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

Abstract

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 (NicX H105F ) and the roles of key residues. In particular, NicX and NicX H105F can catalyze the ring opening degradation of DHP to NFM, but flexible mechanisms are adopted therein. Both reactions of NicX and NicX H105F are initiated by the attack of Fe III 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 NicX H105F 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 NicX H105A 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.

Mechanistic Investigation on C–C Bond Cleavage of Anthraquinone Catalyzed by an Atypical Nonheme Iron-Dependent Dioxygenase BTG13

“…According to our calculation results, the reaction occurs almost exclusively on the quintet state energy surface. This is in fact consistent with the two-state reactivity (TSR) reported for iron-dependent oxidative enzymes and biomimics. ,, In addition, the spin-state ordering of the Fe III –O 2 •– species has been proven to be affected by the coordination structure and the protein environment. − ,− Here, we evaluated the spin-ordering of the lowest-lying spin state by using a large QC model ((E·S·O 2 ) I -large-model, Figure S8). The calculations using the new model give consistent results in the overall trend of energy gaps compared to that from the original model (Table S6), except that the quintet state is now slightly lower in energy by 0.4 kcal/mol.…”

Elucidating the Enzymatic Mechanism of Dihydrocoumarin Degradation: Insight into the Functional Evolution of Methyl-Parathion Hydrolase from QM/MM and MM MD Simulations

“…Furthermore, the same QM regions were considered in QM/MM calculations and QM/MM MD simulations, as shown in Figure S5. The B3LYP treatment of the QM region has been demonstrated to be reliable in our previous studies. − Here, two basis sets were adopted in our calculations. The all-electron basis set Def2-SVP (labeled as B1) , was utilized in the geometry optimization by DL-FIND, and a relatively large basis set, Def2-TZVP (labeled as B2), was applied to determine relative energies.…”

QM/MM and MM MD Simulations on Enzymatic Degradation of the Nerve Agent VR by Phosphotriesterase

“…The AMBER force field was applied for the MM region. The QM subsystem was treated by the B3LYP functional, which has been proven reliable for the metalloenzymes in our previous studies. , The dimer optimizer in the DL-FIND was applied for the geometry optimization without symmetry restraints. The highest point on the potential energy surface along the defined reaction coordinate was used to locate the transition states (TSs), where the energy scanning calculations near TSs are performed using an interval of 0.01 Å.…”