Molecular basis for branched steviol glucoside biosynthesis

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 116; no. 26; pp. 13131 - 13136
• Main Authors: Lee, Soon Goo, Salomon, Eitan, Yu, Oliver, Jez, Joseph M.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 25.06.2019
• Subjects: BASIC BIOLOGICAL SCIENCES; Biochemical analysis; Biological Sciences; Biosynthesis; Biosynthetic Pathways - genetics; Catalysis; Chain branching; Chains; Coenzymes - metabolism; Crystal structure; Crystallography, X-Ray; Diterpenes, Kaurane - chemistry; Diterpenes, Kaurane - metabolism; Enzyme Assays; Glucosides; Glucosides - biosynthesis; Glucosides - chemistry; Glucosyltransferase; Glucosyltransferases - genetics; Glucosyltransferases - isolation & purification; Glucosyltransferases - metabolism; Glucosyltransferases - ultrastructure; Glycosylation; Histidine; Metabolic Engineering - methods; Mutagenesis, Site-Directed; Mutants; Natural products; noncaloric sweetener; plant biochemistry; Plant Proteins - metabolism; Plant Proteins - ultrastructure; Plants, Genetically Modified - metabolism; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Recombinant Proteins - ultrastructure; Regioselectivity; stevia; Stevia - enzymology; Steviol; Stevioside; Sugar; Sweeteners; Sweetening Agents - chemistry; Sweetening Agents - metabolism; Sweetness; Uridine; Uridine Diphosphate - metabolism; X-ray crystal structure; glucosyltransferase; X-ray crystal structure; plant biochemistry; stevia; noncaloric sweetener
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1902104116

Steviol glucosides, such as stevioside and rebaudioside A, are natural products roughly 200-fold sweeter than sugar and are used as natural, noncaloric sweeteners. Biosynthesis of rebaudioside A, and other related stevia glucosides, involves formation of the steviol diterpenoid followed by a series of glycosylations catalyzed by uridine diphosphate (UDP)-dependent glucosyltransferases. UGT76G1 from Stevia rebaudiana catalyzes the formation of the branched-chain glucoside that defines the stevia molecule and is critical for its high-intensity sweetness. Here, we report the 3D structure of the UDP-glucosyltransferase UGT76G1, including a complex of the protein with UDP and rebaudioside A bound in the active site. The X-ray crystal structure and biochemical analysis of site-directed mutants identifies a catalytic histidine and how the acceptor site of UGT76G1 achieves regioselectivity for branched-glucoside synthesis. The active site accommodates a two-glucosyl side chain and provides a site for addition of a third sugar molecule to the C3′ position of the first C13 sugar group of stevioside. This structure provides insight on the glycosylation of other naturally occurring sweeteners, such as the mogrosides from monk fruit, and a possible template for engineering of steviol biosynthesis.