Engineering Catalytic Pathway Selection in Bifunctional Human DNA Glycosylases

Abstract Catalytic function in enzymes is generally considered a fixed property of the protein scaffold. It is commonly modulated by small molecules or post-translational modifications that control the efficacy of the biochemical reaction and leave underlying alternative reaction pathways unaffected. Here, we demonstrate that catalytic pathway selection in human DNA glycosylases can be engineered through manipulation of active-site chemistry. Using a warhead strategy targeting Cys253 in the DNA glycosylase OGG1, we install a permanent chemical base that promotes efficient β-elimination throughout the catalytic cycle, converting OGG1 into a robustly bifunctional enzyme. Building on recent insights into proton-transfer chemistry and conformational control of AP-lyase reactions, we identify conserved catalytic states in the related glycosylase NTHL1 and show that the catalytic pathway can be rewired through rational manipulation of active-site residues. This work demonstrates how protein engineering can be used as a strategy to reprogram enzyme function in human HhH-GPD enzymes.