Structure-function Relationship Of Nicotine N-demethylase

Nicotine N-demethylase (NND) is a cytochrome P450 monooxygenase in tobacco. It catalyzes demethylation of substrate nicotine into nornicotine, a precursor to the N'-nitrosonornicotine (NNN) that is believed to be one of the tobacco-specific nitrosamines (TSNAs) carcinogen. The ubiquitous nature and importance in human health of NND has drawn intense interests in the catalytic reaction mechanism of this enzyme over the past several decades.In this work, we conducted preliminary characterization of NND and exploration of its catalytic mechanism, using molecular biological, biochemical and bioinformatics approaches. The contents of this study are as follow:The cDNA of NND full coding sequence in tobacco (Nicotiana plumbaginifolia) was cloned by using rapid amplication of cDNA (RACE) strategy. Sequence analysis and conserved domain search in National Center for Biotechnology Information (NCBI) suggested that NND in N. plumbaginifolia (namely in this thesis, NNDp) is a novel member of super family of plant cytochrome P450 monooxygenase. NCBI blast result indicated that NNDp share 92%,94%,92% nucleotides identity and 89%, 94%,93% amino acids identity with N. sylvestris, N. tabacum, and N. tomentosformis respectively.The properties and structural information of NNDp was predicted by the methods of bioinformatics. The cDNA of NND was constructed into eukaryotic expression plasmid pYeDP60 and transformed into yeast line WAT(R). Inducible expression and identification of NNDp in yeast is measured by CO difference spectroscopy. Characterization of NNDp was conducted by High Performance Liquid Chromatography with Tandem Mass-Spectroscopy (HPLC-MS/MS). Through molecular docking and quantitative structure activity relationship (QSAR) analysis, we carried out interaction studies of NNDp with its inhibitors. The QSAR model was built up.In conclusion, the research results obtained from the isolation, characterization, bioinformatics, homology modeling and inhibitor interactions of NNDp provided interesting insight into understanding of the reaction mechanism of NNDp and development possibly of new inhibitor with improved functionality and selectivity.