Cloning, expression, and functional characterization of three f 6-hydroxyalizarin glycosyltransferases from Rheum palmatum

Glycosyltransferases are key enzymes responsible for the glycosylation of natural products in plants. The roots and rhizomes of Rheum palmatum Diels, commonly known as rhubarb, are well-established in traditional Chinese medicine and are rich in diverse glycoside natural products. Despite this, no glycosyltransferase from R. palmatum had been molecularly and biochemically characterized until now. In this study, we report the identification and characterization of three novel glycosyltransferases (GTs) - RpUGT1, RpUGT6, and RpUGT30 - that mediate f 6-hydroxyalizarin glycoside biosynthesis. These enzymes exhibit regioselective glycosylation of β-OH anthraquinones and display substrate promiscuity, acting on at least six compounds to form O-glycosides. Molecular modeling and site-directed mutagenesis have identified critical residues essential for substrate binding and glycosylation. These results elucidate the pivotal enzymes and molecular mechanisms underlying 6-hydroxyalizarin biosynthesis in Polygonaceae plants, paving the way for the complete elucidation of this biosynthetic pathway and the construction of an artificial pathway for 6-hydroxyalizarin glycoside production.