LAUSR

2,5-Dihydroxypyridine dioxygenase (NicX) from Pseudomonas putida KT2440 is a mononuclear non-heme iron oxygenase that can catalyze the oxidative pyridine ring cleavage. Recently, the reported crystal structure of NicX has lent support to an apical dioxygen catalytic mechanism, while the mechanistic details remain unclear. In this work, we constructed a Fe(II)-O2-substrate complex model and performed a series of combined quantum mechanics/molecular mechanics (QM/MM) calculations to illuminate the catalysis of NicX. Our results reveal that although the substrate does not directly coordinate with the central iron ion, there is an electron transfer from the substrate to the Fe-coordinated dioxygen, and the active form of the reactant complex can be described as DHP•+-Fe(II)-O2•-, which is different from other similar mononuclear non-heme iron. The NicX-catalyzed pyridine ring degradation contains three parts, including the attack of Fe(II)-superoxo on the activated pyridine ring, the dissociation of the Op-Od bond, and the ring-opening of the seven-membered-ring lactone. Owing to the radical characteristic of the pyridine ring, the first attack of Fe(II)-superoxo on the C6 of the pyridine ring was calculated to be quite easy. In the second step of the reaction, the dissociation of the Op-Od bond leads to the incorporation of the first oxygen atom into the substrate, which is the rate-limiting step of the overall reaction with an energy barrier of 18.0 kcal/mol. The resultant intermediate then undergoes an arrangement by the intramolecular attack of Od• on the carbonyl C5, forming the seven-membered-ring lactone. Finally, the Fe(III)-oxo attacks the carbonyl C5 of lactone, accompanied by the ring-opening to generate N-formylmaleamic acid. His105 can promote reactivity by donating a proton to Fe(III)-oxo, but it is not a necessary residue. In addition to the ligated residues of iron, other pocket residues such as Glu177, His189, and His105 mainly play roles in anchoring the substrate.