Computation‐Guided Tunnel Engineering Enhances O ₂ Transport and H ₂ O ₂ Resistance of Fe(II)/α‐Ketoglutarate‐Dependent Dioxygenases

ABSTRACT Fe(II)/α‐ketoglutarate‐dependent dioxygenases (αKGDs) are versatile biocatalysts whose catalytic activity relies on an efficient supply of O ₂ and αKG. However, in engineering applications, the O ₂ supply is seldom considered, whereas αKG is typically supplied through the oxidation of l ‐glutamate by l ‐glutamate oxidase, which concomitantly produces H ₂ O ₂ that potently inhibits αKGD activity. This study developed a tunnel engineering strategy based on random accelerated molecular dynamics and protein structuromics to precisely modulate the access of small molecules (O ₂ and H ₂ O ₂ ) to isoleucine dioxygenase (IDO), enhancing O ₂ transport while resisting H ₂ O ₂ transport. Notably, mutant 1 (L179C/V225F/I240V) and mutant 2 (N193S/V225I/I240V) displayed markedly higher catalytic activity for five aliphatic amino acid substrates (Leu, Nle, Nva, Met, and Ile), reaching 27.2‐fold greater activity. In addition, mutant 2 exhibited 7.1‐fold higher activity against Ile in the presence of H ₂ O ₂ . This study highlights O ₂ and H ₂ O ₂ tunnel engineering as a new strategy for enhancing αKGDs activity and antioxidative properties.