Identification of a highly promiscuous glucosyltransferase from <i>Penstemon barbatus</i> for natural product glycodiversification

Yang, Yijun; Du, Liangcheng; Zhuang, Yibin; Liu, Tao

doi:10.1039/d3ob00370a

“…UGTs that can accept several UDP-sugars serve as the epitome in glycodiversification ( Wen et al, 2023 ; Wu et al, 2023 ). Hence, it is important to understand which traits lead to UDP-sugar promiscuity.…”

Section: Expanding the Donor Specificityof Ugtsmentioning

confidence: 99%

The sugar donor specificity of plant family 1 glycosyltransferases

Gharabli,

Welner

2024

Front. Bioeng. Biotechnol.

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Plant family 1 glycosyltransferases (UGTs) represent a formidable tool to produce valuable natural and novel glycosides. Their regio- and stereo-specific one-step glycosylation mechanism along with their inherent wide acceptor scope are desirable traits in biotechnology. However, their donor scope and specificity are not well understood. Since different sugars have different properties in vivo and in vitro, the ability to easily glycodiversify target acceptors is desired, and this depends on our improved understanding of the donor binding site. In the aim to unlock the full potential of UGTs, studies have attempted to elucidate the structure-function relationship governing their donor specificity. These efforts have revealed a complex phenomenon, and general principles valid for multiple enzymes are elusive. Here, we review the studies of UGT donor specificity, and attempt to group the information into key concepts which can help shape future research. We zoom in on the family-defining PSPG motif, on two loop residues reported to interact with the C6 position of the sugar, and on the role of active site arginines in donor specificity. We continue to discuss attempts to alter and expand the donor specificity by enzyme engineering, and finally discuss future research directions.

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“…To date, only a limited number of enzymes responsible for assembling PhGs have been documented, facilitating the biosynthesis of compounds like verbascoside and ligupurpuroside B. [24][25][26][27][28][29] These include UGT85AF8, a tyrosol glucosyltransferase (GT); [25] UGT79G7, an osmanthuside A 1,3-rhamnosyltransferase; [25] UGT79 A19, an osmanthuside B 1,4-rhamnosyltransferase; [25] and SiAT1, a salidroside hydroxycinnamoyltransferase [24] (Table 1). Except for UG-T85AF10/11, all other GTs favors for mono-hydroxylated units such as Tyl and Cou.…”

Section: Introductionmentioning

confidence: 99%

Construct Phenylethanoid Glycosides Harnessing Biosynthetic Networks, Protein Engineering and One‐Pot Multienzyme Cascades

Yao,

Wang,

Wang

et al. 2024

Angew Chem Int Ed

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Phenylethanoid glycosides (PhGs) exhibit a multitude of structural variations linked to diverse pharmacological activities. Assembling various PhGs via multienzyme cascades represents a concise strategy over traditional synthetic methods. However, the challenge lies in identifying a comprehensive set of catalytic enzymes. This study explores biosynthetic PhG reconstruction from natural precursors, aiming to replicate and amplify their structural diversity. We discovered 12 catalytic enzymes, including four novel 6'‐OH glycosyltransferases and three new polyphenol oxidases, revealing the intricate network in PhG biosynthesis. Subsequently, the crystal structure of CmGT3 (2.62 Å) was obtained, guiding the identification of conserved residue 144# as a critical determinant for sugar donor specificity. Engineering this residue in PhG glycosyltransferases (FsGT61, CmGT3, and FsGT6) altered their sugar donor recognition. Finally, a one‐pot multienzyme cascade was established, where the combined action of glycosyltransferases and acyltransferases boosted conversion rates by up to 12.6‐fold. This cascade facilitated the reconstruction of 26 PhGs with conversion rates ranging from 5‐100%, and 20 additional PhGs detectable by mass spectrometry. PhGs with extra glycosyl and hydroxyl modules demonstrated notable liver cell protection. This work not only provides catalytic tools for PhGs biosynthesis, but also serves as a proof‐of‐concept for cell‐free enzymatic construction of diverse natural products.

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Construct Phenylethanoid Glycosides Harnessing Biosynthetic Networks, Protein Engineering and One‐Pot Multienzyme Cascades

Yao,

Wang,

Wang

et al. 2024

Angewandte Chemie

0

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Phenylethanoid glycosides (PhGs) exhibit a multitude of structural variations linked to diverse pharmacological activities. Assembling various PhGs via multienzyme cascades represents a concise strategy over traditional synthetic methods. However, the challenge lies in identifying a comprehensive set of catalytic enzymes. This study explores biosynthetic PhG reconstruction from natural precursors, aiming to replicate and amplify their structural diversity. We discovered 12 catalytic enzymes, including four novel 6'‐OH glycosyltransferases and three new polyphenol oxidases, revealing the intricate network in PhG biosynthesis. Subsequently, the crystal structure of CmGT3 (2.62 Å) was obtained, guiding the identification of conserved residue 144# as a critical determinant for sugar donor specificity. Engineering this residue in PhG glycosyltransferases (FsGT61, CmGT3, and FsGT6) altered their sugar donor recognition. Finally, a one‐pot multienzyme cascade was established, where the combined action of glycosyltransferases and acyltransferases boosted conversion rates by up to 12.6‐fold. This cascade facilitated the reconstruction of 26 PhGs with conversion rates ranging from 5‐100%, and 20 additional PhGs detectable by mass spectrometry. PhGs with extra glycosyl and hydroxyl modules demonstrated notable liver cell protection. This work not only provides catalytic tools for PhGs biosynthesis, but also serves as a proof‐of‐concept for cell‐free enzymatic construction of diverse natural products.

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Identification of a highly promiscuous glucosyltransferase from Penstemon barbatus for natural product glycodiversification

Abstract: Glycosylation reactions mediated by UDP-glycosyltransferases (UGTs) are common post-modifications involved in plant secondary metabolism and significantly improve the solubility and bioactivity of aglycones. Penstemon barbatus is rich in phenylethanoid glycosides...

Cited by 4 publications

References 34 publications

The sugar donor specificity of plant family 1 glycosyltransferases

The sugar donor specificity of plant family 1 glycosyltransferases

Construct Phenylethanoid Glycosides Harnessing Biosynthetic Networks, Protein Engineering and One‐Pot Multienzyme Cascades

Construct Phenylethanoid Glycosides Harnessing Biosynthetic Networks, Protein Engineering and One‐Pot Multienzyme Cascades

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