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黄曲霉尿酸酶中二硫键工程的稳定性及功能后果

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

作者信息

Rahbar Mohammad Reza, Nezafat Navid, Morowvat Mohammad Hossein, Savardashtaki Amir, Ghoshoon Mohammad Bagher, Hajizade Mohammad Soroosh, Ghasemi Younes

机构信息

Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.

Department of Pharmaceutical Biotechnology, School of Pharmacy, Shiraz University of Medical Sciences, P.O. Box, Shiraz, 71345-1583, Iran.

出版信息

Sci Rep. 2025 May 26;15(1):18419. doi: 10.1038/s41598-025-01683-y.

DOI:10.1038/s41598-025-01683-y
PMID:40419569
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12106716/
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.

摘要

二硫键工程是一种在治疗和工业应用中提高酶稳定性和功能寿命的有前景的策略。在本研究中，我们应用计算建模在黄曲霉尿酸酶中引入链间二硫键，以提高其稳定性而不影响催化效率。基于能量受挫、结构完整性和通道分析，在选定位置设计了六个含有靶向二硫键的尿酸酶突变体。通过使用受挫密度映射、均方根波动（RMSF）分析和通道分析，我们评估了每个变体的结构稳定性、灵活性和底物可及性。我们的研究结果表明，在诸如Ala6-Cys290和Ser119-Cys220等残基之间含有二硫键的突变体在高度受挫区域有显著减少，增强了酶的结构弹性。RMSF分析表明二硫键位点附近的局部灵活性降低，有助于提高稳定性。通道分析进一步表明，具有策略性放置二硫键的突变体保持了良好的底物可及性和低能量屏障，这对催化周转至关重要。这些结果强调了靶向二硫键工程在优化酶稳定性方面的潜力，为开发适用于治疗和工业用途的稳定、高性能生物催化剂提供了有价值的见解。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/044e/12106716/c7c3f88cc313/41598_2025_1683_Fig10_HTML.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/044e/12106716/18c7f4472e28/41598_2025_1683_Fig4a_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/044e/12106716/425897a673dd/41598_2025_1683_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/044e/12106716/638f46c0009a/41598_2025_1683_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/044e/12106716/ccff5d8848d5/41598_2025_1683_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/044e/12106716/817a18324a13/41598_2025_1683_Fig8_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/044e/12106716/9ded48c9be75/41598_2025_1683_Fig9a_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/044e/12106716/c7c3f88cc313/41598_2025_1683_Fig10_HTML.jpg

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