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.
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Data availability
All data supporting the findings of this study are available within the paper and its Supplementary Information. Ancestral reconstruction and plasmid sequence data are available through Zenodo (https://doi.org/10.5281/zenodo.17965681). Uncropped blot images can be found in Supplementary Fig. 11. Experimental data generated in this study is available in Supplementary Data. Previously published structures used in this study can be accessed using PDB codes 8UE1 and 9CX8.
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Acknowledgements
We thank members of the Binning and Luca labs for their suggestions and technical assistance. The authors also acknowledge the Chemical Biology Core at the H. Lee Moffitt Cancer Center & Research Institute, which is supported in part by a National Cancer Institute (NCI) support grant (P30-CA076292). The protein structure network was adapted using Biorender.com. This study was supported by the U.S. National Institutes of Health grant R35 GM143004 to J.M.B. Figure 6B was created in BioRender. https://BioRender.com/0xc7owf.
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J.K.M. and J.M.B. designed the research and wrote the manuscript. J.K.M. performed all biochemical studies of FN3Ks and variants, ancestral protein reconstruction, structural analysis, and protein structure network analysis. R.E.M. performed the colorimetric assay on glycated substrates and cloned HsFN3K variants. J.L. performed conservation analysis of HsFN3K, cloned HsFN3K variants, and aided in the design of the research. J.M.B. conceptualized, supervised, provided resources, reviewed, and edited the manuscript. All authors read and approved the final manuscript.
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Matlack, J.K., Miner, R.E., Lokhandwala, J. et al. Ancestral protein reconstruction reveals the mechanism of substrate specificity in FN3K-mediated deglycation. Commun Biol (2026). https://doi.org/10.1038/s42003-026-09967-3
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DOI: https://doi.org/10.1038/s42003-026-09967-3
