Fructosamine-3-kinases (FN3Ks) are evolutionarily conserved protein kinase-like (PKL) fold enzymes that play an important role in cellular homeostasis by repairing proteins. Through phosphoryl transfer, the enzyme removes sugar adducts from glycated lysine residues in proteins. Despite being conserved across all domains of life, the family has received very little attention. In this dissertation, I apply a combination of experimental and computational methods to illuminate the structure, function, evolution, and regulation of the enzyme family. By solving the crystal structure of the FN3K ortholog from Arabidopsis thaliana, for the first time, we provide a structural basis for redox mode of regulation. We also demonstrated its conservation across the family, including in humans. Next, by leveraging the FN3K knock out (KO) in HepG2 cells and using a multi-omics (transcriptomics, metabolomics, interactomics, and genomics) approach, we provide new links of FN3K to lipid and co-factor metabolism as well as oxidative stress response. We also find new links between FN3K and Nicotinamide adenine dinucleotide (NAD) and show that human FN3K can specifically bind to different NAD compounds. Moreover, using phylogenetics and Bayesian statistics based evolutionary sequence constraints method, I derived evolutionary relationship between FN3Ks and related families in the PKL superfamily as well as define sequence constraints distinguishing FN3K family. I also identified several divergent bacterial sub-groups within the FN3K family that will require further characterization. Through high resolution crystal structures, I show two distinct modes of adenine binding in the FN3K family. This dissertation sheds light on the multifaceted roles and regulatory mechanisms of FN3K enzymes, expanding our understanding of their function and evolution, and highlighting potential areas for further research within the field.