Molecular Dynamics Analysis Reveals Structural Insights into Mechanism of Nicotine N-Demethylation Catalyzed by Tobacco Cytochrome P450 Mono-Oxygenase

Abstract

CYP82E4, a cytochrome P450 monooxygenase, has nicotine N-demethylase (NND) activity, which mediates the bioconversion of nicotine into nornicotine in senescing tobacco leaves. Nornicotine is a precursor of the carcinogen, tobacco-specific nitrosamine. CYP82E3 is an ortholog of CYP82E4 with 95% sequence identity, but it lacks NND activity. A recent site-directed mutagenesis study revealed that a single amino acid substitution, i.e., cysteine to tryptophan at the 330 position in the middle of protein, restores the NND activity of CYP82E3 entirely. However, the same amino acid change caused the loss of the NND activity of CYP82E4. To determine the mechanism of the functional turnover of the two molecules, four 3D structures, i.e., the two molecules and their corresponding cys–trp mutants were modeled. The resulting structures exhibited that the mutation site is far from the active site, which suggests that no direct interaction occurs between the two sites. Simulation studies in different biological scenarios revealed that the mutation introduces a conformation drift with the largest change at the F-G loop. The dynamics trajectories analysis using principal component analysis and covariance analysis suggests that the single amino acid change causes the opening and closing of the transfer channels of the substrates, products, and water by altering the motion of the F-G and B-C loops. The motion of helix I is also correlated with the motion of both the F-G loop and the B-C loop and; the single amino acid mutation resulted in the curvature of helix I. These results suggest that the single amino acid mutation outside the active site region may have indirectly mediated the flexibility of the F-G and B-C loops through helix I, causing a functional turnover of the P450 monooxygenase.

Distal Effect of Amino Acid Substitutions in CYP2C9 Polymorphic Variants Causes Differences in Interatomic Interactions against (S)-Warfarin

“…Consequently, it could be noted that the non-active-site residues could allosterically regulate catalytic activity of the CYP enzymes as previously proposed [44]. Such effects are not limited to CYP2C9 variants demonstrated by this study and elsewhere [41], [43], [45] but also observable in CYP1A2 [44], CYP82E4 [46] and CYP2B4 [47]. Despite a small change in global conformational fold, the non-active-site residues could disrupt residue-residue contacts, leading to destabilization of active-site conformation [44], [47].…”

Binding free energies for nicotine analogs inhibiting cytochrome P450 2A6 by a combined use of molecular dynamics simulations and QM/MM-PBSA calculations

“…These data suggest that the gate to the inhibitor-binding cavity kept closed through the whole MD simulations on the CYP2A6-inhibitor binding structures. This is different from the observation in a previous report 17 on a CYP82E4-nicotine binding model that there was an open-close process for the substrate-entry gate. As shown in Figures 2 to 4, each of these inhibitors packs tightly with aromatic residues inside the binding cavity, hydrogen-bonded with N297 side chain, and coordinated with the heme iron atom of CYP2A6.…”

“…In general, the dynamic behavior and the mode for CYP2A6 binding with these three inhibitors are quite similar to that of the CYP2A6-Nic2a binding. In a previous report of CYP82E4-nicotine binding structure obtained through homology modeling and MD simulations, 17 nicotine molecule was orientated perpendicularly inside the binding cavity of CYP82E4, with one carbon atom at the pyridine ring of nicotine pointing to the iron atom of the heme group. This binding mode of nicotine with CYP82E4 is totally different from the binding mode for each inhibitor with CYP2A6 as described in the present report.…”

“…Latest studies 8,9,10,11,12,13,14,15,16,17,18 have found that the cytochrome P450 2A6 (CYP2A6) is the primary enzyme for nicotine metabolism. This enzyme metabolizes nicotine into cotinine and nornicotine.…”

Thumb inhibitor binding eliminates functionally important dynamics in the hepatitis C virus RNA polymerase

“…We can relate our observations to two representative models of protein allostery: the conformational selection and induced fit models. In the conformational selection model, ligand binding stabilizes and promotes pre‐existing enzyme conformations that occur at low levels even in the absence of ligand 55, 56. Allostery thus results from a shift in the relative populations of protein conformations when ligand is bound.…”