title: Molybdenum-containing nicotine hydroxylase genes in a nicotine degradation pathway that is a variant of the pyridine and pyrrolidine pathways.
abstract: Ochrobactrum sp. strain SJY1 utilizes nicotine as a sole source of carbon, nitrogen, and energy via a variant of the pyridine and pyrrolidine pathways (the VPP pathway). Several strains and genes involved in the VPP pathway have recently been reported; however, the first catalyzing step for enzymatic turnover of nicotine is still unclear. In this study, a nicotine hydroxylase for the initial hydroxylation step of nicotine degradation was identified and characterized. The nicotine hydroxylase (VppA), which converts nicotine to 6-hydroxynicotine in the strain SJY1, is encoded by two open reading frames (vppAS and vppAL [subunits S and L, respectively]). The vppA genes were heterologously expressed in the non-nicotine-degrading strains Escherichia coli DH5α and Pseudomonas putida KT2440; only the Pseudomonas strain acquired the ability to degrade nicotine. The small subunit of VppA contained a [2Fe-2S] cluster-binding domain, and the large subunit of VppA contained a molybdenum cofactor-binding domain; however, an FAD-binding domain was not found in VppA. Resting cells cultivated in a molybdenum-deficient medium had low nicotine transformation activity, and excess molybdenum was detected in the purified VppA by inductively coupled plasma-mass spectrometry analysis. Thus, it is demonstrated that VppA is a two-component molybdenum-containing hydroxylase.
authors.affiliation: State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, People's Republic of China College of Life Sciences, Qingdao Agricultural University, Qingdao, People's Republic of China.
authors.forename: Hao
authors.initials: H
authors.lastname: Yu
authors.affiliation: State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, People's Republic of China tanghongzhi@sjtu.edu.cn pingxu@sjtu.edu.cn.
authors.forename: Hongzhi
authors.initials: H
authors.lastname: Tang
authors.affiliation: State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, People's Republic of China.
authors.forename: Yangyang
authors.initials: Y
authors.lastname: Li
authors.affiliation: State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, People's Republic of China tanghongzhi@sjtu.edu.cn pingxu@sjtu.edu.cn.
authors.forename: Ping
authors.initials: P
authors.lastname: Xu
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004734:Energy Metabolism / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D008982:Molybdenum / Q000378:metabolism*; D009538:Nicotine / Q000378:metabolism*; D042041:Ochrobactrum / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D016366:Open Reading Frames / Q000235:genetics; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D016958:Pseudomonas putida / Q000235:genetics / Q000378:metabolism; D011725:Pyridines / Q000378:metabolism; D011759:Pyrrolidines / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: D011725:Pyridines; D011759:Pyrrolidines; D009538:Nicotine; D008982:Molybdenum; D010088:Oxidoreductases; C034523:nicotine oxidase; C032519:pyrrolidine; C023666:pyridine