title: 6-hydroxy-3-succinoylpyridine hydroxylase catalyzes a central step of nicotine degradation in Agrobacterium tumefaciens S33.
abstract: Nicotine is a main alkaloid in tobacco and is also the primary toxic compound in tobacco wastes. It can be degraded by bacteria via either pyridine pathway or pyrrolidine pathway. Previously, a fused pathway of the pyridine pathway and the pyrrolidine pathway was proposed for nicotine degradation by Agrobacterium tumefaciens S33, in which 6-hydroxy-3-succinoylpyridine (HSP) is a key intermediate connecting the two pathways. We report here the purification and properties of an NADH-dependent HSP hydroxylase from A. tumefaciens S33. The 90-kDa homodimeric flavoprotein catalyzed the oxidative decarboxylation of HSP to 2,5-dihydroxypyridine (2,5-DHP) in the presence of NADH and FAD at pH 8.0 at a specific rate of about 18.8 ± 1.85 µmol min-1 mg protein-1. Its gene was identified by searching the N-terminal amino acid residues of the purified protein against the genome draft of the bacterium. It encodes a protein composed of 391 amino acids with 62% identity to HSP hydroxylase (HspB) from Pseudomonas putida S16, which degrades nicotine via the pyrrolidine pathway. Considering the application potential of 2,5-DHP in agriculture and medicine, we developed a route to transform HSP into 2,5-DHP with recombinant HSP hydroxylase and an NADH-regenerating system (formate, NAD+ and formate dehydrogenase), via which around 0.53 ± 0.03 mM 2,5-DHP was produced from 0.76 ± 0.01 mM HSP with a molar conversion as 69.7%. This study presents the biochemical properties of the key enzyme HSP hydroxylase which is involved in the fused nicotine degradation pathway of the pyridine and pyrrolidine pathways and a new green route to biochemically synthesize functionalized 2,5-DHP.
authors: State Key Laboratory of Microbial Technology, Shandong University, Jinan, PR China.
authors: Huili
authors: H
authors: Li
authors: State Key Laboratory of Microbial Technology, Shandong University, Jinan, PR China.
authors: Kebo
authors: K
authors: Xie
authors: Institute of Basic Medicine, Shandong Academy of Medical Science, Jinan, PR China.
authors: Haiyan
authors: H
authors: Huang
authors: State Key Laboratory of Microbial Technology, Shandong University, Jinan, PR China.
authors: Shuning
authors: S
authors: Wang
mesh_terms: D016960:Agrobacterium tumefaciens / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D000595:Amino Acid Sequence; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009538:Nicotine / Q000378:metabolism*; D011725:Pyridines / Q000378:metabolism*; D015398:Signal Transduction; D014026:Nicotiana / Q000382:microbiology
chemical_list: D011725:Pyridines; D009243:NAD; D009538:Nicotine; D006899:Mixed Function Oxygenases; C065452:2,5-dihydroxypyridine