title: Systematic unraveling of the unsolved pathway of nicotine degradation in Pseudomonas.
abstract: Microorganisms such as Pseudomonas putida play important roles in the mineralization of organic wastes and toxic compounds. To comprehensively and accurately elucidate key processes of nicotine degradation in Pseudomonas putida, we measured differential protein abundance levels with MS-based spectral counting in P. putida S16 grown on nicotine or glycerol, a non-repressive carbon source. In silico analyses highlighted significant clustering of proteins involved in a functional pathway in nicotine degradation. The transcriptional regulation of differentially expressed genes was analyzed by using quantitative reverse transcription-PCR. We observed the following key results: (i) The proteomes, containing 1,292 observed proteins, provide a detailed view of enzymes involved in nicotine metabolism. These proteins could be assigned to the functional groups of transport, detoxification, and amino acid metabolism. There were significant differences in the cytosolic protein patterns of cells growing in a nicotine medium and those in a glycerol medium. (ii) The key step in the conversion of 3-succinoylpyridine to 6-hydroxy-3-succinoylpyridine was catalyzed by a multi-enzyme reaction consisting of a molybdopeterin binding oxidase (spmA), molybdopterin dehydrogenase (spmB), and a (2Fe-2S)-binding ferredoxin (spmC) with molybdenum molybdopterin cytosine dinucleotide as a cofactor. (iii) The gene of a novel nicotine oxidoreductase (nicA2) was cloned, and the recombinant protein was characterized. The proteins and functional pathway identified in the current study represent attractive targets for degradation of environmental toxic compounds.
authors.affiliation: State Key Laboratory of Microbial Metabolism, and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, People's Republic of China.
authors.forename: Hongzhi
authors.initials: H
authors.lastname: Tang
authors.forename: Lijuan
authors.initials: L
authors.lastname: Wang
authors.forename: Weiwei
authors.initials: W
authors.lastname: Wang
authors.forename: Hao
authors.initials: H
authors.lastname: Yu
authors.forename: Kunzhi
authors.initials: K
authors.lastname: Zhang
authors.forename: Yuxiang
authors.initials: Y
authors.lastname: Yao
authors.forename: Ping
authors.initials: P
authors.lastname: Xu
mesh_terms: D003001:Cloning, Molecular; D003470:Culture Media; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D005990:Glycerol / Q000378:metabolism / Q000494:pharmacology; D017744:Medical Waste Disposal; D009538:Nicotine / Q000378:metabolism / Q000494:pharmacology*; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014176:Protein Biosynthesis / Q000235:genetics*; D016958:Pseudomonas putida / Q000187:drug effects / Q000235:genetics*; D011725:Pyridines / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D003470:Culture Media; D017744:Medical Waste Disposal; D011725:Pyridines; D009538:Nicotine; D010088:Oxidoreductases; D005990:Glycerol