Characterization of ancestral L-arginine and L-lysine oxidase

Abstract L-amino acid oxidase (LAAO) is an FAD-dependent enzyme catalyzing the oxidative deamination of L-amino acids. Owing to their diverse enzymatic properties, such as varied substrate specificities, LAAOs are promising for biotechnological applications. Broadening their functional repertoire by exploring LAAOs with distinct properties is essential for expanding their applicability. Here, we performed an in silico enzyme screening based on bioinformatic analysis and ancestral sequence reconstruction, a method by which designed ancestral enzymes are known to possess superior properties. An ancestral LAAO identified in our screening exhibited high thermal stability and activity toward both L-arginine and L-lysine. To investigate its substrate selectivity distinct from those of previously characterized ancestral LAAOs, we structurally compared the substrate recognition site, identifying a key residue responsible for the unique selectivity. These findings demonstrate that the in silico enzyme screening is a powerful strategy for exploring the LAAO family as a source of enzymes with diverse characteristics.