The flavin-dependent amine oxidase superfamily contains various l-amino acid oxidases (LAAOs) bearing different substrate specificities and enzymatic properties. LAAOs catalyze the oxidation of the α-amino group of l-amino acids (L-AAs) to produce imino acids and H2O2. In this study, an ancestral l-Lys α-oxidase (AncLLysO2) was designed utilizing genome-mined sequences from the Caulobacter species. The AncLLysO2 exhibited high specificity toward l-Lys; the k cat/K m values toward l-Lys were one and two orders larger than those of l-Arg and l-ornithine, respectively. Liquid chromatography-high resolution mass spectrometry analysis indicated that AncLLysO2 released imino acid immediately from the active site after completion of oxidation of the α-amino group. Crystal structures of the ligand-free, l-Lys- and l-Arg-bound forms of AncLLysO2 were determined at 1.4-1.6 Å resolution, indicating that the active site of AncLLysO2 kept an open state during the reaction and more likely to release products. The structures also indicated the substrate recognition mechanism of AncLLysO2; ε-amino, α-amino, and carboxyl groups of l-Lys formed interactions with Q357, A551, and R77, respectively. Biochemical and molecular dynamics simulation analysis of AncLLysO2 indicated that active site residues that indirectly interact with the substrate are also important to exhibit high activity; for example, the aromatic group of Y219 is important to ensure that the l-Lys substrate is placed in the correct position to allow the reaction to proceed efficiently. Taken together, we propose the reaction mechanism of AncLLysO2.
© 2022 The Authors. Published by American Chemical Society.