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Title: Molecular basis for branched steviol glucoside biosynthesis

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.

Authors:
 [1];  [2];  [3];  [4]
  1. Washington Univ., St. Louis, MO (United States); North Carolina State Univ., Wilmington, NC (United States)
  2. Washington Univ., St. Louis, MO (United States); National Center for Mariculture, Israel Oceanographic and Limnological Research, Eilat (Israel)
  3. Conagen, Inc., Bedford, MA (United States)
  4. Washington Univ., St. Louis, MO (United States)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1545888
Grant/Contract Number:  
AC02-06CH11357
Resource Type:
Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 116; Journal Issue: 26; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; glucosyltransferase; noncaloric sweetener; plant biochemistry; stevia; X-ray crystal structure

Citation Formats

Lee, Soon Goo, Salomon, Eitan, Yu, Oliver, and Jez, Joseph M. Molecular basis for branched steviol glucoside biosynthesis. United States: N. p., 2019. Web. doi:10.1073/pnas.1902104116.
Lee, Soon Goo, Salomon, Eitan, Yu, Oliver, & Jez, Joseph M. Molecular basis for branched steviol glucoside biosynthesis. United States. doi:10.1073/pnas.1902104116.
Lee, Soon Goo, Salomon, Eitan, Yu, Oliver, and Jez, Joseph M. Mon . "Molecular basis for branched steviol glucoside biosynthesis". United States. doi:10.1073/pnas.1902104116.
@article{osti_1545888,
title = {Molecular basis for branched steviol glucoside biosynthesis},
author = {Lee, Soon Goo and Salomon, Eitan and Yu, Oliver and Jez, Joseph M.},
abstractNote = {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.},
doi = {10.1073/pnas.1902104116},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 26,
volume = 116,
place = {United States},
year = {2019},
month = {6}
}

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