Functional characterization of a Flavonol 3-O-rhamnosyltransferase and two UDP-rhamnose synthases from Hypericum...

Rhamnosyltransferase (RT) and rhamnose synthase (Rhs) are the key enzymes that are responsible for the biosynthesis of rhamnosides and UDP-l-rhamnose (UDP-Rha) in plants, respectively. How to discover such enzymes efficiently for use is still a problem to be solved. Here, we identified HmF3RT, HmRhs1, and HmRhs2 from Hypericum monogynum, which is abundant in flavonol rhamnosides, with the help of a full-length and high throughput transcriptome sequencing platform. HmF3RT could regiospecifically transfer the rhamnose moiety of UDP-Rha onto the 3-OH position of flavonols and has weakly catalytic for UDP-xylose (UDP-Xyl) and UDP-glucose (UDP-Glc). HmF3RT showed well quercetin substrate affinity and high catalytic efficiency with K_m of 5.14 μM and k_cat/K_m of 2.21 × 10⁵ S^-1 M^-1, respectively. Docking, dynamic simulation, and mutagenesis studies revealed that V129, D372, and N373 are critical residues for the activity and sugar donor recognition of HmF3RT, mutant V129A, and V129T greatly enhance the conversion rate of catalytic flavonol glucosides. HmRhs1 and HmRhs2 convert UDP-Glc to UDP-Rha, which could be further used by HmF3RT. The HmF3RT and HmRhs1 co-expressed strain RTS1 could produce quercetin 3-O-rhamnoside (quercitrin), kaempferol 3-O-rhamnoside (afzelin), and myricetin 3-O-rhamnoside (myricitrin) at yields of 85.1, 110.7, and 77.6 mg L^-1, respectively. It would provide a valuable reference for establishing a better and more efficient biocatalyst for preparing bioactive flavonol rhamnosides by identifying HmF3RT and HmRhs.