Reaction Mechanism and Chemoselectivity of an Enzymatic PAS Domain: A Combined MD and QM/MM Study

A Per-ARNT-Sim (PAS) domain exhibits oxidative N-demethylation activity toward dimethylamine (DMA); however, the detailed reaction mechanism remains elusive, thus hindering the rational engineering of the enzyme. In this work, we conducted molecular dynamics (MD) simulations and quantum mechanical/molecular mechanics (QM/MM) calculations to investigate the PAS domain-catalyzed DMA N-demethylation reaction. Our results indicate that the protonated substrate DMA is the most plausible proton donor during the formation of compound I (Cpd I, iron(IV)-oxo porphyrin cation radical). The calculated energy barrier for Cpd I generation is 14.4 kcal/mol, which is the rate-determining step of the reaction. Following its generation, Cpd I can rapidly oxidize the substrate via an electron-transfer-initiated hydrogen atom abstraction. The chemoselectivity favoring methyl hydroxylation over amine oxidation is mainly determined by the protein environment. Our computations provide mechanistic insights into the heme-dependent oxidative N-demethylase (HODM)-catalyzed reaction and offer information for the rational design of new PAS enzymes.