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

Abstract

bOchrobactrum sp. strain SJY1 utilizes nicotine as a sole source of carbon, nitrogen, and energy via a variant of the pyridine and pyrrolidine pathways (the VPP pathway). Several strains and genes involved in the VPP pathway have recently been reported; however, the first catalyzing step for enzymatic turnover of nicotine is still unclear. In this study, a nicotine hydroxylase for the initial hydroxylation step of nicotine degradation was identified and characterized. The nicotine hydroxylase (VppA), which converts nicotine to 6-hydroxynicotine in the strain SJY1, is encoded by two open reading frames (vppA S and vppA L [subunits S and L, respectively]). The vppA genes were heterologously expressed in the non-nicotine-degrading strains Escherichia coli DH5␣ and Pseudomonas putida KT2440; only the Pseudomonas strain acquired the ability to degrade nicotine. The small subunit of VppA contained a [2Fe-2S] cluster-binding domain, and the large subunit of VppA contained a molybdenum cofactor-binding domain; however, an FADbinding domain was not found in VppA. Resting cells cultivated in a molybdenum-deficient medium had low nicotine transformation activity, and excess molybdenum was detected in the purified VppA by inductively coupled plasma-mass spectrometry analysis. Thus, it is demonstrated that VppA is a two-component molybdenum-containing hydroxylase. N icotine is the main toxic compound in tobacco, and it accumulates with tobacco wastes during the processing of tobacco products. Nicotine can easily spread into the environment due to its water solubility and endanger the health of humans and other organisms (1). Microbial biodegradation is one of the best remediation strategies to remove nicotine from the environment (2-4). A number of bacteria and fungi which use nicotine as a sole source of carbon, nitrogen, and energy for their growth have been isolated and identified (3, 4). 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) (6-9). 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. However, the genes involved in several catalyzing steps of the VPP pathway are still unknown.Thus far, several bacteria that are able to degrade nicotine via the VPP pathway have been reported, including Ochrobactrum sp. strain SJY1 (6, 7), Agrobacterium tumefaciens S33 (12), Shinella sp. strain HZN7 (8), and a Pusillimonas strain (13). In the VPP pathway, nicotine degradation is initiated by a hydroxylation reaction to form 6-hydroxynicotine (6HN), which is then converted to 6-hydroxy-N-methylmyosmine (6HMM) and 6-hydroxypseudooxynicotine (6HPON). Subsequently, 6HPON is oxidized to form 6-hydroxy-3-succinoylpyridine (HSP), which is catabolized to fumaric acid through 2,5-dihy...

Nicotine Dehydrogenase Complexed with 6-Hydroxypseudooxynicotine Oxidase Involved in the Hybrid Nicotine-Degrading Pathway in Agrobacterium tumefaciens S33

“…BLAST analysis showed that NdhAB is almost identical to the most recently reported nicotine hydroxylase from Ochrobactrum sp. strain SJY1 (24), which mediates the first step in the same pathway, too. However, the function of the nicotine hydroxylase from Ochrobactrum sp.…”

“…Just like heterologous expression of nicotine hydroxylase from Ochrobactrum sp. strain SJY1 in E. coli (24), ndhAB was not functionally expressed (data not shown), likely because the molybdopterin cofactor in NdhAB was not synthesized by E. coli (29), which normally synthesizes the molybdopterin guanosine dinucleotide (29,61). In contrast, NdhAB from strain S33 may bind a molybdopterin cytosine dinucleotide like Ndh from A. nicotinovorans (29) and isoquinoline 1-oxidoreductase from B. diminuta 7 (48).…”

“…Based on BLAST analysis of protein sequence, NdhA is 99% identical to the large subunit of nicotine hydroxylase from Ochrobactrum sp. strain SJY1 (24), 27% identical to the large subunit of isoquinoline 1-oxidoreductase from B. diminuta 7 (47, 48), 14.0% identical to NdhL, and 14.4% identical to KdhL (the large subunit of 6-hydroxypseudooxynicotine dehydrogenase, also named ketone dehydrogenase) from A. nicotinovorans, which contain the conserved molybdopterin-binding domain. NdhB is 100% identical to the small subunit of nicotine hydroxylase from Ochrobactrum sp.…”

“…NdhB is 100% identical to the small subunit of nicotine hydroxylase from Ochrobactrum sp. strain SJY1 (24) (25,49,50), except for the missing of flavin-binding subunit. The pno gene is annotated as histamine dehydrogenase or NADH:flavin oxidoreductase in RAST or GenBank, respectively, and is 48% identical to the histamine dehydrogenase of Pimelobacter simplex (formerly Nocardioides simplex) (51)(52)(53), 46% identical to histamine dehydrogenase of Rhizobium sp.…”

“…strain HZN7 (21,22) and Ochrobactrum sp. strain SJY1 (23,24) was found recently using genome sequencing, intermediates analysis, and characterization of key enzymes such as nicotine hydroxylase, 6-hydroxynicotine oxidase (Hno), HSP hydroxylase (Hsh), and 2,5-dihydroxypyridine dioxygenase (Hpo), which are similar to the corresponding enzymes from Arthrobacter nicotinovorans and Pseudomonas putida S16. However, other enzymes involved in the hybrid pathway are still unknown.…”