Graduation Year

2025

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Medical Sciences

Major Professor

Jennifer Binning, Ph.D.

Co-Major Professor

Yu Chen, Ph.D.

Committee Member

Robert Deschenes, Ph.D.

Committee Member

Laura Blair, Ph.D.

Committee Member

Vladimir Uversky, Ph.D.

Keywords

Ancestral Protein Reconstruction, Structural Biology, Kinases, Glycation, Post-translational modifications, Evolution

Abstract

Protein glycation is a universal, non-enzymatic modification that occurs when a sugar covalently attaches to a primary amine. These spontaneous modifications may have deleterious or regulatory effects on protein function, and their removal is mediated by the conserved metabolic kinase fructosamine-3-kinase (FN3K). Despite its crucial role in protein repair, we currently have a poor understanding of how FN3K engages or phosphorylates its substrates. Additionally, the molecular basis for the distinct yet overlapping substrate specificities of FN3K and its paralog, ketosamine-3 kinase (KT3K), remains unresolved. By integrating structural biology, biochemistry, and ancestral protein reconstruction (APR), this thesis elucidates the catalytic mechanism for FN3K-mediated protein deglycation and its biochemical differences from paralog KT3K. We identified key amino acids required for binding and phosphorylating glycated substrates. Structural-functional studies revealed unique structural features of human FN3K as well as differences in the dimerization behavior and regulation of FN3K family members. Through APR and systematic mutagenesis, we uncover an evolutionarily tuned allosteric network that mediates fructosamine recognition via long-range intramolecular communication. These findings improve our understanding of the structure of HsFN3K, its catalytic mechanism, and how allostery allows for fructosamine recognition. These insights provide a new mechanistic framework for understanding FN3K-mediated catalysis and substrate selection and opens avenues for rational design of FN3K-selective therapeutics targeting protein glycation in metabolic disease and aging.

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