title: 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.
authors.affiliation: Key Lab for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, Changchun, China.
authors.forename: Shan
authors.initials: S
authors.lastname: Wang
authors.forename: Shuo
authors.initials: S
authors.lastname: Yang
authors.forename: Baiyi
authors.initials: B
authors.lastname: An
authors.forename: Shichen
authors.initials: S
authors.lastname: Wang
authors.forename: Yuejia
authors.initials: Y
authors.lastname: Yin
authors.forename: Yang
authors.initials: Y
authors.lastname: Lu
authors.forename: Ying
authors.initials: Y
authors.lastname: Xu
authors.forename: Dongyun
authors.initials: D
authors.lastname: Hao
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D055162:Biocatalysis; D020134:Catalytic Domain / Q000235:genetics; D003198:Computer Simulation; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008745:Methylation; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009538:Nicotine / Q000737:chemistry* / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D007527:Isoenzymes; D010940:Plant Proteins; D009538:Nicotine; D003577:Cytochrome P-450 Enzyme System; C516919:nicotine demethylase, tobacco; D010089:Oxidoreductases, N-Demethylating