Functional Identification of Two Novel Genes from Pseudomonas sp. Strain HZN6 Involved in the Catabolism of Nicotine

Qiu, Jiguo; Ma, Yi; Wen, Yuezhong; Chen, Liansheng; Li-ping, WU; Liu, Weiping

doi:10.1128/aem.07025-11

Cited by 55 publications

(63 citation statements)

References 32 publications

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“…Three types of degradation are mediated through the pyridine pathway of the Gram-positive bacterium Arthrobacter sp., the pyrrolidine pathway of the Gram-negative bacterium Pseudomonas sp., and the demethylation pathway present in fungi such as Aspergillus oryzae (13,14,18,19). The biochemical mechanisms involved in the pyridine and pyrrolidine pathways have been characterized in detail (13,15,16,20).We discovered that A. tumefaciens S33 catabolizes nicotine via a hybrid between the pyridine and pyrrolidine pathways by investigating the intermediates and the key enzymes activities in cell extracts (5). In the hybrid pathway (Fig.…”

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confidence: 99%

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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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).…”

Section: Discussionmentioning

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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“…Based on conserved motif of certain enzymes, chromosome walking provided a new method to identify functional enzymes [44]. A functional P450 monooxygenase from Rhodococcus sp.…”

Section: Chromosome Walkingmentioning

confidence: 99%

Bioreductive preparation of ACE inhibitors precursor (R)-2-hydroxy-4-phenylbutanoate esters: Recent advances and future perspectives

2015

Bioresour. Bioprocess.

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Optically active (R)-2-hydroxy-4-phenylbutanoate esters ((R)-HPBE) are key precursors for the production of angiotension-converting enzyme (ACE) inhibitors, which are important prescriptive drugs for preventing the formation of angiotensin II and lowering the blood pressure. The biocatalytic asymmetric reduction of ethyl 2-oxo-4-phenylbutanoate (OPBE) to (R)-HPBE with carbonyl reductases has several advantageous attributes, including high enantioselectivity, mild reaction condition, high catalytic efficiency, and environmental benignity. An increasing number of OPBE reductases have been discovered owing to the drastic achievements in genomics, screening and evolution technologies, and process engineering. The potential of (R)-HPBE production process has also been intensively evaluated. This review covers recent progress on the bioreductive preparation of (R)-HPBE, especially on various screening approaches for the identification of OPBE reductases and their characterization.

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Functional Identification of Two Novel Genes from Pseudomonas sp. Strain HZN6 Involved in the Catabolism of Nicotine

Cited by 55 publications

References 32 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

Bioreductive preparation of ACE inhibitors precursor (R)-2-hydroxy-4-phenylbutanoate esters: Recent advances and future perspectives

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