A Novel (
            <i>S</i>
            )-6-Hydroxynicotine Oxidase Gene from Shinella sp. Strain HZN7

Qiu, Jiguo; Wei, Yin; Ma, Yi; Wen, Rongti; Wen, Yuezhong; Liu, Weiping

doi:10.1128/aem.01312-14

Cited by 32 publications

(26 citation statements)

References 35 publications

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“…These genes form a gene cluster with an ORF encoding a protein with 99% identity to 6-hydroxynicotine oxidase (Hno) from Shinella sp. strain HZN7 (22 The ndhA and ndhB genes are annotated to encode the isoquinoline 1-oxidoreductase alpha and beta subunits, respectively, which is a molybdenum-containing hydroxylase catalyzing the hydroxylation of isoquinoline to 1-oxo-1,2-dihydroisoquinoline from Brevundimonas diminuta 7 (formerly Pseudomonas diminuta 7) (47, 48) with the same conserved domains of NdhAB from S33. Based on BLAST analysis of protein sequence, NdhA is 99% identical to the large subunit of nicotine hydroxylase from Ochrobactrum sp.…”

Section: Purification Of Ndh Frommentioning

confidence: 99%

“…The in-frame deletion of ndhA, ndhB, and pno of A. tumefaciens S33 was performed using the suicide plasmid pJQ200SK and a two-step homologous recombination method (22,31). First, the in-frame-deleted gene fragments were obtained using crossover PCR as described previously (32).…”

Section: Chemicals and Reagentsmentioning

confidence: 99%

“…The same pathway present in Shinella 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.…”

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confidence: 99%

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

Xie

et al. 2016

Appl Environ Microbiol

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c Nicotine, a major toxic alkaloid in tobacco wastes, is degraded by bacteria, mainly via pyridine and pyrrolidine pathways. Previously, we discovered a new hybrid of the pyridine and pyrrolidine pathways in Agrobacterium tumefaciens S33 and characterized its key enzyme 6-hydroxy-3-succinoylpyridine (HSP) hydroxylase. Here, we purified the nicotine dehydrogenase initializing the nicotine degradation from the strain and found that it forms a complex with a novel 6-hydroxypseudooxynicotine oxidase. The purified complex is composed of three different subunits encoded by ndhAB and pno, where ndhA and ndhB overlap by 4 bp and are ϳ26 kb away from pno. As predicted from the gene sequences and from chemical analyses, NdhA (82.4 kDa) and NdhB (17.1 kDa) harbor a molybdopterin cofactor and two [2Fe-2S] clusters, respectively, whereas Pno (73.3 kDa) harbors an flavin mononucleotide and a [4Fe-4S] cluster. Mutants with disrupted ndhA or ndhB genes did not grow on nicotine but grew well on 6-hydroxynicotine and HSP, whereas the pno mutant did not grow on nicotine or 6-hydroxynicotine but grew well on HSP, indicating that NdhA and NdhB are responsible for initialization of nicotine oxidation. We successfully expressed pno in Escherichia coli and found that the recombinant Pno presented 2,6-dichlorophenolindophenol reduction activity when it was coupled with 6-hydroxynicotine oxidation. The determination of reaction products catalyzed by the purified enzymes or mutants indicated that NdhAB catalyzed nicotine oxidation to 6-hydroxynicotine, whereas Pno oxidized 6-hydroxypseudooxynicotine to 6-hydroxy-3-succinoylsemialdehyde pyridine. These results provide new insights into this novel hybrid pathway of nicotine degradation in A. tumefaciens S33.A grobacterium tumefaciens is well known for its ability to induce crown gall tumors in dicotyledonous plants and mediate interkingdom genetic transfer, for that it is widely used in plant molecular biology and biotechnology (1). Interestingly, some strains of this species are also able to degrade xenobiotics such as cyanuric acid, iminodisuccinate, and methylene urea (2-4). We isolated A. tumefaciens strain S33, which has the strong ability to degrade the natural alkaloid nicotine from the rhizospheric soil of a tobacco plant (5, 6).Nicotine is a major alkaloid in tobacco, which causes tobacco addiction and may result in diseases such as pulmonary disease and cancer (7,8), and it is the primary toxic compound in tobacco wastes. The tobacco-manufacturing process and all activities using tobacco produce a large amount of solid or liquid waste containing high concentrations of nicotine, which are classified as "toxic and hazardous wastes" by the European Union (9). Therefore, detoxification of tobacco wastes is a major concern for public health and the environment. The discovery of nicotine degradation by microorganisms provides an alternative way to dispose of such wastes (10-12).Microbial degradation of nicotine attracts attention because it represents a method to treat the tobacco waste...

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Section: Purification Of Ndh Frommentioning

confidence: 99%

Section: Chemicals and Reagentsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Xie

et al. 2016

Appl Environ Microbiol

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“…The pyridine pathway was mainly discovered in Arthrobacter species (3), and the pyrrolidine pathway was typically carried by Pseudomonas strains (2,33). In contrast, the VPP pathway was reported in at least four strains belonging to four different genera (7,8,12,13), and genes of the VPP pathway from different strains have high sequence identity to each other (9,14). On the other hand, VppA shows low sequence identity to its isozyme Ndh from Arthrobacter (13.7% and 36.4% sequence identity to the large and small subunits, respectively).…”

Section: Discussionmentioning

confidence: 99%

“…More recently, the function of nctB (GenBank accession number AGS16700), a gene that encodes (S)-6-hydroxynicotine oxidase, in Shinella sp. HZN7, was also investigated (14). However, the gene responsible for nicotine hydroxylation in the VPP pathway is still unknown.…”

mentioning

confidence: 99%

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

Tang

et al. 2015

Appl Environ Microbiol

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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...

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Genome-wide investigation of the genes involved in nicotine metabolism in Pseudomonas putida J5 by Tn5 transposon mutagenesis

Xia

Zhang

Lei

et al. 2015

Appl Microbiol Biotechnol

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Pseudomonas putida J5 is an efficient nicotine-degrading bacterial strain isolated from the tobacco rhizosphere. We successfully performed a comprehensive whole-genome analysis of nicotine metabolism-associated genes by Tn5 transposon mutagenesis in P. putida J5. A total of 18 mutants with unique insertions screened from 16,324 Tn5-transformants failed to use nicotine as the sole carbon source. Flanking sequences of the Tn5 transposon were cloned with a shotgun method from all of the nicotine-growth-deficient mutants. The potentially essential products of mutated gene were classified as follows: oxidoreductases, protein and metal transport systems, proteases and peptidases, transcriptional and translational regulators, and unknown proteins. Bioinformatic analysis of the Tn5 insertion sites indicated that the nicotine metabolic genes were separated and widely distributed in the genome. One of the mutants, M2022, was a Tn5 insert into a gene encoding a homolog of 6-hydroxy-L-nicotine oxidase, the second enzyme of nicotine metabolism in Arthrobacter nicotinovorans. Genetic and biochemical analysis confirmed that three open reading frames (ORFs) from an approximately 13-kb fragment recovered from the mutant M2022 were responsible for the transformation of nicotine to 3-succinoyl-pyridine via pseudooxynicotine and 3-succinoyl semialdehyde-pyridine, the first three steps of nicotine degradation. Further research on these mutants and the Tn5-inserted genes will help us characterize nicotine metabolic processes in P. putida J5.

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A Novel ( S )-6-Hydroxynicotine Oxidase Gene from Shinella sp. Strain HZN7

Cited by 32 publications

References 35 publications

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

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

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

Genome-wide investigation of the genes involved in nicotine metabolism in Pseudomonas putida J5 by Tn5 transposon mutagenesis

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