Dataset Information

Structural Insights into the Catalytic Mechanism of a Plant Diterpene Glycosyltransferase SrUGT76G1.

ABSTRACT: Diterpene glycosyltransferase UGT76G1 from Stevia rebaudiana (SrUGT76G1) is key to the generation of economically important steviol glycosides (SGs), a group of natural sweeteners with high-intensity sweetness. SrUGT76G1 accommodates a wide range of steviol-derived substrates and many other small molecules. We report here the crystal structures of SrUGT76G1 in complex with multiple ligands to answer how this enzyme recognizes diterpenoid aglycones and catalyzes the 1,3-sugar chain branching. A spacious pocket for sugar-acceptor binding was observed from the determined SrUGT76G1 structures, which can explain its broad substrate spectrum. Residues Gly87 and Leu204 lining the pocket play key roles in switching between diterpenoid and flavonoid glucosylation. An engineered mutant of SrUGT76G1, T284S, could catalyze a selectively increased production of next-generation sweetener rebaudioside M, with diminished side product of rebaudioside I. Taken together, these resutls provide significant insights into molecular basis of the substrate specificity of scarcely documented diterpenoid glycosyltransferases and would facilitate the structure-guided glycoengineering to produce diversified diterpenoids with new activities.

SUBMITTER: Liu Z

PROVIDER: S-EPMC7747997 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Structural Insights into the Catalytic Mechanism of a Plant Diterpene Glycosyltransferase SrUGT76G1.

Liu Zhifeng Z Li Jianxu J Sun Yuwei Y Zhang Peng P Wang Yong Y

Plant communications 20190928 1

Diterpene glycosyltransferase UGT76G1 from <i>Stevia rebaudiana</i> (SrUGT76G1) is key to the generation of economically important steviol glycosides (SGs), a group of natural sweeteners with high-intensity sweetness. SrUGT76G1 accommodates a wide range of steviol-derived substrates and many other small molecules. We report here the crystal structures of SrUGT76G1 in complex with multiple ligands to answer how this enzyme recognizes diterpenoid aglycones and catalyzes the 1,3-sugar chain branchi ...[more]

PMID: 33404544

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Project description:Glycosyltransferases constitute a large family of enzymes across all domains of life, but knowledge of their biochemical function remains largely incomplete, particularly in the context of plant specialized metabolism. The labdane diterpenes represent a large class of phytochemicals with many pharmacological benefits, such as anti-inflammatory, hepatoprotective, and anticarcinogenic. The medicinal plant kalmegh (Andrographis paniculata) produces bioactive labdane diterpenes; notably, the C19-hydroxyl diterpene (andrograpanin) is predominantly found as C19-O-glucoside (neoandrographolide), whereas diterpenes having additional hydroxylation(s) at C3 (14-deoxy-11,12-didehydroandrographolide) or C3 and C14 (andrographolide) are primarily detected as aglycones, signifying scaffold-selective C19-O-glucosylation of diterpenes in planta. Here, we analyzed UDP-glycosyltransferase (UGT) activity and diterpene levels across various developmental stages and tissues and found an apparent correlation of UGT activity with the spatiotemporal accumulation of neoandrographolide, the major diterpene C19-O-glucoside. The biochemical analysis of recombinant UGTs preferentially expressed in neoandrographolide-accumulating tissues identified a previously uncharacterized UGT86 member (ApUGT12/UGT86C11) that catalyzes C19-O-glucosylation of diterpenes with strict scaffold selectivity. ApUGT12 localized to the cytoplasm and catalyzed diterpene C19-O-glucosylation in planta. The substrate selectivity demonstrated by the recombinant ApUGT12 expressed in plant and bacterium hosts was comparable to native UGT activity. Recombinant ApUGT12 showed significantly higher catalytic efficiency using andrograpanin compared with 14-deoxy-11,12-didehydroandrographolide and trivial activity using andrographolide. Moreover, ApUGT12 silencing in plants led to a drastic reduction in neoandrographolide content and increased levels of andrograpanin. These data suggest the involvement of ApUGT12 in scaffold-selective C19-O-glucosylation of labdane diterpenes in plants. This knowledge of UGT86 function might help in developing plant chemotypes and synthesis of pharmacologically relevant diterpenes.

Dataset Information

Structural Insights into the Catalytic Mechanism of a Plant Diterpene Glycosyltransferase SrUGT76G1.

Publications

Structural Insights into the Catalytic Mechanism of a Plant Diterpene Glycosyltransferase SrUGT76G1.

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