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Updated: Jul 7, 2026

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition
Published on: October 3, 2018
Computation-Guided Tunnel Engineering Enhances O2 Transport and H2O2 Resistance of Fe(II)/α-Ketoglutarate-Dependent
Huan Liu1, Lunjie Wu1,2, Liying Mao3
1Lab of Brewing Microbiology and Applied Enzymology, School of Biotechnology, The Key Laboratory of Industrial Biotechnology, Key Laboratory of Industrial Synthetic Biology of Jiangsu Province, Ministry of Education, Jiangnan University, Wuxi, China.
Abstract:
Fe(II)/α-ketoglutarate-dependent dioxygenases (αKGDs) are versatile biocatalysts whose catalytic activity relies on an efficient supply of O2 and αKG. However, in engineering applications, the O2 supply is seldom considered, whereas αKG is typically supplied through the oxidation of l-glutamate by l-glutamate oxidase, which concomitantly produces H2O2 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 (O2 and H2O2) to isoleucine dioxygenase (IDO), enhancing O2 transport while resisting H2O2 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 H2O2. This study highlights O2 and H2O2 tunnel engineering as a new strategy for enhancing αKGDs activity and antioxidative properties.
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