title: A novel glucuronosyltransferase has an unprecedented ability to catalyse continuous two-step glucuronosylation of glycyrrhetinic acid to yield glycyrrhizin.
abstract: Glycyrrhizin is an important bioactive compound that is used clinically to treat chronic hepatitis and is also used as a sweetener world-wide. However, the key UDP-dependent glucuronosyltransferases (UGATs) involved in the biosynthesis of glycyrrhizin remain unknown. To discover unknown UGATs, we fully annotated potential UGATs from Glycyrrhiza uralensis using deep transcriptome sequencing. The catalytic functions of candidate UGATs were determined by an in vitro enzyme assay. Systematically screening 434 potential UGATs, we unexpectedly found one unique GuUGAT that was able to catalyse the glucuronosylation of glycyrrhetinic acid to directly yield glycyrrhizin via continuous two-step glucuronosylation. Expression analysis further confirmed the key role of GuUGAT in the biosynthesis of glycyrrhizin. Site-directed mutagenesis revealed that Gln-352 may be important for the initial step of glucuronosylation, and His-22, Trp-370, Glu-375 and Gln-392 may be important residues for the second step of glucuronosylation. Notably, the ability of GuUGAT to catalyse a continuous two-step glucuronosylation reaction was determined to be unprecedented among known glycosyltransferases of bioactive plant natural products. Our findings increase the understanding of traditional glycosyltransferases and pave the way for the complete biosynthesis of glycyrrhizin.
authors.affiliation: School of Chinese Material Medica, Beijing University of Chinese Medicine, Beijing, 100102, China.
authors.forename: Guojie
authors.initials: G
authors.lastname: Xu
authors.affiliation: School of Chinese Material Medica, Beijing University of Chinese Medicine, Beijing, 100102, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Cai
authors.affiliation: School of Traditional Chinese Medicine, Capital Medical University, Beijing, 100069, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Gao
authors.affiliation: School of Chinese Material Medica, Beijing University of Chinese Medicine, Beijing, 100102, China.
authors.forename: Chunsheng
authors.initials: C
authors.lastname: Liu
mesh_terms: D055162:Biocatalysis; D014453:Glucuronosyltransferase / Q000378:metabolism*; D006031:Glycosylation; D006034:Glycyrrhetinic Acid / Q000737:chemistry / Q000378:metabolism*; D006035:Glycyrrhiza / Q000201:enzymology / Q000235:genetics; D019695:Glycyrrhizic Acid / Q000737:chemistry / Q000378:metabolism*; D059014:High-Throughput Nucleotide Sequencing; D062105:Molecular Docking Simulation; D058977:Molecular Sequence Annotation; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D018517:Plant Roots / Q000235:genetics; D059467:Transcriptome / Q000235:genetics; D014530:Uridine Diphosphate / Q000378:metabolism
chemical_list: D014530:Uridine Diphosphate; D019695:Glycyrrhizic Acid; D014453:Glucuronosyltransferase; D006034:Glycyrrhetinic Acid
keywords: Glycyrrhiza uralensis; biosynthesis; glucuronosylation; glucuronosyltransferase; glycosyltransferase; glycyrrhetinic acid; glycyrrhizin