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

Protein glycation is a detrimental byproduct of living cells’ reliance on 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 kinase (FN3K) and Ketosamine-3 kinase (KT3K) enzymes which share conserved catalytic mechanisms but differ in substrate specificity. Recent structural studies defined key active site residues required for FN3K activity on a model substrate, yet the molecular basis for differential substrate recognition by FN3K and KT3K remains unresolved. Here, we integrate phylogenetic analysis, ancestral protein reconstruction (APR), and mutational biochemistry to to elucidate how substrate specificity evolved within the fructosamine-3 kinase family. We show that conserved substrate-binding residues are required for the phosphorylation of both fructosamines and ketosamines, but do not contribute to substrate specificity. Using APR, we resurrected four ancestral fructosamine kinases that recapitulate the distinct substrate preferences of FN3K and KT3K despite differing by only 12 amino acids. Through mutational studies and structural analysis, we show that substrate specificity is modulated by an evolutionarily tuned allosteric network that enables long-range intramolecular communication. These insights provide a new mechanistic framework for understanding FN3K substrate selection and open avenues for rational design of FN3K-selective therapeutics targeting protein glycation in metabolic disease and aging.