammals, serving as an additional layer of chromatin regulation. In this review we explore recent advances in our understanding of the complex interplays between histone variants and DNA methylation in plants, and discuss the role that chromatin remodeling plays in coordinating histone exchange and methylation of DNA.

Evaluating Changes in the VOC Profile of Different Types of Food Products After Electron Beam Irradiation

During the development of food radiation processing protocols, one of the aims is to find an optimal dose range for a specific type of product in which pathogenic microflora are inhibited while biochemical and organoleptic properties are not disturbed. When various food products are exposed to ionizing radiation, volatile organic compounds (VOCs) are formed. Depending on the radiation dose, the list of VOCs and their content change, so they could be considered marker compounds for the description of irradiation-related processes. This work proposes a universal way to study and compare the profile of volatile compounds in products of animal and plant origin using GC-MS in combination with various data representation techniques, including unsupervised machine learning methods. The VOC profiles of beef, chicken, turkey, fish, and potatoes were examined.

The Gut Bacteria of Gampsocleis gratiosa (Orthoptera: Tettigoniidae) by Culturomics

Gampsocleis gratiosa Brunner von Wattenwyl, 1862, is a type of omnivorous chirping insect with a long history of artificial breeding. It has high economic value and is also an excellent orthopteran model organism. In this study, 12 types of culture media combined with 16S rRNA sequencing were employed to isolate 838 bacterial strains from the gut of G. gratiosa. After sequence comparison, a total of 98 species of bacteria were identified, belonging to 3 phyla, 5 classes, 11 orders, 20 families, and 45 genera. Firmicutes and Proteobacteria accounted for the majority (92.86%). At the order level, Enterobacteriaceae, Bacillales, and Lactobacillales predominated (79.59%). At the genus level, Klebsiella (11.22%) and Enterococcus (7.14%) predominated. This study also enumerated the strain morphological, physiological and biochemical properties of 98 species of bacteria, including colony morphology, Gram staining, bacterial motility test, temperature gradient growth, pH gradient growth, citrate utilization test, temperature oxidase test, contact enzyme test, methyl red test, V-P test, indole test, gelatin liquefaction test, nitrate reduction test, hydrogen sulfide test, starch hydrolysis test, cellulose decomposition test, esterase (corn oil) test and antibiotic susceptibility testing. Additionally, 16 antibiotics were utilized to test the bacterial susceptibility of the strains. This study explored the types and community structure of some culturable microorganisms in the intestinal tract of G. gratiosa and recorded their physiological characteristics. These data reflect the physiological functions of the intestinal microorganisms of G. gratiosa and provide support for subsequent research on the interaction mechanism between microorganisms and their hosts.

Assessing the Impact of Residue Retention on Soil Biochemical Properties of Vertisols in a Maize-Chickpea Cropping System under Different Tillage Practices in Central India

This study examined, the potential effect of different residue retention practices on soil biochemical properties in Vertisol of central India. Experiment was conducted in Randomized Block Design (RBD) with three different levels of crop residue retention (RR); 0%, 30%,90% in maize chickpea cropping system over conventional tillage (CT). The parameters assessed were total organic carbon (TOC), β-glucosidase activity, dehydrogenase activity (DHA), fluorescein diacetate hydrolysis activity (FDA), and stratification ratio. Soil samples were collected from 0–15 cm and 15–30 cm depths at the end of the cropping cycle to evaluate the effects of different residue management strategies. At the surface soil (0–15 cm), TOC was significantly higher under 90% RR (15.24 g kg⁻¹) and 30% RR (12.16 g kg⁻¹) compared to CT (9.39 g kg⁻¹). Enzymatic activities also showed significant improvements with increased residue retention. DHA at 0–15 cm was highest under 90% RR (103.57 µg TPF g⁻¹ day⁻¹), followed by 30% RR (84.63 µg TPF g⁻¹ day⁻¹) and CT (70.75 µg TPF g⁻¹ day⁻¹). A similar trend was observed for FDA, where 90% RR recorded 26.13 µg fluorescein g⁻¹ h⁻¹, exceeding CT (22.91 µg fluorescein g⁻¹ h⁻¹). β-glucosidase activity was also highest under 90% RR (169.60 µg PNG g⁻¹ soil h⁻¹), with reduced values at greater soil depths. Enzymatic activities (β-glucosidase, DHA, and FDA) exhibited a strong correlation (p < 0.01) with TOC content and were also strongly correlated, confirming their sensitivity to management practices. Stratification ratios did not vary significantly across residue retention levels, likely due to the high clay content protecting TOC and enzymes. These findings highlight the potential of residue retention to enhance soil health and serve as reliable indicators of soil quality in sustainable cropping systems.

Soil Texture Mediates the Toxicity of ZnO and Fe3O4 Nanoparticles to Microbial Activity

The widespread use of metal oxide nanoparticles (NPs) in industrial and household products has raised concerns about their potential soil contamination and its ecological consequences. The purpose of this study was to examine and compare the effects of iron oxide nanoparticles (FeONPs) and zinc oxide nanoparticles (ZnONPs) on the microbial activity and biochemical properties of differently textured soils. A mesocosm experiment was conducted using three soil types–clay loam (CL), sandy clay loam (SCL), and sandy loam (SL) amended with farmyard manure (FYM), ZnONPs and/or FeONPs. The results revealed significant differences in microbial colony-forming units (CFUs) and carbon dioxide (CO2) emissions in the order of SL > SCL > CL. Compared with those from the unfertilized control, the CO2 emissions from the FYM increased by 112%, 184% and 221% for CL, SCL and SL, respectively. The addition of ZnONPs and FeONPs notably increased the microbial biomass Zn/Fe, which reflected their consumption by the soil microbes. As a result, microbial CFUs were considerably reduced, which led to a 24%, 8% and 12% reduction in cumulative CO2 emissions after the addition of ZnONPs to the CL, SCL and SL soils, respectively. The respective decrements in the case of FeONPs were 19%, 2% and 12%. The temporal dynamics of CO2 emissions revealed that the CO2 emissions from CL with or without FYM/NPs did not differ much during the first few days and later became pronounced with time. Almost all the studied chemical characteristics of the soils were not strongly affected by the ZnONPs/FeONPs, except EC, which decreased with the addition of these nanomaterials to the manure-amended soils. Principal component analysis revealed that the ZnONPs and FeONPs are negatively corelated with microbial CFUs, and CO2 emission, with ZnONPs being more toxic to soil microbes than FeONPs, though their toxicity is strongly influenced by soil texture. Hence, these findings suggest that while both these NPs have the potential to impair microbial activity, their effects are mediated by soil texture.

Structural insights into glucose-6-phosphate recognition and hydrolysis by human G6PC1

The glucose-6-phosphatase (G6Pase) is an integral membrane protein that catalyzes the hydrolysis of glucose-6-phosphate (G6P) in the endoplasmic reticulum lumen and plays a vital role in glucose homeostasis. Dysregulation or genetic mutations of G6Pase are associated with diabetes and glycogen storage disease 1a (GSD-1a). Studies have characterized the biophysical and biochemical properties of G6Pase; however, the structure and substrate recognition mechanism of G6Pase remain unclear. Here, we present two cryo-EM structures of the 40-kDa human G6Pase: a wild-type apo form and a mutant G6Pase-H176A with G6P bound, elucidating the structural basis for substrate recognition and hydrolysis. G6Pase comprises nine transmembrane helices and possesses a large catalytic pocket facing the lumen. Unexpectedly, G6P binding induces substantial conformational rearrangements in the catalytic pocket, which facilitate the binding of the sugar moiety. In conjunction with functional analyses, this study provides critical insights into the structure, substrate recognition, catalytic mechanism, and pathology of G6Pase.

Effects of different light intensity on the growth, physiological and biochemical properties, and stomatal ultrastructure of Rhododendron micranthum saplings

Effects of different light intensity on the growth, physiological and biochemical properties, and stomatal ultrastructure of Rhododendron micranthum saplings

Cofactors facilitate bona fide prion misfolding in vitro but are not necessary for the infectivity of recombinant murine prions.

Prion diseases, particularly sporadic cases, pose a challenge due to their complex nature and heterogeneity. The underlying mechanism of the spontaneous conversion from PrPC to PrPSc, the hallmark of prion diseases, remains elusive. To shed light on this process and the involvement of cofactors, we have developed an in vitro system that faithfully mimics spontaneous prion misfolding using minimal components. By employing this PMSA methodology and introducing an isoleucine residue at position 108 in mouse PrP, we successfully generated recombinant murine prion strains with distinct biochemical and biological properties. Our study aimed to explore the influence of a polyanionic cofactor in modulating strain selection and infectivity in de novo-generated synthetic prions. These results not only validate PMSA as a robust method for generating diverse bona fide recombinant prions but also emphasize the significance of cofactors in shaping specific prion conformers capable of crossing species barriers. Interestingly, once these conformers are established, our findings suggest that cofactors are not necessary for their infectivity. This research provides valuable insights into the propagation and maintenance of the pathobiological features of cross-species transmissible recombinant murine prion and highlights the intricate interplay between cofactors and prion strain characteristics.

Multifunctional Mycobacterial Topoisomerases with Distinctive Features.

Tuberculosis (TB) continues to be a major cause of death worldwide despite having an effective combinatorial therapeutic regimen and vaccine. Being one of the most successful human pathogens, Mycobacterium tuberculosis retains the ability to adapt to diverse intracellular and extracellular environments encountered by it during infection, persistence, and transmission. Designing and developing new therapeutic strategies to counter the emergence of multidrug-resistant and extensively drug-resistant TB remains a major task. DNA topoisomerases make up a unique class of ubiquitous enzymes that ensure steady-state level supercoiling and solve topological problems occurring during DNA transactions in cells. They continue to be attractive targets for the discovery of novel classes of antibacterials and to develop better molecules from existing drugs by virtue of their reaction mechanism. The limited repertoire of topoisomerases in M. tuberculosis, key differences in their properties compared to topoisomerases from other bacteria, their essentiality for the pathogen's survival, and validation as candidates for drug discovery provide an opportunity to exploit them in drug discovery efforts. The present review provides insights into their organization, structure, function, and regulation to further efforts in targeting them for new inhibitor discovery. First, the structure and biochemical properties of DNA gyrase and Topoisomerase I (TopoI) of mycobacteria are described compared to the well-studied counterparts from other bacteria. Next, we provide an overview of known inhibitors of DNA gyrase and emerging novel bacterial topoisomerase inhibitors (NBTIs). We also provide an update on TopoI-specific compounds, highlighting mycobacteria-specific inhibitors.

The potential of seaweed-derived polysaccharides as sustainable biostimulants in agriculture.

Seaweed polysaccharides such as alginate, carrageenan, agar, and ulvan are emerging as key bioresources in sustainable agriculture due to their unique structural characteristics and functional properties. This review highlights their potential as eco-friendly biostimulants capable of enhancing soil health, plant growth, and stress resilience. Specific mechanisms, including the gel-forming capacity of alginate, ion exchange abilities, and the hydrophilic nature of these polysaccharides, enable improved water retention, nutrient uptake, and plant productivity under adverse conditions, including drought, salinity, and extreme temperatures. Moreover, their role as hydrogels and bio-elicitors introduces novel approaches to addressing global challenges in agriculture, such as climate change and food security. Real-world applications, such as the use of Ascophyllum nodosum extract for drought tolerance and Gracilaria tenuistipitata var. liui to boost grain yields, underscore the practicality and success of these biostimulants. Despite their promising applications, challenges like variability in seaweed quality, high extraction costs, and limited product standardization hinder their scalability. This review provides an integrated analysis of their biochemical properties, agricultural applications, and commercial products while proposing solutions to optimize their use for advancing sustainable farming practices.

Explaining deuterium-depleted water as a cancer therapy: a narrative review

Deuterium is a natural heavy isotope of hydrogen, containing a neutron and a proton. This gives it distinct biophysical and biochemical properties, compared with hydrogen. Deuterium alters enzymatic activity in significant ways. Human metabolic processes minimize the amount of deuterium in mitochondrial water, because it causes a dysfunction in mitochondrial ATPase pumps, leading to excessive reactive oxygen species (ROS) and loss of ATP production. Mitochondrial dysfunction is a characteristic feature of cancer and many other diseases. Lactate plays an important role in cancer progression, and a central role holds also for vacuolar ATPases (V-ATPases). In the presence of excess deuterium, cancer cells show a remarkably altered metabolic policy, enabling invasion and proliferation. Cancer cells protect their mitochondria from excessive ROS by minimizing the use of ATPase to synthesize ATP. Instead, they rely on glycolysis to supply ATP and support the massive synthesis of lactate, which is excreted into the microenvironment. They also use V-ATPases in an unusual way at the plasma membrane to pump deuterium-depleted protons out of the cell, enriching cytoplasmic deuterium. These complex processes suggest that cancer cells are able to sense deuterium levels in the medium and commit apoptosis when deuterium levels are low or proliferate when they are high. Tumorigenesis involves a metabolic switch that supports increased cellular deuterium levels, decreasing the deuterium burden overall in the organism. Strong clinical evidence supports deuterium-depleted water (DDW) as an anticancer treatment. More investigations on cancer autophagic behavior are needed to guide DDW clinical use.

Intermittent deep tillage increases soil quality and ecosystem multifunctionality in a Fluvo-aquic soil on the North China Plain.

Intensive tillage operations often disrupt soil structure and accelerate the decomposition of organic matter, resulting in negative long-term impacts on soil health. Thus, identifying sustainable tillage practices is key for enhancing soil nutrient cycling and improving soil biochemical and biological properties. This study evaluated the effects of five tillage modes on soil quality and ecosystem multifunctionality (EMF) over three years in the North China Plain, where rotary tillage (RT) has degraded soil structure and hindered wheat yield increases. The treatments combined deep tillage (DT), RT, and shallow rotary tillage (SRT): (1) RT-RT-RT; (2) DT-RT-RT; (3) DT-RT-SRT; (4) DT-SRT-SRT; (5) DT-SRT-RT. Measurements included soil nutrients, microbial biomass carbon and nitrogen (Cmic and Nmic), enzyme activities, soil quality index (SQI), EMF, across 0-50cm soil layers, as well as crop yields. We found that DT significantly improved various soil properties compared to continuous RT, thereby improving crop yield. In the first year of the cycle, DT increased available nitrogen (AN), available phosphorus (AP), potassium (K), Cmic, Nmic, enzyme activities, SQI, and EMF in the 20-40cm soil layer. These improvements persisted in the following two years. Compared to RT-RT-RT, the largest increases in SQI (13.0%-22.2%) and EMF (11.2%-126.9%) were found in the 0-30cm layer under DT-SRT-RT. Random forest analysis identified Cmic, AN, AP, Corg, and Ntotal as key EMF drivers. Meanwhile, wheat yield under DT-SRT-RT was 14.6% higher than under RT-RT-RT. These findings demonstrate that incorporating occasional DT enhances soil properties and crop yields, offering a sustainable strategy for cropping system management.

Testing the Purity of Limnospira fusiformis Cultures After Axenicity Treatments.

Contaminations are challenging for monocultures, as they impact the culture conditions and thus influence the growth of the target organism and the overall biomass composition. In phycology, axenic cultures comprising a single living species are commonly strived for both basic research and industrial applications, because contaminants reduce significance for analytic purposes and interfere with the safety and quality of commercial products. We aimed to establish axenic cultures of Limnospira fusiformis, known as the food additive "Spirulina". Axenicity is strived because it ensures that pathogens or harmful microorganisms are absent and that the harvested biomass is consistent in terms of quality and composition. For the axenic treatment, we applied sterile filtration, ultrasonication, pH treatment, repeated centrifugation, and administration of antibiotics. For testing axenicity, we considered the most common verification method plate tests with Lysogeny Broth (LB) medium, which indicated axenicity after treatments were performed. In addition, we included plate tests with Reasoner's 2A (R2A) agar and modified Zarrouk+ medium, the latter comparable to the biochemical properties of L. fusiformis' cultivation medium. In contrast to LB plates, the other media, particularly Zarrouk+, indicated bacterial contamination. We conclude that LB-agar plates are inappropriate for contamination screening of extremophiles. Contamination was also verified by cultivation-independent methods like flow cytometry and 16S rRNA genome amplicon sequencing. We detected taxa of the phyla Proteobacteria, Bacteriodota, Firmicutes and to a lesser extent Verrucomicrobiota. Contaminants are robust taxa, as they survived aggressive treatments. Sequencing data suggest that some of them are promising candidates for in-depth studies to commercially exploit them.

Zooming into CTC black-tea wine metabolites: A GC-MS-based study

This research was designed to propose a report on fermentation metabolomics of CTC (crush-tear-curl) tea wine (TW), a yeast-fermented broth of sugared CTC black tea infusion. The gas chromatography-mass spectrometry analysis of the tea wine revealed the presence of thirty-five metabolites, including the major compound glycerine with some potential antioxidant molecules and other bioactive agents (4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-; furfural; furfuryl alcohol; succinic acid; levulinic acid; palmitic acid; tyrosol, pyruvaldehyde; and 1-hexadecanol). The role of metabolites in the physicochemical, biochemical, and medicinal properties of TW has been discussed. Biomolecules responsible for the flavour of TW were as follows: glycerine derivatives; pyruvaldehyde; furfural; furfuryl alcohol; acetic, levulinic, succinic, and palmitic acids, etc. – which might develop a sweet, caramel-like, astringent, slightly sour and wine-like flavour and taste. Furthermore, on the basis of yeast metabolism, possible biosynthesis pathways of metabolites were designed aiming for fermentation metabolomics. The outcome of this study cross-verified physicochemical, biochemical, and medicinal properties of TW suggesting its acceptability. As the fields of both wine research and tea science continue to evolve, the findings of this study may encourage fermentation technology for product development from tea that may also boost the growth of the tea industry.

Rad5 and Ubc4 directly ubiquitinate PCNA at Lys164 in vitro.

Ubiquitination of the proliferating cell nuclear antigen (PCNA) by the budding yeast protein Rad5 have important functions in replication stress responses. Rad5 together with the Ubc13-Mms2 complex attaches Lys63-linked ubiquitin chain to a highly conserved Lys164 residue in PCNA. The reaction requires prior PCNA mono-ubiquitination by the Rad6-Rad18 complex and signals for error-free DNA damage tolerance responses. Cellular studies suggested that Rad5 also cooperates with Ubc4 to catalyze PCNA ubiquitination in response to Okazaki fragment ligation defects, but biochemical evidence of this reaction is lacking. Here we reconstituted this reaction and studied its biochemical properties. We found that Rad5 and Ubc4 directly ubiquitinate PCNA and the reaction requires a coordination of Rad5's HIRAN and RING domains. Most interestingly, we found that the reaction ubiquitinates PCNA at multiple sites among which Lys164 is a major ubiquitination site. These findings suggest that Rad5 may contribute to replication stress responses through a novel mechanism by directly ubiquitinating Lys164 in PCNA.

Correction: Comparative Effect of a Feedstock and its Biochar on Soil Biochemical Properties under Low and Optimum Moisture Availability in C Poor and C Rich Soils

Correction: Comparative Effect of a Feedstock and its Biochar on Soil Biochemical Properties under Low and Optimum Moisture Availability in C Poor and C Rich Soils

The Role of nsp5 in SARS-CoV-2

SARS-CoV-2, the causative agent of the COVID-19 pandemic, is a single-stranded positive-sense RNA virus belonging to the coronavirus family. It emerged at the end of 2019 and has swiftly disseminated globally, presenting an unparalleled threat to public health worldwide. The non-structural protein 5 (nsp5) is a key component in the SARS-CoV-2 replication process, also known as the main protease (Mpro or 3CLpro), which is essential for cleaving viral polyproteins into functional proteins required for replication and transcription. The conserved characteristics and essential role of nsp5 render it an attractive target for antiviral pharmacological development. Recent developments in nsp5-targeted treatments, like Paxlovid, exhibit significant effectiveness in decreasing COVID-19-associated hospitalizations and fatalities. However, the development of nsp5 inhibitors continues to face challenges due to viral mutations and specific administration requirements. This review explores the structural and biochemical properties of nsp5, with particular attention to its catalytic dyad, substrate recognition sites, and three-domain architecture that underpin its role in viral replication. It highlights the potential impact of nsp5 research on advancing antiviral strategies, aiding in the fight against current SARS-CoV-2 infections, and laying the groundwork for future coronavirus outbreak preparedness.