Ali Mohamed Yassin, Abdalla Mohnad, Roumia Ahmed F, Tammam Mohamed A, Ramadan Mohamed Fawzy, Edrees Mohmmed Abdelssalam Hassan, Kabra Atul, Zhu Daochen
Biofuels Institute, School of Emergency Management, School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang, 212013, China.
Jiangsu Collaborative Innovation Center of Technology and Material of Water Treatment, Suzhou University of Science and Technology, Suzhou, 215009, China.
Folia Microbiol (Praha). 2025 Jan 22. doi: 10.1007/s12223-025-01241-z.
Ginsenoside Rh2(S) is well-known for its therapeutic potential against diverse conditions, including some cancers, inflammation, and diabetes. The enzymatic activity of uridine diphosphate glycosyltransferase 51 (UGT51) from Saccharomyces cerevisiae plays a pivotal role in the glycosylation process between UDP-glucose (donor) and protopanaxadiol (acceptor), to form ginsenoside Rh2. However, the catalytic efficiency of the UGT51 has remained a challenging task. To this end, we employed site-directed mutagenesis on UGT51 to improve its catalytic efficiency for enhanced production of ginsenoside Rh2. The mutated structure, featuring four key mutations (E805A, S998A, R1031A, and L1032A), exhibited heightened stability, binding affinity, and active site accessibility for protopanaxadiol (PPD) compared to the wild type. Under in vitro conditions, three mutants (E805A, R1031A, and L1032A) demonstrated 10%, 58%, and 65% higher enzymatic activities compared to the wild strain. Notably, the double mutant R1031A/L1032A exhibited an 85% increase in activity. Employing a fed-batch technology with PPD as the substrate yielded a Rh2 production of 4.663 g/L. The molecular dynamics (MD) simulations were employed to investigate the movements and dynamic dynamics of UGT51 mutations and PPD complexes. The root mean square deviation (RMSD) analysis revealed substantial alterations in structural conformation, particularly in the R1031A/L1032A mutations, correlating with boosted catalytic efficiency. Furthermore, the root mean square fluctuation (RMSF) simulation study aligned with both the RMSD and the solvent-accessible surface area (SASA) analyses. The computationally guided site-directed mutagenesis approach holds promise for extending its application to the development of commercially significant enzymes.
人参皂苷Rh2(S)因其对多种病症具有治疗潜力而闻名，这些病症包括某些癌症、炎症和糖尿病。来自酿酒酵母的尿苷二磷酸糖基转移酶51（UGT51）的酶活性在UDP-葡萄糖（供体）和原人参二醇（受体）之间的糖基化过程中起着关键作用，以形成人参皂苷Rh2。然而，提高UGT51的催化效率仍然是一项具有挑战性的任务。为此，我们对UGT51进行了定点诱变，以提高其催化效率，从而增强人参皂苷Rh2的产量。与野生型相比，具有四个关键突变（E805A、S998A、R1031A和L1032A）的突变结构对原人参二醇（PPD）表现出更高的稳定性、结合亲和力和活性位点可及性。在体外条件下，三个突变体（E805A 、R1031A和L1032A）的酶活性比野生菌株分别高10%、58%和65%。值得注意的是，双突变体R1031A/L1032A的活性提高了85%。以PPD为底物采用补料分批技术，人参皂苷Rh2的产量为4.663 g/L。利用分子动力学（MD）模拟来研究UGT51突变体与PPD复合物的运动和动力学。均方根偏差（RMSD）分析揭示了结构构象的显著变化，特别是在R1031A/L1032A突变中，这与催化效率的提高相关。此外，均方根波动（RMSF）模拟研究与RMSD和溶剂可及表面积（SASA）分析结果一致。这种计算指导的定点诱变方法有望将其应用扩展到具有商业意义的酶的开发中。
