Functional and Structural Dissection of a Plant Steroid 3-O-Glycosyltransferase Facilitated the Engineering Enhancement of Sugar Donor Promiscuity

Cardiotonic steroids (CTSs) are ancient and effective clinical drugs for treating heart failure. Glycosylation of the CTS, especially at the C-3 position, is of great significance in nowadays pharmacological applications because it can lead to increased solubility, reduced toxicity, and expanded bioactivity of the CTS. Plant steroid 3-O-glycosyltransferase (S3GT) is a practical tool to achieve C-3 glycosylation of the CTS. However, a S3GT with both substrate and sugar donor promiscuity remains unidentified. Mechanisms of the plant S3GT for CTS recognition and catalysis are still not clear. In this study, a plant steroid 3-O-glycosyltransferase, UGT74AN2, from the medicinal plant Calotropis gigantea was identified and characterized. UGT74AN2 exhibited substrate regiospecificity and moderate sugar donor promiscuity toward the 3-hydroxyl group of structurally diverse CTSs, as well as N-/S-glycosylation activities. To disclose enzymatic mechanisms of the plant S3GT and guide its engineering, a series of complex structures of UGT74AN2 representing various activity states were solved at atomic resolutions. Key residues were identified for the sugar donor recognition and preference, and a unique V-shaped hydrophobic pocket was revealed, accounting for CTS recognition and regiospecificity. In addition, the conserved residue Gly23 has been proven essential for enzyme catalysis. Based on these findings, an engineered variant UGT74AN2 I284R/W390H/V391G was constructed, which showed enhanced sugar donor promiscuity. The mutant exhibited approximately 1.8-, 7.6-, or 11.4-fold increase in the catalytic activities in comparison with the wild type using UDP-Glc, UDP-GlcNAc, or UDP-Gal as the sugar donor, respectively. In addition, UDP-Rha was accepted as a supplemented sugar donor with a conversion rate of around 55%. Overall, this study revealed the in-depth molecular basis for 3-O-glycosylation of CTS and provided a potent biocatalyst capable of generating diverse CTS 3-O-glycosides for drug discovery.