title: Identification of nicotine biotransformation intermediates by Agrobacterium tumefaciens strain S33 suggests a novel nicotine degradation pathway.
abstract: Nicotine, a major alkaloid in tobacco plants and the main toxic chemical in tobacco wastes, can be transformed by bacteria into hydroxylated-pyridine intermediates, which are important precursors for the chemical synthesis of valuable drugs and insecticides. Such biotransformation could be a useful approach to utilize tobacco and its wastes. In this study, we explored nicotine degradation by a recently isolated Agrobacterium tumefaciens S33 by identifying the intermediates during its growth on nicotine and during transformation of nicotine with its resting cells. Five hydroxylated-pyridine intermediates were detected through multiple approaches, including GC-HR-MS, HPLC, and ESI-Q-TOF MS analyses. Surprisingly, these identified intermediates suggest that strain S33 employs a novel pathway that is different from the two characterized pathways described in Arthrobacter and Pseudomonas. Based on these findings, we propose that strain S33 is able to transform nicotine to 6-hydroxy-pseudooxynicotine first via the pyridine pathway through 6-hydroxy-L-nicotine and 6-hydroxy-N-methylmyosmine, and then, it turns to the pyrrolidine pathway with the formation of 6-hydroxy-3-succinoylpyridine and 2,5-dihydroxypyridine. The activities of the key enzymes, nicotine dehydrogenase, 6-hydroxy-L-nicotine oxidase, and 6-hydroxy-3-succinoylpyridine hydroxylase, were demonstrated in the cell extract of strain S33 and by partially enriched enzymes. Moreover, the cell extract could transform 6-hydroxy-pseudooxynicotine into 6-hydroxy-3-succinoylpyridine by coupling with 6-hydroxy-L-nicotine oxidation reaction by 6-hydroxy-L-nicotine oxidase. These results indicated that strain S33 can transform nicotine into renewable hydroxylated-pyridine intermediates by the special pathway, in which at least three intermediates, 6-hydroxy-L-nicotine, 6-hydroxy-3-succinoylpyridine, and 2,5-dihydroxypyridine, have potential to be further chemically modified into useful compounds.
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People's Republic of China, shuningwang@sdu.edu.cn
authors.forename: Shuning
authors.initials: S
authors.lastname: Wang
authors.forename: Haiyan
authors.initials: H
authors.lastname: Huang
authors.forename: Kebo
authors.initials: K
authors.lastname: Xie
authors.forename: Ping
authors.initials: P
authors.lastname: Xu
mesh_terms: D016960:Agrobacterium tumefaciens / Q000201:enzymology / Q000254:growth & development / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D001673:Biodegradation, Environmental; D001711:Biotransformation; D002074:Butanones / Q000737:chemistry / Q000378:metabolism; D015394:Molecular Structure; D009538:Nicotine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D002074:Butanones; C081694:pseudooxynicotine; D009538:Nicotine