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.

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Journal Article

Authors

Ali MY, Abdalla M, Roumia AF, Tammam MA, Ramadan MF, Edrees MAH, Kabra A, Zhu D

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