Biodegradation of 2,5-Dihydroxypyridine by 2,5-Dihydroxypyridine Dioxygenase and Its Mutants: Insights into O–O Bond Activation and Flexible Reaction Mechanisms from QM/MM Simulations

2,5-Dihydroxypyridine dioxygenase (NicX) from Pseudomonas putida KT2440 is a mononuclear non-heme iron oxygenase responsible for the biodegradation of 2,5-dihydroxypyridine (DHP) to N-formylmaleamic acid (NFM). Here, extensive quantum mechanical–molecular mechanical (QM/MM) calculations and molecular dynamics (MD) simulations are used to elucidate the degradation mechanism of DHP by wild-type NicX and its H105F variant (NicXH105F) and the roles of key residues. In particular, NicX and NicXH105F can catalyze the ring opening degradation of DHP to NFM, but flexible mechanisms are adopted therein. Both reactions of NicX and NicXH105F are initiated by the attack of FeIII superoxide species onto the substrate, during which a proton-coupled electron transfer (PCET) process is involved. For wild-type NicX, the PCET reaction is mediated by the adjacent His105, while the further proton transfer from His105 to the peroxo species can remarkably enhance the following O–O cleavage. However, for the NicXH105F mutant, a water molecule replaces the role of residue His105, which not only stabilizes the substrate binding via a H bonding network but also functions as a base to mediate the PCET process. For the NicXH105A mutant, MD simulations show that the disruption of the H bonding network can displace the substrate binding, leading to the loss of enzyme activity. These findings can expand our understanding of the PCET-mediated O–O bond activation and the flexible catalytic routes in various mutants, which have general implications on enzyme catalysis