Abstract
Diets high in sugar are recognized as a serious health problem, and there is a drive to reduce their consumption. Steviol glycosides are natural zero-calorie sweeteners, but the most desirable ones are biosynthesized with low yields. UGT76G1 catalyzes the β (1–3) addition of glucose to steviol glycosides, which gives them the preferred taste. UGT76G1 is able to transfer glucose to multiple steviol substrates yet remains highly specific in the glycosidic linkage it creates. Here, we report multiple complex structures of the enzyme combined with biochemical data, which reveal that the enzyme utilizes hydrophobic interactions for substrate recognition. The lack of a strict three-dimensional recognition arrangement, typical of hydrogen bonds, permits two different orientations for β (1–3) sugar addition. The use of hydrophobic recognition is unusual in a regio- and stereo-specific catalysis. Harnessing such non-specific hydrophobic interactions could have wide applications in the synthesis of complex glycoconjugates.
Highlights
Diets high in sugar are recognized as a serious health problem, and there is a drive to reduce their consumption
By combining multiple structures of UGT76G1 with the reactivity profile of the available authentic and two self-made steviol glucosides, we have revealed how the enzyme relies on the hydrophobic diterpenoid steviol aglycone to allow flexibility in binding orientation but specificity in catalysis, which provide a rational route for the development of the enzyme in sugar biotechnology
UGT76G1 was characterized with a range of steviol substrates, and the products were confirmed by comparison with authentic standards using high-performance liquid chromatography (HPLC) (Supplementary Fig. 1) and highresolution mass spectrometry (MS)
Summary
Diets high in sugar are recognized as a serious health problem, and there is a drive to reduce their consumption. The sustained consumption of steviol glucosides has been reported to potentiate TRPM5 ion channel activity and glucose-induced insulin secretion, preventing the development of diabetes in mice[14]. The combination of these properties has made steviol glucosides the attractive sugar substitutes. The most abundant steviol glucosides in S. rebaudiana leaves, stevioside (ST) and rebaudioside A (Reb A) (Fig. 1a) are 200-fold sweeter than sucrose and were the first commercially available steviol sweeteners[13,15] Both ST and Reb A activate the bitter taste receptors hTAS2R4 and hTAS2R14, limiting their acceptance[16].
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