Enzymology and structural basis of glycosyltransferases involved in saponin C28 carboxylic acid O-d-fucosylation

Hudson, Graham A., Pereira, Jose H., Winegar, Peter H., Fitzgerald, David M., Degiovanni, Andy, Chen, Xiaoyue, Zhao, Xixi, Astolfi, Maria C. T., Reed, James, El-Demerdash, Amr, Rejzek, Martin, Kikuchi, Shingo, Osbourn, Anne, Scheller, Henrik V., Adams, Paul D. and Keasling, Jay D. (2025) Enzymology and structural basis of glycosyltransferases involved in saponin C28 carboxylic acid O-d-fucosylation. JACS Au, 5 (12). 6011–6024. ISSN 2691-3704

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Abstract

Saponins are a class of natural products composed of an oxidized triterpene core adorned with glycosylations, ultimately giving rise to medicinally important compounds bearing bioactivity that includes, but is not limited to, anti-inflammatory, antimicrobial, antifungal, antiarrhythmic, and immunostimulatory activities. QS-21 is a prominent immunostimulatory saponin and is a critical adjuvant component of several FDA-approved vaccines. One linchpin modification in the biosynthesis and bioactivity of several saponins, including QS-21, is O-d-fucosylation via an ester linkage. In QS-21, the C28-COOH O-d-fucose residue is part of a linear oligosaccharide that is an integral component of the “core pharmacophore” responsible for its immunomodulatory activity. In this work, we performed in-depth in vitro enzymological characterization of two glycosyltransferases involved in C28-COOH O-d-fucosylation during the maturation of two saponin natural products: QsFucT from QS-21 biosynthesis and SvFucT from vaccaroside biosynthesis. QsFucT was previously shown to be a UDP-4-keto-6-deoxy-d-glucosyltransferase; our data reveal that the taxonomically distant SvFucT also functions as a UDP-4-keto-6-deoxy-d-glucosyltransferase and that both glycosyltransferases act on a triterpene acceptor with low-micromolar affinity. Substrate scope studies demonstrate that both enzymes are highly permissive with regard to both the triterpene acceptor and, unexpectedly, the UDP-sugar donor. These data also reveal that the conserved C3-OH branched trisaccharide of QS-21 and other saponins may serve an unusual biosynthetic role in protecting the C23 aldehyde from spurious reduction during biosynthesis. In addition, we crystallized and solved the structures of QsFucT and SvFucT, providing the first structural characterization of 4-keto-6-deoxy-d-glucosyltranferases in the glycosyltransferase family 1 (GT1) class of enzymes and used these structures to explore the importance of conserved residues in the active site. These data suggest that both QsFucT and SvFucT could be leveraged to rapidly explore saponin chemical space and glycodiversify these important medicinal compounds through engineered biosynthesis or in vitro enzymatic synthesis, possibly leading to novel analogs with enhanced physicochemical or pharmacological properties.

Item Type:	Article
Additional Information:	Funding information: Research reported in this publication was supported by the National Center for Complementary & Alternative Medicine (NCCAM) of the National Institutes of Health under Award 1R01AT010593-01, the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award 1R01AI186111-01 (to J.D.K.), and by the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health under Award 1F32GM153046-01 (to P.H.W.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This work was also supported by the National Institute of Standards and Technology under Award 70NANB22H017 (to J.D.K.) and made possible, in part, with the support of the Bioindustrial Manufacturing and Design Ecosystem (BioMADE); the content expressed herein is that of the authors and does not necessarily reflect the views of BioMADE. Crystallography work was conducted at the Advanced Light Source (ALS), a national user facility operated by Lawrence Berkeley National Laboratory on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences through Contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the U.S. Department of Energy. Additional support for beamline access came from the National Institute of Health Project ALS-ENABLE (P30 GM124169). This work was also supported by the Biotechnological and Biological Sciences Research Council (BBSRC) Institute Strategic Programme Grant ‘Harnessing Biosynthesis for Sustainable Food and Health (HBio) (Grant BB/X01097X/1), the John Innes Foundation, BBSRC Super Follow-on-Fund Award BB/R005508/1, the Novozymes Prize 2023 (Novo Nordisk Foundation), Wellcome Discovery Award 227375/Z/23/Z, and BBSRC Responsive Mode Award APP3941 (to A.O.).
Faculty \ School:	Faculty of Science > School of Chemistry, Pharmacy and Pharmacology Faculty of Science > School of Chemistry (former - to 2024) Faculty of Science > School of Biological Sciences
UEA Research Groups:	Faculty of Science > Research Groups > Synthetic and Medicinal Chemistry
Depositing User:	LivePure Connector
Date Deposited:	17 Dec 2025 17:30
Last Modified:	05 Jan 2026 12:30
URI:	https://ueaeprints.uea.ac.uk/id/eprint/101457
DOI:	10.1021/jacsau.5c00907

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