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.

Molecular Mechanism of Nicotine Degradation by a Newly Isolated Strain, Ochrobactrum sp. Strain SJY1

“…The biotransformation of 6HN to 6HMM and 6HPON was confirmed by using 6HN as the substrate; HSP was transformed to form 2,5-DHP, and 2,5-DHP could be further converted (data not shown). However, SP could not be transformed by resting cells of strain SJY1, which confirmed that HSP was not transformed from SP as in the pyrrolidine pathway (13). No blue pigment was produced during nicotine degradation, indicating that 6HPON was not transformed into 2,3,6-trihydroxypyridine.…”

“…2,5-DHP is a central metabolic intermediate in the catabolism of many pyridine derivatives (23,30). Genome sequencing promotes the study of molecular mechanisms of xenobiotic degradation (13,22), because genes can be identified through sequence alignment with isoenzymes or enzymes with similar functions. Genes responsible for the conversion of nicotine to 6HN and for the conversion from 6HPON to HSP have still not been found in the genome of strain SJY1.…”

“…The pathways of nicotine degradation have been widely investigated by researchers. Pyridine and pyrrolidine pathways are the two best-elucidated nicotine metabolic pathways, and most of the catalytic enzymes in these two pathways have been functionally characterized (6,13). In the pyrrolidine pathway, nicotine degradation is initiated by dehydrogenation of the pyrrolidine ring to form N-methylmyosmine, which is then converted to 6-hydroxynicotine (6HN), 6-hydroxy-N-methylmyosmine (6HMM), 6-hydroxypseudonicotine (6HPON), 2,6-dihydroxypseudooxynicotine, 2,6-dihydroxypyridine, and 2,3,6-trihydroxypyridine.…”

“…The pyridine pathway in Pseudomonas putida S16 is nicotine, N-methylmyosmine, pseudoxynicotine, 3-succinoyl-pyridine (SP), HSP, and 2,5-dihydroxypyridine (2,5-DHP). The 2,5-DHP is converted via a ring cleavage reaction to render N-formylmaleamic acid, maleamic acid, maleic acid, and fumaric acid (13). In addition to the pyridine and pyrrolidine pathways, some other nicotine degradation pathways were also proposed, including a variant of the pyridine and pyrrolidine pathways, which is designated the VPP pathway in bacteria (10,11).…”

“…Genome sequencing has the potential to accelerate the study of molecular mechanisms rather than requiring significant production scale searching (13,22). A 97.6-kbp DNA fragment designated the vpp cluster was assembled by gap closing from the draft genome sequence of strain SJY1.…”