Catalytic Mechanism of Salicylate Dioxygenase: QM/MM Simulations Reveal the Origin of Unexpected Regioselectivity of the Ring Cleavage

Abstract

Salicylate 1,2-dioxygenase (SDO) was the first enzyme discovered, in the family of iron dioxygenases, to catalyze the ring cleavage of a monohydroxylated aromatic compound, salicylate, without a proton donor. Salicylate is not electron-rich like the familiar dihydroxy or aromatic substrates with an electron-donating group that are utilized in the well-known dioxygenases. SDO carries out the intramolecular C-C bond cleavage in salicylate bearing the OH and COOH groups with high regioselectivity in comparison with the extradiol and intradiol dioxygenases. The catalytic cleavage of a nonactivated substrate like salicylate that lacks an electron-donating group, also in the absence of a proton source, raises many puzzling questions about the oxy intermediates in the reaction pathway of dioxygenase enzymes in general. To answer these fundamental queries, we have investigated the full catalytic mechanism of SDO by a combination of quantum mechanics and molecular mechanics (QM/MM) calculations. Herein, our QM/MM study has several unexpected and interesting implications for the mechanistic pathway of SDO in comparison to the experimental observations. Importantly, it unravels the basis for the unexpected "intra"-cleavage regioselectivity in SDO. Ostensibly a similar alkylperoxo intermediate is formed in SDO much like in the extradiol and intradiol dioxygenases. In stark contrast to the two diol enzymes, the O-O bond breaking leads to an unprotonated gem-hydroxy carboxylate intermediate, a paradigm analogue of the elusive gem diol intermediate. This unprotonated gem-hydroxy carboxylate intermediate exclusively dictates the C-C cleavage regiospecificity in SDO, which is unprecedented in the family of dioxygenases. It forms a seven-membered lactone species, which eventually forms the ring-cleavage final product by incorporation of two oxygen atoms in the salicylate. Thus, our computational study unravels a detailed reaction pathway of the oxidative cleavage of salicylate without a proton source by identifying the hitherto elusive intermediates in the catalytic cycle of SDO with testable predictions. Moreover, we describe the atomistic origin of the new catalytic role of Arg127 in the catalysis and regioselectivity of SDO. It also rationalizes the formation of a side product without invoking a dioxetane intermediate. This study is important from a fundamental perspective and opens up a new window in the mechanism of the family of dioxygenase enzymes.

Catalytic Mechanism for 2,3-Dihydroxybiphenyl Ring Cleavage by Nonheme Extradiol Dioxygenases BphC: Insights from QM/MM Analysis

“…The dioxygen group has a superoxide character, and the opposite spin between the substrate radical and the superoxide (DHBP •↑ –Fe II –O 2 •–↓ ) can be the reactive state for the subsequent catalytic reaction. The above results are similar to many previous theoretical studies of the nonheme iron-containing extradiol dioxygenases. ,,,,− To better understand the further insight into the quintet reactive state, molecular orbital diagram was obtained (Figure ), which indicates that the iron 3d orbitals could interact with the surrounding ligands and split into a pair of two π* orbitals (π xz * , π yz * ) and a pair of two σ* orbitals ( ). As shown in Figure , the π yz * and π xz * of the 3d orbitals in Fe II represent the antibonding combinations with the 2p orbital of dioxygen and DHBP, whereas the d xy orbital is not changed as there is not any antibonding interaction between iron and DHBP.…”

N-Nitrosation Mechanism Catalyzed by Non-heme Iron-Containing Enzyme SznF Involving Intramolecular Oxidative Rearrangement

“…Our calculations indicated that this reaction is the rate-limiting step in the catalytic process. 3 studies on the non-heme iron-containing enzymes SDO and HPCD also obtained relatively high-energy barriers in the similar superoxo addition step (19.8 kcal/mol in SDO and 17.4 kcal/mol in HPCD), 28,31,38 which should be ascribed to that the substrates of these two enzymes, as well as the substrate 1 of SznF, are all featured with a conjugation π system in them.…”

“…It is very common among the non-heme iron-containing enzymes such as SDO, 2,3dihydroxybiphenyl dioxygenase (BphC), and homoprotocatechuate 2,3-dioxygenase (HPCD) that simultaneously binding of the substrate and the dioxygen to the iron center generates Fe II −superoxo species and cationic substrate radical. 22,23,[25][26][27][28]38 It should be ascribed to that the electron-rich substrates of these enzymes are bound up with the iron center so that they can donate electrons to reduce the dioxygen through the iron center. As shown in Figure 2a, in 3 IntA HA , the Fe−O bond length is 2.01 Å.…”

Mechanistic Insights into a Stibene Cleavage Oxygenase NOV1 from Quantum Mechanical/Molecular Mechanical Calculations

“…It has been extensively showed by theoretical studies that in many mononuclear non-heme enzymes the quintet FeÀ O 2 is the reactive species. [15,29,30,33,35,[38][39][40][41][42][43][44][45][46][47][48] As such, we took 5 1 side-on as the catalytically relevant state for the oxidative cleavage of resveratrol. The subsequent QM/MM calculations were carried out on the quintet surface.…”