Ancestral protein reconstruction reveals the mechanism of substrate specificity in FN3K-mediated deglycation

Ancestral protein reconstruction reveals the mechanism of substrate specificity in FN3K-mediated deglycation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Ancestral protein reconstruction reveals the mechanism of substrate specificity in FN3K-mediated deglycation Jennifer Binning, Jenet Matlack, Robert Miner III, Jameela Lokhandwala This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7263937/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Protein glycation is a detrimental byproduct of carbohydrate metabolism, and nearly all organisms encode kinases that facilitate the removal of early glycation products. In humans, these repair functions are performed by Fructosamine-3 and Ketosamine-3 kinases (FN3K, KT3K). Recent structural studies identified conserved residues essential for FN3K activity, but the molecular basis for substrate discrimination between FN3K and KT3K remains unknown. Here, we show that substrate-binding residues are highly conserved and do not confer specificity in the FN3K family. Using APR, we resurrected four ancestral fructosamine kinases that recapitulate the distinct substrate preferences of FN3K and KT3K. Through mutational studies and structural analysis, we identify an evolutionarily tuned allosteric network that modulates substrate selection through long-range intramolecular interactions. Our findings define the mechanism of substrate selectivity within the FN3K family and establish a framework for the development of selective FN3K inhibitors. Biological sciences/Biochemistry/Enzyme mechanisms Biological sciences/Molecular biology/Post-translational modifications Biological sciences/Biochemistry/Kinases Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementalFigureLegends.pdf Supplemental Figure Legends ManuscriptSupplementalFigures.pdf Supplemental Figures Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7263937","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":499566218,"identity":"44b2b2c0-50bf-4c7b-8f6f-aee81b1f3d50","order_by":0,"name":"Jennifer Binning","email":"data:image/png;base64,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","orcid":"","institution":"H. 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