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In nature, coenzyme-independent oxidases have evolved in selective catalysis using isolated substrate-binding pockets. Single-atom nanozymes (SAzymes), an emerging type of non-protein artificial enzymes, are promising to simulate enzyme active centers, but owing to the lack of recognition sites, realizing substrate specificity is a formidable task. Here we report a metal-ligand dual-site SAzyme (Ni-DAB) that exhibited selectivity in uric acid (UA) oxidation. Ni-DAB mimics the dual-site catalytic mechanism of urate oxidase, in which the Ni metal center and the C atom in the ligand serve as the specific UA and O 2 binding sites, respectively, characterized by synchrotron soft X-ray absorption spectroscopy, in situ near ambient pressure X-ray photoelectron spectroscopy, and isotope labeling. The theoretical calculations reveal the high catalytic specificity is derived from not only the delicate interaction between UA and the Ni center but also the complementary oxygen reduction at the beta C site in the ligand. As a potential application, a Ni-DAB-based biofuel cell using human urine is constructed. This work unlocks an approach of enzyme-like isolated dual sites in boosting the selectivity of non-protein artificial enzymes. Single-atom nanozymes are a type of non-protein artificial enzymes and promising for mimicking enzyme active centers, but lack recognition sites to confer substrate specificity. Here, the authors report on a metal-ligand dual-site single-atom nanozyme (Ni-DAB) that mimics the dual-site catalytic mechanism of urate oxidase and has high selectivity in uric acid (UA) oxidation.

Uricase is an enzyme involved in purine catabolism and is found in all three domains of life. Curiously, uricase is not functional in some organisms despite its role in converting highly insoluble uric acid into 5-hydroxyisourate. Of particular interest is the observation that apes, including humans, cannot oxidize uric acid, and it appears that multiple, independent evolutionary events led to the silencing or pseudogenization of the uricase gene in ancestral apes. Various arguments have been made to suggest why natural selection would allow the accumulation of uric acid despite the physiological consequences of crystallized monosodium urate acutely causing liver/kidney damage or chronically causing gout. We have applied evolutionary models to understand the history of primate uricases by resurrecting ancestral mammalian intermediates before the pseudogenization events of this gene family. Resurrected proteins reveal that ancestral uricases have steadily decreased in activity since the last common ancestor of mammals gave rise to descendent primate lineages. We were also able to determine the 3D distribution of amino acid replacements as they accumulated during evolutionary history by crystallizing a mammalian uricase protein. Further, ancient and modern uricases were stably transfected into HepG2 liver cells to test one hypothesis that uricase pseudogenization allowed ancient frugivorous apes to rapidly convert fructose into fat. Finally, pharmacokinetics of an ancient uricase injected in rodents suggest that our integrated approach provides the foundation for an evolutionarily-engineered enzyme capable of treating gout and preventing tumor lysis syndrome in human patients.

Uricase (UOx)-based gout treatments are generally limited due in part to the accumulation of H 2 O 2 in an arthrosis environment characterized by a sluggish metabolism. Here we develop a self-cascade nanomotor with high efficiency toward simultaneous uric acid (UA) degradation and H 2 O 2 elimination on the basis of UOx and sodium citrate loading in amine functionalized hollow mesoporous silica nanoparticles (AHMSNs). Due to the inherent asymmetry of AHMSNs, the developed nanomotors can be actuated by ionic diffusiophoresis induced by the enzymatic UA degradation, thus enlarging the diffusion range within the joint cavity. H 2 O 2 generated during UA decomposition can be simultaneously eliminated by the loaded sodium citrate, resulting in a considerable improvement in mammalian cell viability. In vivo studies display powerful therapeutic effect in gouty models using this self-cascade nanomotor system. These results present the potential of nanomotors as active therapeutic agents for gout, providing a perspective on the application of enzyme-driven micro/nanomotors. Uricase-based gout treatments are generally limited due in part to the accumulation of H 2 O 2 in an arthrosis microenvironment. Here the authors develop a nanomotor-based strategy that simultaneously degrades uric acid and eliminate H 2 O 2 for efficient gouty therapy.

Hyperuricemia is associated with an increased risk of gout, hypertension, diabetes, and cardiovascular diseases. Most mammals maintain normal serum uric acid (SUA) via urate oxidase (Uox), an enzyme that metabolizes poorly-soluble UA to highly-soluble allantoin. In contrast, Uox became a pseudogene in humans and apes over the long course of evolution. Here we demonstrate an atavistic strategy for treating hyperuricemia based on endogenous expression of Uox in hepatocytes mediated by mRNA (mUox) loaded with an ionizable lipid nanoparticle termed iLAND. mUox@iLAND allows effective transfection and protein expression in vitro. A single dose of mUox@iLAND lowers SUA levels for several weeks in two female murine models, including a novel long-lasting model, which is also confirmed by metabolomics analysis. Together with the excellent safety profiles observed in vivo, the proposed mRNA agent demonstrates substantial potential for hyperuricemia therapy and the prevention of associated conditions. Urate oxidase (Uox) lost its function during evolution. Here the authors propose an atavistic strategy to treat hyperuricemia by using a proprietary lipid nanoparticle to load Uox mRNA, which reduced the serum uric acid levels effectively in two animal models.

Aspergillus flavus Urate oxidase (AFUOX) is promising for potential therapeutic applications, particularly in gout treatment. However, the enzyme’s low thermostability and solubility limit its efficacy. A targeted mutation, substituting Gln with Leu at position 269 (Q269L) has been proposed to enhance its stability. The turnover number, catalytic efficiency, and specific activity of Q269L were 3.7 (s -1 ), 53.2 (mM -1 . s -1 ), and 3.926 U/mg, respectively. In comparison, for the wild type, these were 3.1 (S -1 ), 35.1 (mM -1 . s -1 ), and 4.018 U/mg, respectively. Notably, the wild type exhibited maximum activity at pH 9 and 25 °C, whereas the activity of Q269L was obtained at pH 9.5 and 30 °C. Furthermore, the half-life of Q269L at 40 °C is significantly longer (85.55 min) compared to the wild-type (49.85 min). The thermodynamic parameters ΔH ≠ , ΔS ≠ , and ΔG ≠ at 40 °C for Q269L were 60.9 kJ.mol -1 , -276 J.mol -1 , and 147.3 kJ.mol -1 , respectively. Intrinsic fluorescence reductions and ANS fluorescence increases suggest that tryptophan resides in a polar environment with augmented hydrophobic pockets. FTIR analysis of Q269L reveals a decrease in β-sheet and an increase in α-helix structures, supporting molecular dynamics simulations. Collectively, MD and experimental results underscore Q269L’s enhanced thermostability and localized structural alterations, advancing AFUOX’s therapeutic potential.

Biologic drugs have transformed the standard of care for many diseases. However, many biologics induce the formation of anti-drug antibodies (ADAs), which can compromise their safety and efficacy. Preclinical studies demonstrate that biodegradable nanoparticles-encapsulating rapamycin (ImmTOR), but not free rapamycin, mitigate the immunogenicity of co-administered biologic drugs. Here we report the outcomes from two clinical trials for ImmTOR. In the first ascending dose, open-label study (NCT02464605), pegadricase, an immunogenic, pegylated uricase enzyme derived from Candida utilis , is assessed for safety and tolerability (primary endpoint) as well as activity and immunogenicity (secondary endpoint); in the second single ascending dose Phase 1b trial (NCT02648269) composed of both a double-blind and open-label parts, we evaluate the safety of ImmTOR (primary endpoint) and its ability to prevent the formation of anti-drug antibodies against pegadricase and enhance its pharmacodynamic activity (secondary endpoint) in patients with hyperuricemia. The combination of ImmTOR and pegadricase is well tolerated. ImmTOR inhibits the development of uricase-specific ADAs in a dose-dependent manner, thus enabling sustained enzyme activity and reduction in serum uric acid levels. ImmTOR may thus represent a feasible approach for preventing the formation of ADAs to a broad range of immunogenic biologic therapies. Anti-drug antibodies (ADA) induced by biologic drugs may hamper the efficacy of treatment, so inhibiting ADA induction is desirable. Here, in two clinical trials, the authors show that ImmTOR, previously reported to reduce drug immunogenicity in animal studies, helps mitigate ADA induced by pegylated uricase for treating patients with hyperuricemia.

Disulfide bond engineering is a promising strategy for enhancing the stability and functional lifespan of enzymes in therapeutic and industrial applications. In this study, we applied computational modeling to introduce interchain disulfide bonds in Aspergillus flavus uricase to increase its stability without compromising catalytic efficiency. Six uricase muteins were engineered with targeted disulfide bonds at positions selected based on energetic frustration, structural integrity, and tunnel profiling analyses. By employing frustration density mapping, Root Mean Square Fluctuation (RMSF) profiling, and tunnel analysis, we evaluated the structural stability, flexibility, and substrate accessibility of each variant. Our findings revealed that muteins with disulfide bonds between residues such as Ala6-Cys290 and Ser119-Cys220 exhibited significant reductions in highly frustrated regions, enhancing the enzyme’s structural resilience. RMSF analysis indicated decreased local flexibility near disulfide sites, contributing to increased stability. Tunnel profiling further demonstrated that muteins with strategically placed disulfide bonds maintained favorable substrate access and low-energy barriers, critical for catalytic turnover. These results underscore the potential of targeted disulfide bond engineering for optimizing enzyme stability, offering valuable insights for the development of stable, high-performance biocatalysts suitable for therapeutic and industrial use.

With the demand for mass production of protein drugs, solubility has become a serious issue. Extrinsic and intrinsic factors both affect this property. A homotetrameric cofactor-free urate oxidase (UOX) is not sufficiently soluble. To engineer UOX for optimum solubility, it is important to identify the most effective factor that influences solubility. The most effective feature to target for protein engineering was determined by measuring various solubility-related factors of UOX. A large library of homologous sequences was obtained from the databases. The data was reduced to six enzymes from different organisms. On the basis of various sequence- and structure-derived elements, the most and the least soluble enzymes were defined. To determine the best protein engineering target for modification, features of the most and least soluble enzymes were compared. Metabacillus fastidiosus UOX was the most soluble enzyme, while Agrobacterium globiformis UOX was the least soluble. According to the comparison-constant method, positive surface patches caused by arginine residue distribution are appropriate targets for modification. Two Arg to Ala mutations were introduced to the least soluble enzyme to test this hypothesis. These mutations significantly enhanced the mutant’s solubility. While different algorithms produced conflicting results, it was difficult to determine which proteins were most and least soluble. Solubility prediction requires multiple algorithms based on these controversies. Protein surfaces should be investigated regionally rather than globally, and both sequence and structural data should be considered. Several other biotechnological products could be engineered using the data reduction and comparison-constant methods used in this study.

Xanthine oxidase inhibitors such as allopurinol and febuxostat have been the mainstay urate-lowering therapy (ULT) for treating hyperuricaemia in patients with gout. However, not all patients receiving oral ULT achieve the target serum urate level, in part because some patients cannot tolerate, or have actual or misconceived contraindications to, their use, mainly due to comorbidities. ULT dosage is also limited by formularies and clinical inertia. This failure to sufficiently lower serum urate levels can lead to difficult-to-treat or uncontrolled gout, usually due to poorly managed and/or under-treated gout. In species other than humans, uricase (urate oxidase) converts urate to allantoin, which is more soluble in urine than uric acid. Exogenic uricases are an exciting therapeutic option for patients with gout. They can be viewed as enzyme replacement therapy. Uricases are being used to treat uncontrolled gout, and can achieve rapid reduction of hyperuricaemia, dramatic resolution of tophi, decreased chronic joint pain and improved quality of life. Availability, cost and uricase immunogenicity have limited their use. Uricases could become a leading choice in severe and difficult-to-treat gout as induction and/or debulking therapy (that is, for lowering of the urate pool) to be followed by chronic oral ULT. This Review summarizes the evidence regarding available uricases and those in the pipeline, their debulking effect and their outcomes related to gout and beyond.In this Review, the authors provide an overview of the therapeutic effects of uricases, outcomes related to gout and its comorbidities in clinical trials and challenges surrounding the use of uricases as potent urate-lowering therapy for the treatment of gout.

For the first time, this study reports extracellular uricase enzyme isolation and characterization from strain UR1 of spp. from Western Saudi Arabia. The strain efficiently produced highly active extracellular uricase for therapeutic applications. It offers a simplified enzyme purification approach rather than complicated intracellular enzyme purification from other microbes. Strain UR1 exhibited significantly higher uricase synthesis potential [916 U/mg (specific activities) and 275 U/ml (volume)]. The study optimized the conditions (37°C and pH 7.4) for 10% enhanced uricase production in the BT medium where sucrose served as the carbon source. Uricase enzyme remained stable at various pH levels (5–9) up to 50°C, however, the optimal activity was noted at 40°C and pH 7.5. The strain was sensitive to EDTA-like inhibitors. Ca improved the strain activity, which could yield potent formulations for clinical and industrial applications. This novel aspect presents spp. strain UR1 as a promising candidate for the treatment of hyperuricemia and gout. It offers an efficient and inexpensive alternative for uricase synthesis at the industrial scale. These findings encourage further investigations regarding genetic aspects of uricase for improved bioprocessing and therapeutic applications.