Kyohei Hanaoka

A QM/MM study of nitric oxide reductase-catalysed N2O formation

Nitrous oxide (N2O), with a greenhouse effect 300 times that of CO2, is increasingly eliminated into the atmosphere. Using a hybrid quantum mechanics/molecular mechanics (QM/MM) method, we examined nitric oxide reductase-catalysed N2O formation, which includes two important chemical reactions of N–N bond formation and N–O bond cleavage. The N–N bond formation has no activation barrier, but N–O bond cleavage exhibits an activation barrier of 20.9 kcal·mol−1 at the QM/MM level. We show that the N–O bond cleavage occurs via a hyponitrous intermediate (FeB (II; s = 4/2)/N2O2 (−1; s = 1/2)/ (III; s = −1/2)), with bidentate coordination between Glu211 and a non-heme iron atom. The Glu211 coordination decreases the N–O bond cleavage energy barrier by inhibiting the formation of stable, five-membered ring intermediate (FeB–O1–N1–N2–O2–).

Substrate-mediated proton relay mechanism for the religation reaction in topoisomerase II.

The DNA religation reaction of yeast type II topoisomerase (topo II) was investigated to elucidate its metal-dependent general acid/base catalysis. Quantum mechanical/molecular mechanical calculations were performed for the topo II religation reaction, and the proton transfer pathway was examined. We found a substrate-mediated proton transfer of the topo II religation reaction, which involves the 3′ OH nucleophile, the reactive phosphate, water, Arg781, and Tyr782. Metal A stabilizes the transition states, which is consistent with a two-metal mechanism in topo II. This pathway may be required for the cleavage/religation reaction of topo IA and II and will provide a general explanation for the catalytic mechanism in the topo IA and II.

Molecular Mechanism of the Reaction Specificity in Threonine Synthase: Importance of the Substrate Conformations

Threonine synthase (ThrS) catalyzes the final chemical reaction of l-threonine biosynthesis from its precursor, O-phospho-l-homoserine. As the phosphate ion generated in its former half reaction assists its latter reaction, ThrS is recognized as one of the best examples of product-assisted catalysis. In our previous QM/MM study, the chemical reactions for the latter half reactions, which are critical for the product-assisted catalysis, were revealed. However, accurate free energy changes caused by the conformational ensembles and entrance of water molecules into the active site are unknown. In the present study, by performing long-time scale MD simulations, the free energy changes by the divalent anions (phosphate or sulfate ions) and conformational states of the intermediate states were theoretically investigated. We found that the calculated free energy double differences are in good agreement with the experimental results. We also revealed that the phosphate ion contributes to forming hydrogen bonds that are suitable for the main reaction progress. This means that the conformation of the active site amino acid residues and the substrate, and hence, the tunable catalysis, are controlled by the product phosphate ion, and this clearly demonstrates a molecular mechanism of the product-assisted catalysis in ThrS.