Evolution of human IL4I1 preference for aromatic amino acids from a broad-specificity L-amino acid oxidase ancestor

IL4I1, a human L-amino acid oxidase (LAAO), has drawn considerable interest for its role in catabolism with implications for cancer biology and immune modulation. The function and activity of this enzyme in humans contrast with the cytotoxic effects exerted by LAAOs from snake venoms. The inherent instability of IL4I1 has hindered detailed biochemical characterization and its exploration as a drug target. In this study, we present a comprehensive biochemical and structural investigation of tetrapod LAAOs, with a focus on human IL4I1 and its evolutionary relationship to venomous snake proteins. By exploiting ancestral sequence reconstruction, we inferred, expressed, and characterized the tetrapodal ancestral LAAO and mammalian ancestral IL4I1 and compared their properties to recombinant human IL4I1 purified from human cells. The tetrapodal ancestor displays a broad substrate profile, acting on hydrophobic, polar, and positively charged amino acids, whereas human IL4I1 prefers aromatic amino acids. Guided by crystal structures, we engineered eight ancestral protein variants to mimic mammalian- and snake-like active site configurations. Kinetic analyses of these variants reveal distinct evolutionary trajectories: one leading to the high-activity venomous LAAOs of reptiles, and another to the aromatic-selective IL4I1 of mammals. Overall, our findings demonstrate that the functional divergence between human IL4I1 and venomous snake LAAOs is underpinned by evolutionary shifts in substrate specificity and catalytic efficiency.