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.

FIG 1

Proposed hybrid pathway of nicotine degradation by A. tumefaciens S33. Ndh, nicotine dehydrogenase; Hno, 6-hydroxynicotine oxidase; Pno, 6-hydroxypseudooxynicotine oxidase; Hsh, 6-hydroxy-3-succinoylpyridine hydroxylase.

FIG 2

SDS-PAGE (A and B) and native-PAGE (C) analysis of the purification of Ndh from A. tumefaciens S33 (A, 12.5% gel; B, 6.0% gel). Lanes: M, protein marker; 1, cell extract; 2, ammonium sulfate precipitate; 3, Phenyl Sepharose; 4, DEAE-Sepharose; 5, Superdex 200. The left part of panel C was stained by Coomassie brilliant blue, and the right part shows specific activity staining with a solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and nicotine.

FIG 3

Region of A. tumefaciens S33 genome around the ndhAB and pno genes encoding nicotine dehydrogenase and 6-hydroxypseudooxynicotine oxidase, respectively. Open arrows without labels indicate genes encoding hypothetical proteins. Cofactor binding sites were deduced from the amino acid sequences of the proteins. Mo, molybdopterin that binds molybdenum. qor, quinone oxidoreductase; paz, pseudoazurin; hno, 6-hydroxynicotine oxidase; hsh, 6-hydroxy-3-succinoylpyridine hydroxylase. Between hno and pno, there are 12 ORFs predicted to encode six mobile element proteins and six hypothetical proteins.

FIG 4

RT-PCR analysis of ndhAB and pno transcription in A. tumefaciens S33. Lanes 1 and 2, cDNAs synthesized from total RNAs isolated from cells cultured in nicotine medium and in glucose and ammonium medium, respectively; lane 3, negative control (total RNA isolated from cells cultured in nicotine medium); lane 4, positive control (genomic DNA from the culture grown in glucose and ammonium medium).

FIG 5

Growth of strain S33 mutants in the presence of nicotine (A), 6-hydroxy-3-succinoylpyridine (B), and 6-hydroxynicotine (C) as the sole source of carbon and nitrogen. Symbols (A and B) and numbers (C): ■ and 1, wild-type strain; ● and 2, S33-ΔndhA; ▲ and 3, S33-ΔndhB; ▼ and 4, S33-Δpno.

FIG 6

Purification (A) and UV-visible absorption spectrum (B) of recombinant Pno from A. tumefaciens S33. M, marker; lane 1, cell extract of recombinant E. coli; lane 2, HisTrap HP column; lane 3, HiTrap Q HP column. The sample used for UV-visible absorption spectrum analysis contained 0.38 mg of purified protein/ml in 50 mM sodium phosphate buffer (pH 7.0).