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

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.