Molecular basis for branched steviol glucoside biosynthesis

Soon Goo Lee, Eitan Salomon, Oliver Yu, and Joseph M. Jez

PNAS June 25, 2019 116 (26) 13131-13136

PLANT BIOLOGY

Significance

The naturally occurring noncaloric sweetener stevia is a plant natural product consisting of a core terpene structure decorated with a specific pattern of glucose molecules, including a branched three-sugar unit. Stevia and other related molecules are being explored as noncaloric dietary sweeteners because they can help maintain the health of diabetic, phenylketonuric, and obese patients. Here, we describe the three-dimensional structure of the plant enzyme (UGT76G1) that forms the branched group of sugars that defines the stevia molecule and is critical for its high-intensity sweetness. Understanding how this enzyme forms this chemical group provides insight on how the stevia plant makes this sweetener and suggests how to alter the protein to generate new versions of the noncaloric sweetener.

Abstract

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.

See https://www.pnas.org/content/116/26/13131

Fig. 1.

Role of UGT in steviol glucoside biosynthesis and the overall structure of UGT76G1. (A) Role of UGT in the steviol biosynthesis pathway. The C13 and C19 positions of the steviol aglycone are indicated. (B) Three-dimensional structure of UGT76G1. The ribbon diagram for the UGT76G1•UDP complex shows the secondary structure with α-helices (blue) and β-strands (gold). UDP is shown as a space-filling model.