Enzymology and Structural Basis of Glycosyltransferases Involved in Saponin C28 Carboxylic Acid O - d -Fucosylation
Year: 2025
Type: article
Abstract: High Resolution Image Download MS PowerPoint Slide 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.
Source: JACS Au
Authors: Graham A. Hudson, Jose H. Pereira, Peter H. Winegar, David M. FitzGerald, Andy DeGiovanni, Xiaoyue Chen, Xixi Zhao, Maria C. T. Astolfi, James Reed, Amr El-Demerdash, Martin Rejzek, Shingo Kikuchi, Anne Osbourn, Henrik V. Scheller, Paul D. Adams, Jay D. Keasling
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $3,500
Funders: U.S. Department of Energy, U.S. Department of Health and Human Services, Wellcome Trust, University of East Anglia, John Innes Foundation, Novo Nordisk, Mansoura University, National Institutes of Health, Directorate for Biological Sciences, National Institute of Standards and Technology, Biotechnology and Biological Sciences Research Council, National Institute of General Medical Sciences, National Institute of Allergy and Infectious Diseases, National Center for Complementary and Alternative Medicine, Basic Energy Sciences, National Center for Complementary and Integrative Health, Lawrence Berkeley National Laboratory
Awards: G1011385809, G1079818582, G1286236842, G1286251239, G1973870177, G2469927280, Biosynthesis of Cyclopamine and New-to-Nature Triterpenoids in Yeast, G2539837713, Harnessing plant metabolic diversity for human health, G30714459, G3083819904, G334911014, Engineering Quillaja saponin biosynthesis pathways for bio-production of QS-21, G382641700, G4249915386, G4361631179, G4632065618, G4950536974, G5250805592, G5518633504, G6206027980, G650090426, G7354628648, G786782417, Synthetic biology tools for scalable production of medicinal plant terpenes, G8229677252, Harnessing plant metabolic diversity for human health, TITLE: DISTRIBUTED MANUFACTURING OF ANTIGEN FOR SEROLOGICAL TESTING & COUNTERMEASURES? PURPOSE: THE PURPOSE OF THIS PROJECT IS TO DEVELOP TECHNOLOGIES CURRENTLY AT PROOF-OF-CONCEPT STAGE FOR AGILE AND DISTRIBUTED MANUFACTURING OF CORONAVIRUS FAMILY SPIKE PROTEIN VARIANTS BY DEPLOYING EASILY SCALABLE PROTEIN EXPRESSION SYSTEMS. ACTIVITIES TO BE PERFORMED: THE PROJECT TEAM WILL DEVELOP METHODS TO RAPIDLY SCREEN SARS-COV-2 ANTIGENS THAT BIND TO PATIENT SERA AND/OR ANTIBODIES THAT TARGET DIFFERENT VIRAL PROTEINS; IDENTIFY PRODUCTION PLATFORMS THAT CAN MOST EFFICIENTLY PRODUCE THE TARGET ANTIGENS THAT MEET QUALITY SPECIFICATIONS AT A SPEED, SCALE, AND COST THAT IS NOT ACHIEVABLE WITH CURRENT ANTIGEN PRODUCTION PROCESSES; CONDUCT RESEARCH TO UNDERSTAND HOW THE CHOICE OF HOST-CELL PRODUCTION PLATFORM IMPACTS FUNCTIONALITY OF THE ANTIGENS PRODUCED; AND DEVELOP CAPABILITIES FOR SCALEUP AND POST-PRODUCTION MODIFICATION OF GLYCOFORMS ON PURIFIED ANTIGENS; AND PROVIDE A TECHNO-ECONOMIC ASSESSMENT OF PROMISING PRODUCTION PLATFORMS. EXPECTED OUTCOMES: THIS RESEARCH WILL ENABLE AGILE DOMESTIC BIOMANUFACTURING OF REAGENTS FOR CORONAVIRUS RESEARCH AND DIAGNOSTICS, DEVELOP NOVEL PRODUCTION TECHNOLOGIES THAT ENHANCE U.S. BIOINDUSTRIAL COMPETITIVENESS, DE-RISK INVESTMENT IN ANTIGEN PRODUCTION INFRASTRUCTURE, AND EXPAND THE BIOMANUFACTURING WORKFORCE. BY ESTABLISHING METHODS FOR RAPID, DISTRIBUTED ANTIGEN PRODUCTION, THIS PROJECT WILL SIGNIFICANTLY SPEED DISTRIBUTED PRODUCTION AND DEPLOYMENT OF REAGENTS FOR DIAGNOSTICS AND COUNTERMEASURES IN RESPONSE TO ANY NEW DISEASE OUTBREAK. INTENDED BENEFICIARIES: AS NEW CORONAVIRUSES AND VARIANTS ARISE, HEALTHCARE RESEARCHERS WILL BENEFIT BY HAVING THE ABILITY TO QUICKLY DESIGN AND PRODUCE ANTIGENS NEEDED TO ACCELERATE DIAGNOSTICS AND COUNTERMEASURE DEVELOPMENT. THE PUBLIC WILL BENEFIT THROUGH FASTER RESPONSES TO FUTURE PANDEMICS, AND THE NATION WILL BENEFIT MORE BROADLY BY THE INCREASED CAPABILITIES OF THE U.S. BIOINDUSTRIAL SECTOR. SUBRECIPIENT ACTIVITIES: THE RECIPIENT PLANS TO SUBAWARD FUNDS TO ACADEMIC PARTNERS TO CONDUCT RESEARCH FOR ANTIGEN DISCOVERY, STANDARDIZED CLONING, PRODUCTION OF ANTIGENS IN DIFFERENT HOST CELLS, AND CHARACTERIZATION AND ?GLYCO-TAILORING? OF THE TARGET ANTIGENS PRODUCED. THE RECIPIENT WILL ALSO SUBAWARD FUNDS FOR AND TECHNO-ECONOMIC ANALYSIS FOR THIS PROJECT., G9000263589, ALS Efficiently Networking Advanced Beam Line Experiments (ALS-ENABLE), G969889393
OpenAlex ID: W4416568798