Abstract 1696 Molecular mechanism of protein deglycation by Fructosamine-3-kinase

Protein glycation is a universal non-enzymatic modification that arises when a sugar covalently attaches to a primary amine. Unchecked glycation has been associated with multiple diseases including diabetes, neurodegenerative disorders, osteoarthritis, cancer, and atherosclerosis. Fructose-amine-3-kinase (FN3K) is a highly conserved metabolic kinase that mediates deglycation by phosphorylating the sugar moieties to facilitate its removal. Despite its crucial role in protein repair, FN3K's molecular architecture and molecular mechanism of deglycation remain elusive. Recently, there has been a growing interest in targeting FN3K for cancer therapy. It has been shown that the transcriptional activity of nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of oxidative stress, depends on deglycation by FN3K and depletion of FN3K reduced tumor growth in NRF2-driven lung and liver tumors. To realize the full therapeutic potential of FN3K, we need to develop a comprehensive understanding of FN3K enzymatic activity and cellular regulation. In this study, we integrate structural biology and biochemistry approaches, to elucidate the molecular mechanism for FN3K-mediated protein deglycation. We report the first crystal structure of human FN3K at 2.9Å. Through molecular docking of a substrate mimetic representing a protein-bound fructosamine, we identified key residues involved in FN3K substrate binding and catalysis. Further, mutational and enzymological studies validate amino acids crucial for kinase activity. Together, our data provide a deeper understanding of how FN3K executes deglycation at the molecular level and reveal an evolutionarily conserved protein repair mechanism. Importantly, our findings suggest promising avenues for the development of structure-based small molecule inhibitors of FN3K for the treatment of NRF2-driven tumors and glycation-related disorders. This study was supported by the U.S. National Institutes of Health grant R35 GM143004 to Dr. Jennifer. Binning.