Stability and functional consequences of disulfide bond engineering in Aspergillus flavus uricase

Abstract

Abstract 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.

Keywords

• Disulfide Bond Engineering
• Uricase Stability
• Energetic Frustration
• Protein Folding Dynamics
• Enzyme Tunnel Profiling
• In Silico Protein Design