A Novel Gene, Encoding 6-Hydroxy-3-Succinoylpyridine Hydroxylase, Involved in Nicotine Degradation by Pseudomonas putida Strain S16

Abstract

Previous research suggested that Pseudomonas spp. may attack the pyrrolidine ring of nicotine in a way similar to mammalian metabolism, resulting in the formation of pseudooxynicotine, the direct precursor of a potent tobacco-specific lung carcinogen. In addition, the subsequent intermediates, 6-hydroxy-3-succinoylpyridine (HSP) and 2,5-dihydroxypyridine (DHP) in the Pseudomonas nicotine degradation pathway are two important precursors for drug syntheses. However, there is little information on the molecular mechanism for nicotine degradation via the pyrrolidine pathway until now. In this study we cloned and sequenced a 4,879-bp gene cluster involved in nicotine degradation. Intermediates N-methylmyosmine, pseudooxynicotine, 3-succinoylpyridine, HSP, and DHP were identified from resting cell reactions of the transformant containing the gene cluster and shown to be identical to those of the pyrrolidine pathway reported in wild-type strain Pseudomonas putida S16. The gene for 6-hydroxy-3-succinoylpyridine hydroxylase (HSP hydroxylase) catalyzing HSP directly to DHP was cloned, sequenced, and expressed in Escherichia coli, and the purified HSP hydroxylase (38 kDa) is NADH dependent. DNA sequence analysis of this 936-bp fragment reveals that the deduced amino acid shows no similarity with any protein of known function.

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

“…Cells were collected by centrifugation at 6,000 ϫ g for 5 min and then washed twice with 50 mM phosphate buffer (pH 7.0). The cells were suspended with the same phosphate buffer to 3.6 g liter Ϫ1 dry cell weight (as resting cells [18]), and the resting-cell reactions were performed at 30°C at 120 rpm.…”

Characterization of 2,5-dihydroxypyridine dioxygenases from Sinorhizobium sp. L1

“…The purified enzymes are similar by molecular masses to 2,5-dihydroxypyridine 5,6-dioxygenase from Pseudomonas putida N-9, KT2440 and S16 (Jimenez et al, 2008;Gauthier, Rittenberg, 1971;Tang et al, 2008).…”

Genome Sequence of a Nicotine-Degrading Strain of Arthrobacter

“…However, motility genes are absent from this bacterium, which suggests that the strain's competitiveness does not require movement (1). Also, the Pseudomonas nicotine degradation-related genes were not found (10)(11)(12)(13). This information about the genome of strain M2012083 will help in the study of the molecular mechanism of nicotine degradation by Arthrobacter.…”