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

Abstract

A newly isolated strain, SJY1, identified as Ochrobactrum sp., utilizes nicotine as a sole source of carbon, nitrogen, and energy. Strain SJY1 could efficiently degrade nicotine via a variant of the pyridine and pyrrolidine pathways (the VPP pathway), which highlights bacterial metabolic diversity in relation to nicotine degradation. A 97-kbp DNA fragment containing six nicotine degradation-related genes was obtained by gap closing from the genome sequence of strain SJY1. Three genes, designated vppB, vppD, and vppE, in the VPP pathway were cloned and heterologously expressed, and the related proteins were characterized. The vppB gene encodes a flavin-containing amine oxidase converting 6-hydroxynicotine to 6-hydroxy-N-methylmyosmine. Although VppB specifically catalyzes the dehydrogenation of 6-hydroxynicotine rather than nicotine, it shares higher amino acid sequence identity with nicotine oxidase (38%) from the pyrrolidine pathway than with its isoenzyme (6-hydroxy-L-nicotine oxidase, 24%) from the pyridine pathway. The vppD gene encodes an NADH-dependent flavin-containing monooxygenase, which catalyzes the hydroxylation of 6-hydroxy-3-succinoylpyridine to 2,5-dihydroxypyridine. VppD shows 62% amino acid sequence identity with the hydroxylase (HspB) from Pseudomonas putida strain S16, whereas the specific activity of VppD is ϳ10-fold higher than that of HspB. VppE is responsible for the transformation of 2,5-dihydroxypyridine. Sequence alignment and phylogenetic analysis suggested that the VPP pathway, which evolved independently from nicotinic acid degradation, might have a closer relationship with the pyrrolidine pathway. The proteins and functional pathway identified here provide a sound basis for future studies aimed at a better understanding of molecular principles of nicotine degradation. N icotine, which easily passes biological membranes, is significantly harmful to humans and is considered a potentially addictive drug (1, 2). Large quantities of tobacco waste with high concentrations of nicotine are generated annually by cigarette and cigar manufacturing (3). Due to its water solubility, nicotine can easily spread in the environment, emerging as a threat to public health (3). As a result, the U.S. Environmental Protection Agency classified nicotine as a "toxic release inventory" chemical in 1994 (4). Microbes play significant roles in removing nicotine from the environment, and in the meantime, they serve as the catalysts for biotransformation. Nicotine could potentially be used for the production of value-added chemicals. 6-Hydroxy-3-succinoylpyridine (HSP), an important precursor for the synthesis of compounds with biological activities, was efficiently produced by an engineered nicotine-degrading strain (5). Therefore, the study of the mechanism of nicotine degradation provides useful information for both bioremediation and biocatalysis.Many microorganisms that use various nicotine degradation pathways have been isolated from the environment, including Arthrobacter (6), Pseudomonas (7, 8), Ochroba...

Molybdenum-Containing Nicotine Hydroxylase Genes in a Nicotine Degradation Pathway That Is a Variant of the Pyridine and Pyrrolidine Pathways

“…NAD(P)H, FAD, 2,6-dichlorophenolindophenol sodium (DCIP), and phenazine ethosulfate (PES) were obtained from Sangon Biotech, Shanghai, China. The 6-hydroxynicotine oxidase was purified according to the method described previously (7). All other reagents and solvents were of analytical or chromatographic grade and were commercially available.…”

“…Ochrobactrum sp. SJY1 and Pseudomonas putida strains were grown at 30°C in LB medium (7) or MSM with different carbon and nitrogen sources (21).…”

“…Three nicotine degradation pathways have been proposed in bacteria based on the identification of intermediates: the pyridine pathway (3), the pyrrolidine pathway (2,5), and the variant of pyridine and pyrrolidine pathways (the VPP pathway) ( Fig. 1) (6)(7)(8)(9). The molecular mechanisms of nicotine degradation by the pyridine pathway and the pyrrolidine pathway have been elucidated in detail by the research groups of Brandsch (3) and Xu et al (2,10,11), respectively.…”

“…strain SJY1 (6,7), Agrobacterium tumefaciens S33 (12), Shinella sp. strain HZN7 (8), and a Pusillimonas strain (13).…”

“…In an early study, Ochrobactrum sp. strain SJY1 was isolated, and a 97.6-kb gene cluster (the vpp cluster, GenBank accession number KM065745), which is responsible for nicotine degradation, was characterized (7). Six genes (vppBDEFGH) involved in the VPP pathway were identified in the vpp cluster, and three of them were functionally characterized using in vitro experiments.…”