title: Nicotine Dehydrogenase Complexed with 6-Hydroxypseudooxynicotine Oxidase Involved in the Hybrid Nicotine-Degrading Pathway in Agrobacterium tumefaciens S33.
abstract: Nicotine, a major toxic alkaloid in tobacco wastes, is degraded by bacteria, mainly via pyridine and pyrrolidine pathways. Previously, we discovered a new hybrid of the pyridine and pyrrolidine pathways in Agrobacterium tumefaciens S33 and characterized its key enzyme 6-hydroxy-3-succinoylpyridine (HSP) hydroxylase. Here, we purified the nicotine dehydrogenase initializing the nicotine degradation from the strain and found that it forms a complex with a novel 6-hydroxypseudooxynicotine oxidase. The purified complex is composed of three different subunits encoded by ndhAB and pno, where ndhA and ndhB overlap by 4 bp and are ∼26 kb away from pno. As predicted from the gene sequences and from chemical analyses, NdhA (82.4 kDa) and NdhB (17.1 kDa) harbor a molybdopterin cofactor and two [2Fe-2S] clusters, respectively, whereas Pno (73.3 kDa) harbors an flavin mononucleotide and a [4Fe-4S] cluster. Mutants with disrupted ndhA or ndhB genes did not grow on nicotine but grew well on 6-hydroxynicotine and HSP, whereas the pno mutant did not grow on nicotine or 6-hydroxynicotine but grew well on HSP, indicating that NdhA and NdhB are responsible for initialization of nicotine oxidation. We successfully expressed pno in Escherichia coli and found that the recombinant Pno presented 2,6-dichlorophenolindophenol reduction activity when it was coupled with 6-hydroxynicotine oxidation. The determination of reaction products catalyzed by the purified enzymes or mutants indicated that NdhAB catalyzed nicotine oxidation to 6-hydroxynicotine, whereas Pno oxidized 6-hydroxypseudooxynicotine to 6-hydroxy-3-succinoylsemialdehyde pyridine. These results provide new insights into this novel hybrid pathway of nicotine degradation in A. tumefaciens S33.
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, People's Republic of China.
authors.forename: Huili
authors.initials: H
authors.lastname: Li
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, People's Republic of China.
authors.forename: Kebo
authors.initials: K
authors.lastname: Xie
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, People's Republic of China.
authors.forename: Wenjun
authors.initials: W
authors.lastname: Yu
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, People's Republic of China.
authors.forename: Liejie
authors.initials: L
authors.lastname: Hu
authors.affiliation: Institute of Basic Medicine, Shandong Academy of Medical Science, Jinan, People's Republic of China.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Huang
authors.affiliation: Environment Research Institute, Shandong University, Jinan, People's Republic of China.
authors.forename: Huijun
authors.initials: H
authors.lastname: Xie
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, People's Republic of China shuningwang@sdu.edu.cn.
authors.forename: Shuning
authors.initials: S
authors.lastname: Wang
mesh_terms: D016960:Agrobacterium tumefaciens / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism*; D020816:Amino Acid Motifs; D001711:Biotransformation; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D055786:Gene Knockout Techniques; D053858:Metabolic Networks and Pathways; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D009538:Nicotine / Q000378:metabolism*; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D009097:Multienzyme Complexes; D009538:Nicotine; D010088:Oxidoreductases; D000587:Oxidoreductases Acting on CH-NH Group Donors; C066274:nicotine dehydrogenase