Thebaine is Selectively Demethylated by Thebaine 6-<i>O</i>-Demethylase and Codeine-3-<i>O</i>-demethylase at Distinct Binding Sites: A Computational Study

Two homologous 2-oxoglutarate-dependent (ODD) nonheme enzymes thebaine 6-O-demethylase (T6ODM) and codeine-3-O-demethylase (CODM), are involved in the morphine biosynthesis pathway from thebaine, catalyzing O-demethylation reaction with precise regioselectivity at C6 C3 positions of respectively. We investigated origin these by combined molecular dynamics (MD) quantum mechanics/molecular mechanics (QM/MM) calculations found that Thebaine binds two distinct sites T6ODM CODM, which determines enzymes. A remarkable oxo rotation is observed decarboxylation process. Starting closed pentacoordinate configuration, C-terminal lid adopts an open conformation octahedral Fe(IV) = O complex to facilitate subsequent demethylation. Phe241 Phe311 stabilize substrate binding pocket, while Arg219 acts as a gatekeeper residue substrate. Our results unravel 2-OG dependent may shed light for exploring scope developing novel biotechnology biosynthesis.