c 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 ϳ26 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.A grobacterium tumefaciens is well known for its ability to induce crown gall tumors in dicotyledonous plants and mediate interkingdom genetic transfer, for that it is widely used in plant molecular biology and biotechnology (1). Interestingly, some strains of this species are also able to degrade xenobiotics such as cyanuric acid, iminodisuccinate, and methylene urea (2-4). We isolated A. tumefaciens strain S33, which has the strong ability to degrade the natural alkaloid nicotine from the rhizospheric soil of a tobacco plant (5, 6).Nicotine is a major alkaloid in tobacco, which causes tobacco addiction and may result in diseases such as pulmonary disease and cancer (7,8), and it is the primary toxic compound in tobacco wastes. The tobacco-manufacturing process and all activities using tobacco produce a large amount of solid or liquid waste containing high concentrations of nicotine, which are classified as "toxic and hazardous wastes" by the European Union (9). Therefore, detoxification of tobacco wastes is a major concern for public health and the environment. The discovery of nicotine degradation by microorganisms provides an alternative way to dispose of such wastes (10-12).Microbial degradation of nicotine attracts attention because it represents a method to treat the tobacco waste...

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