Molecular basis for branched steviol glucoside biosynthesis.

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Steviol glucosides, like stevioside and rebaudioside A, are natural, noncaloric sweeteners derived from Stevia.
• Their biosynthesis involves the formation of steviol followed by glycosylation reactions catalyzed by UDP-dependent glucosyltransferases (UGTs).
• UGT76G1 from Stevia rebaudiana is crucial for synthesizing the specific branched-chain glucoside responsible for high-intensity sweetness.

Purpose of the Study:

• To elucidate the 3D structure of the UGT76G1 enzyme.
• To understand the mechanism of regioselective glycosylation in steviol glucoside biosynthesis.
• To provide structural insights for engineering stevia biosynthesis and other natural sweeteners.

Main Methods:

• X-ray crystallography was used to determine the 3D structure of UGT76G1.
• The structure was determined for both the apoenzyme and a complex with UDP and rebaudioside A.
• Biochemical analysis of site-directed mutants was performed.

Main Results:

• The 3D structure of UGT76G1, including its active site with bound UDP and rebaudioside A, was determined.
• A catalytic histidine residue was identified.
• The structural basis for regioselective synthesis of branched steviol glucosides was revealed, showing accommodation of a two-glucosyl side chain and a site for adding a third sugar.

Conclusions:

• The determined structure provides critical insights into the mechanism of UGT76G1.
• Understanding UGT76G1's active site aids in comprehending the biosynthesis of high-intensity stevia sweeteners.
• The findings offer a structural template for engineering steviol biosynthesis and potentially other glycosyltransferases involved in natural sweetener production.