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. 2014 Sep;80(18):5552–5560. doi: 10.1128/AEM.01312-14

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

Jiguo Qiu a, Yin Wei b, Yun Ma b, Rongti Wen b, Yuezhong Wen a, Weiping Liu a,✉
Editor: R M Kelly
PMCID: PMC4178596  PMID: 25002425

Abstract

Nicotine is an important environmental toxicant in tobacco waste. Shinella sp. strain HZN7 can metabolize nicotine into nontoxic compounds via variations of the pyridine and pyrrolidine pathways. However, the catabolic mechanism of this variant pathway at the gene or enzyme level is still unknown. In this study, two 6-hydroxynicotine degradation-deficient mutants, N7-M9 and N7-W3, were generated by transposon mutagenesis. The corresponding mutant genes, designated nctB and tnp2, were cloned and analyzed. The nctB gene encodes a novel flavin adenine dinucleotide-containing (S)-6-hydroxynicotine oxidase that converts (S)-6-hydroxynicotine into 6-hydroxy-N-methylmyosmine and then spontaneously hydrolyzes into 6-hydroxypseudooxynicotine. The deletion and complementation of the nctB gene showed that this enzyme is essential for nicotine or (S)-6-hydroxynicotine degradation. Purified NctB could also convert (S)-nicotine into N-methylmyosmine, which spontaneously hydrolyzed into pseudooxynicotine. The kinetic constants of NctB toward (S)-6-hydroxynicotine (Km = 0.019 mM, kcat = 7.3 s−1) and nicotine (Km = 2.03 mM, kcat = 0.396 s−1) indicated that (S)-6-hydroxynicotine is the preferred substrate in vivo. NctB showed no activities toward the R enantiomer of nicotine or 6-hydroxynicotine. Strain HZN7 could degrade (R)-nicotine into (R)-6-hydroxynicotine without any further degradation. The tnp2 gene from mutant N7-W3 encodes a putative transposase, and its deletion did not abolish the nicotine degradation activity. This study advances the understanding of the microbial diversity of nicotine biodegradation.

INTRODUCTION

Nicotine is an important environmental toxicant in tobacco and tobacco waste that endangers the health of humans (1–3). Several bacterial strains distributed in different genera have the ability to eliminate this toxic compound or transform it into commercially valuable compounds (4–7). Different strains have shown a widely diverse set of metabolic mechanisms. Until now, four distinct metabolic pathways have been reported. First, the pyridine pathway and its related metabolic mechanism were elucidated at the molecular and enzymatic levels in strain Arthrobacter nicotinovorans pAO1 (8–11). This pathway is composed of five main intermediates, 6-hydroxynicotine (6HN), 6-hydroxy-N-methylmyosmine (6HMM), 6-hydroxypseudooxynicotine (6HPON), 2,6-dihydroxypseudooxynicotine, and 2,6-dihydroxypyridine. This type of catabolic mechanism was then found in Nocardioides sp. strain JS614 and Rhodococcus opacus (12, 13). Second, the pyrrolidine pathway has been well studied in Pseudomonas putida S16 (14–18) and Pseudomonas sp. strain HZN6 (19–21). The main intermediates of this pathway include N-methylmyosmine (NMM), pseudooxynicotine (PN), 3-succinoyl semialdehyde-pyridine (SAP), 3-succinoyl-pyridine (SP), 6-hydroxy-3-succinoyl-pyridine (HSP), and 2,5-dihydroxypyridine (2,5-DHP). The genes and enzymes involved in the pyrrolidine pathway have been cloned and characterized. Third, a variant of the pyridine and pyrrolidine pathways which contains the 6HN, 6HMM, 6HPON, HSP, and 2,5-DHP intermediates has been reported in Agrobacterium tumefaciens S33 (22) and Shinella sp. strain HZN7 (Fig. 1) (23). Additionally, a fourth pathway, a new variant of the pyrrolidine pathway which involves the PN and 4-hydroxy-1-(3-pyridyl)-1-butanone intermediates, was recently found in strain Pseudomonas plecoglossicida TND35 (5). The pyridine pathway and the pyrrolidine pathway have been well studied at the pathway level and at the gene and enzymatic levels. However, the genes or enzymes involved in the variant pathways in the newly isolated strains have been only poorly characterized or not characterized at all.

FIG 1.

FIG 1

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Proposed degradation pathways of (S)-nicotine (A; bold arrow), PN (B; slender arrow), and (R)-nicotine (C; hollow arrow) by strain Shinella sp. HZN7. Red and blue, intermediates involved in the pyridine pathway and pyrrolidine pathway, respectively; dotted arrow, nicotine can be oxidized into NMM (spontaneously hydrolyzed into PN) by purified NctB in vitro; red cross, strain HZN7 cannot degrade (R)-6-hydroxynicotine anymore.

Shinella sp. strain HZN7 is a Gram-negative bacterium that can mineralize nicotine using a variant of the pyridine and pyrrolidine pathways. Our previous study showed that the orf2 gene was crucial for 6HPON degradation. However, none of the enzymes responsible for the degradation of nicotine or its intermediates were studied in strain HZN7 (23). In this study, the degradation of 6HN, the first intermediate in the degradation pathway, was investigated. Two mutants deficient in 6HN degradation, N7-M9 and N7-W3, were generated by random mutagenesis. A novel (S)-6-hydroxynicotine oxidase gene (nctB) from mutant N7-M9 was cloned, expressed, and functionally identified. An unexpected putative transposase gene from mutant N7-W3 was also cloned and analyzed. The data presented in this study indicate that Shinella sp. HZN7 has a novel catabolic mechanism that is different from any mechanism reported in other species (Fig. 1). This is the first study to report a functional enzyme involved in this type of catabolic mechanism in bacteria.

MATERIALS AND METHODS

Chemicals and reagents.

(S)-Nicotine (purity, >99%) was purchased from Sigma-Aldrich (St. Louis, MO). (R)-Nicotine (purity, >99%), (S)-6HN (purity, >98%), PN (purity, 98%), and SP (purity, 98%) were obtained from Toronto Research Chemicals, Inc. (Toronto, Canada). Unless otherwise noted, nicotine and 6HN were specific for (S)-nicotine and (S)-6HN, respectively. 2,5-DHP (98%) was purchased from SynChem OHG (Altenburg, Germany). (R)-6HN was not commercially available and was prepared using the method described below. Flavin adenine dinucleotide (FAD) was obtained from Sangon Biotech, Shanghai, China. All other chemicals were of analytical or chromatographic grade and were commercially available.

Bacterial strains, plasmids, and growth conditions.

The bacterial strains and plasmids used in this study are listed in Table 1. Shinella sp. strain HZN7 was isolated and identified as a nicotine-degrading bacterium and was deposited in the China Center for Type Culture Collection (CCTCC M 2013060) (23). Strain A. nicotinovorans pAO1 (DSM 420) is a well-studied, nicotine-degrading bacterium and was a gift from Roderich Brandsch (Institute of Biochemistry and Molecular Biology, University of Freiburg, Freiburg, Germany) (8). Escherichia coli, Shinella sp. strains, and A. nicotinovorans pAO1 were routinely grown at 37°C, 30°C, and 30°C, respectively, in LB medium (10 g/liter tryptone, 10 g/liter NaCl, 5 g/liter yeast extract). E. coli DH5α and SM10λpir were used for the cloning procedures and plasmid maintenance. Mineral salts medium (MSM) containing 1.5 g/liter K2HPO4, 0.5 g/liter KH2PO4, 0.2 g/liter MgSO4, 1.0 g/liter (NH4)2SO4, 1.0 g/liter NaCl, and 1 ml/liter trace elements solution, as previously described (19), was used for the physiological characterization of the strains. Unless otherwise noted, the cultures were grown in 250-ml Erlenmeyer flasks containing medium at up to one-fifth of their nominal volume with rotary shaking at 180 rpm. Growth was estimated by measuring the optical density at 600 nm (OD600), as assessed in a UV-visible spectrophotometer (V-550; JASCO, Japan). All solid media used in this work contained 2.0 g/liter agar. Ampicillin (Ap; 100 μg/ml), kanamycin (Km; 50 μg/ml), or gentamicin (Gm; 50 μg/ml) was filter sterilized and added when appropriate.

TABLE 1.

Strains and plasmids used in this study

Strain or plasmid Description Source
Strains
    Arthrobacter nicotinovorans pAO1 Nicotine-degrading strain, Gram positive DSM 420
    Shinella species
        HZN7 Apr, wild type, nicotine-degrading strain, Gram negative CCTCC M 2013060
        N7-M9 Apr Kmr, mutant of HZN7 obtained with transposon Tn5, 6HN degradation-deficient mutant This study
        N7-W3 Apr Kmr, mutant of HZN7 obtained with transposon Tn5, 6HN degradation-deficient mutant This study
        N7-M9-Com Apr Kmr Gmr, N7-M9 containing pBB-nctB This study
        N7-ΔnctB Apr Kmr, nctB::Kmr mutant of HZN7 This study
        N7-ΔnctB-Com Apr Kmr Gmr, N7-ΔnctB containing pBB-nctB This study
        N7-W3-tnp2 Apr Kmr Gmr, N7-W3 containing pBB-tnp2 This study
        N7-Δtnp2 Apr Kmr, tnp2::Kmr mutant of HZN7 This study
        N7-W3-nctB Apr Kmr Gmr, N7-W3 containing pBB-nctB This study
    Escherichia coli
        DH5α F− recA1 endA1 thi-1 hsdR17 supE44 relA1 deoRΔ(lacZYA-argF)U169 ϕ80dlacZΔM15 TaKaRa
        SM10λpir Donor strain for biparental mating Lab stock
        BL21(DE3) F− ompT hsdSB(rB− mB−) gal dcm lacY1 (DE3) Novagen
Plasmids
    pMD18-T Apr, T-A cloning vector TaKaRa
    pET29a(+) Kmr, expression vector Novagen
    pSC123 Cmr Kmr, mariner transposon Lab stock
    pJQ200SK Gmr mob+ orip15A lacZα+ sacB, suicide vector Lab stock
    pBBR1-MCS5 Gmr, broad-host-range cloning vector Lab stock
    pJQ-ΔnctB Gmr, BamHI-SacI fragment containing nctB inserted into pJQ200SK where nctB was disrupted by the Kmr gene This study
    pJQ-Δtnp2 Gmr, BamHI-SacI fragment containing tnp2 inserted into pJQ200SK where tnp2 was disrupted by the Kmr gene This study
    pBB-nctB Gmr, BamHI-SacI fragment containing the nctB gene inserted into pBBR1-MCS5 This study
    pBB-tnp2 Gmr, BamHI-SacI fragment containing the tnp2 gene inserted into pBBR1-MCS5 This study
    pET-nctB Kmr, NdeI-XhoI fragment containing nctB inserted into pET29a(+) This study
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Molecular genetics procedures.

Standard techniques were used for the DNA manipulations. The oligonucleotides were synthesized by Sangon Biotech (Shanghai, China). PCRs were set up using either LA Taq DNA polymerase (for self-formed adaptor PCR [SEFA-PCR] or cloning PCR) or PrimeSTAR HS DNA polymerase (for overlap PCR) (TaKaRa, Dalian, China), on the basis of the experimental requirements. Restriction endonucleases and T4 DNA ligase were purchased from TaKaRa and were used according to the manufacturer's specifications. The amplified fragments were purified using an AxyPrep PCR cleanup kit (Axygen, Hangzhou, China). DNA fragments were isolated from agarose gels using a DNA gel extraction kit (Axygen). All cloned inserts and DNA fragments were confirmed by DNA sequencing. Standard procedures (24) were used to transform the E. coli or Shinella strains, and the resulting transformants were selected on LB plates containing the appropriate antibiotics.

Transposon mutagenesis and screen for 6HN degradation-deficient mutants.

A library of nicotine growth-deficient mutants of the Shinella sp. HZN7 strain was constructed by transposon mutagenesis as previously described (23). The generated mutants were streaked onto MSM plates containing 2.81 mM 6HN, or a large inoculum was added to liquid MSM containing 0.11 mM 6HN. The mutants that could not grow on the 6HN plate or could not degrade 6HN in liquid culture were selected for further study.

Sequencing and analysis of the interrupted DNA.

The genomic regions flanking the transposon were amplified using the SEFA-PCR method (25). The primers specific to the transposon were described in our previous report (19, 23). LA Taq DNA polymerase with GC buffer II was used in the SEFA-PCRs. The PCR products were purified and cloned into the pMD18-T vector and sequenced. The analysis of the open reading frames (ORFs) and comparisons of the amino acid and nucleotide sequences were performed using the ORF Finder and BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) programs on the NCBI website. Conserved protein domains were predicted and analyzed using the Conserved Domain Database (CDD; http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). DNA and amino acid sequences were aligned using the ClustalW2 program (http://www.ebi.ac.uk/Tools/clustalw2/index.html).

Cloning, overexpression, and purification of NctB.

The original nctB gene contains one NdeI site (964 bp after the start codon) and one XhoI site (1,025 bp after the start codon) (see Fig. S1 in the supplemental material). To omit the NdeI and XhoI sites but maintain the original amino acid sequences, the primer pairs nctB-p1/nctB-p2 and nctB-p3/nctB-p4 (see Table S1 in the supplemental material) were designed to amplify these two parts of the nctB gene. In primers nctB-p2 and nctB-p3, the codons GCA and CTC were replaced with GCT and CTG, respectively (Fig. S1). The complete nctB gene was fused from these two fragments using the overlap PCR method and inserted into the NdeI-XhoI sites of the pET29a(+) plasmid to produce pET-nctB. The recombinant NctB protein contained a C-terminal His6 tag. The overexpression and purification procedures were the same as those previously described for pseudooxynicotine amine oxidase (PNAO) from Pseudomonas sp. HZN6 (20). The protein concentrations were determined using the Bradford method, with bovine serum albumin used as a standard (26).

NctB enzyme assay.

To identify the enzyme function of the NctB quickly, rapid degradations were induced by the addition of high concentrations of enzyme and low concentrations of substrates and were monitored by determination of spectrophotometric changes. The reactions were performed in 50 mM phosphate-buffered saline (PBS; pH 7.0) containing 0.0562 mM 6HN or 0.123 mM nicotine at room temperature. The reactions were initiated by the addition of purified NctB at 10 μg/ml for the 6HN reactions or 30 μg/ml for the nicotine reactions. The reference cuvette contained all of these compounds except the substrate.

To determine the enzyme activity of NctB, a standard enzyme assay was performed in a 1-ml reaction mixture containing 50 mM potassium phosphate buffer (PBS, pH 7.0), substrates (0.0562 mM 6HN or 0.123 mM nicotine), and purified NctB (0.1 μg/ml for 6HN, 5 μg/ml for nicotine) at 40°C. The reactions were initiated by the addition of purified NctB. The enzymatic activities were monitored by determination of the formation of H2O2. One unit of enzyme activity was defined as the amount of enzyme required to produce 1 μmol of H2O2 per min. To determine whether the production of H2O2 was oxygen dependent, the reaction was performed under both aerobic and anaerobic conditions. PBS was purged with high-purity nitrogen (99.99% N2) for 20 min to remove the oxygen before the substrate was added. PBS without the oxygen removed served as a positive control. The samples were periodically removed to measure H2O2 formation.

For the kinetic studies, the substrates were appropriately diluted to seven different concentrations around the Km (0.0281, 0.0562, 0.112, 0.169, 0.225, 0.281, and 0.337 mM for 6HN; 0.926, 1.23, 1.54, 1.85, 2.16, 2.47, and 3.09 mM for nicotine). The reactions were initiated by the addition of purified NctB. No more than 10% of the substrate was transformed during the assay. The kinetic values were obtained using the Hanes-Woolf equation against the various H2O2 concentrations.

Biochemical characterization of NctB.

The molecular mass of denatured NctB was determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The molecular mass of the native protein was determined by gel filtration chromatography. The pH range of the purified NctB was determined by incubating the enzyme with 0.0562 mM 6HN as the substrate for 30 min at 40°C between pH 4 and 9.8. Three different buffering systems were used: 50 mM citric acid-sodium citrate (pH 4 to 6), 50 mM KH2PO4-K2HPO4 buffer (pH 6 to 8), and 50 mM glycine-NaOH (pH 8.6 to 9.8). To determine the stability of the protein at different pHs, the enzyme was preincubated in buffers at pH 4 to 9.8 for 1 h at 40°C, and the remaining activity was assayed. The optimal reaction temperature was determined in assays performed under standard conditions at pH 7.0 and different temperatures (4, 20, 30, 40, 50, and 60°C). To determine the thermostability of the protein, the enzyme was preincubated in a water bath at different temperatures for 1 h, and the remaining activity was assayed. The effects of potential inhibitors on the enzyme were determined by the addition of various chemical agents (1.0 mM) to the reaction mixture, followed by incubation at 40°C for 1 h. The activity was assayed as described above and was expressed as a percentage of the activity obtained in the absence of the added compound.

Generation of strains with deletions and genetic complementation.

The in-frame disruption of the nctB and tnp2 genes in strain Shinella sp. HZN7 was performed using the suicide plasmid pJQ200SK and a two-step homologous recombination method, as described previously (20). Briefly, to disrupt the nctB gene, a 2.1-kb fusion product was constructed using the overlap PCR method; this product consisted of the sequence upstream from the nctB gene, the kanamycin resistance gene, and the downstream sequence and was constructed using the primers KO-nctB-p1/p2, KmF/KmR, and KO-nctB-p3/p4 (see Table S1 in the supplemental material), respectively. Similarly, a 1.9-kb fusion product was constructed by PCR to disrupt the tnp2 gene using primers KO-tnp2-p1/p2, KmF/KmR, and KO-tnp2-p3/p4 (see Table S1 in the supplemental material). These two fusion products were ligated into pJQ200SK at the BamHI-SacI sites, yielding the pJQ-ΔnctB and pJQ-Δtnp2 plasmids, respectively. The resulting plasmids were introduced into the Shinella sp. HZN7 strain via SM10λpir. Single-crossover clones were selected on LB plates containing Ap, Km, and Gm. Double-crossover mutants were screened on LB plates containing 10% (wt/vol) sucrose and Ap and Km. The genotypes of the recombinants were confirmed by PCR using primers nctBFw/nctBRv and tnp2Fw/tnp2Rv (see Table S1 in the supplemental material), which amplify 2.3-kb and 2.02-kb products, respectively, when the gene is successfully deleted. The nctB and tnp2 in-frame-disrupted strains were designated N7-ΔnctB and N7-Δtnp2, respectively.

A 1.68-kb fragment carrying the coding region of the nctB gene and a 1.51-kb fragment carrying the coding region of the tnp2 gene were amplified from the genomic DNA of HZN7 using the primers nctBFw/nctBRv and tnp2Fw/tnp2Rv, respectively. These two fragments were inserted into BamHI-SacI-digested pBBR1-MCS5, yielding the pBB-nctB and pBB-tnp2 plasmids, respectively. The pBB-nctB plasmid was transferred into the N7-M9 and N7-ΔnctB strains by biparental mating to generate the complementation strains N7-M9-Com and N7-ΔnctB-Com, respectively. The pBB-nctB and pBB-tnp2 plasmids were transferred into mutant N7-W3, generating recombinant N7-W3-nctB and N7-W3-tnp2, respectively.

Preparation of (R)-6HN.

(R)-6HN was prepared using semipreparative high-pressure liquid chromatography (HPLC) after the degradation of (R)-nicotine by strain A. nicotinovorans pAO1. According to the pyridine pathway, (R)-nicotine was hydroxylated into (R)-6HN (8). Strain A. nicotinovorans pAO1 was added into MSM containing 6.17 mM (R)-nicotine. After incubation for 2 h at 30°C with shaking at 180 rpm, the reaction mixture was centrifuged at 12,000 rpm for 20 min and filtered through a 0.22-μm-pore-size Millipore filter. The semipreparative HPLC was equipped with an Agilent SB-C18 column (9.4 by 250 mm, 5 μm) and a UV detector operating at a wavelength of 295 nm. The collection was performed on the HPLC, liquid chromatography-mass spectrometry (LC-MS) (see “Analytical methods” below) was performed to confirm the formation of (R)-6HN, and (R)-6HN was used as a standard.

Biodegradation assay.

The Shinella sp. HZN7 strain and its derivative mutants were grown overnight in LB medium, and the cells were collected by centrifugation, washed twice with MSM, and resuspended in MSM. The cell suspension was transferred to a 100-ml flask containing 40 ml of MSM, and the OD600 was adjusted to 1.5.

To investigate the degradation of nicotine and its related compounds by HZN7 and its derivative mutants, 1.0 mM each substrate, including (S)-nicotine, (R)-nicotine, (S)-6HN, (R)-6HN, 6HPON, HSP, 2,5-DHP, and PN, was added to a 100-ml flask and incubated in a rotary shaker at 30°C and 180 rpm. After inoculation, the concentrations of the substrates were determined as described in “Analytical methods” below. Negative controls without bacterial inoculation were included for each substrate. The cultures were withdrawn periodically, and the remaining substrate was measured.

Analytical methods.

Qualitative and quantitative analyses of (R)- and (S)-nicotine, (R)- and (S)-6HN, 6HMM, 6HPON, HSP, 2,5-DHP, and PN were performed by LC-MS or HPLC as described previously (23). Qualitative and quantitative analyses of the FAD released from purified NctB were performed by the same methods used to determine the release of FAD from PNAO in Pseudomonas sp. HZN6 described previously (20). Analysis of the hydrogen peroxide was performed with an H2O2 quantitative assay kit (Water Compatible; Sangon Biotech, Shanghai, China) following the manufacturer's instructions.

All assays in this study were performed independently three times, and the means and standard deviations were calculated.

Nucleotide sequence accession numbers.

The nucleotide sequences of the 3,777 bp containing the nctB gene and the 4,266 bp containing the tnp2 gene from the Shinella sp. HZN7 strain were deposited in the GenBank database under accession numbers KF306095 and KC912565, respectively.

RESULTS

Screening for 6HN degradation-deficient mutants.

Of more than 12,000 mutant colonies screened, two transposon mutants, designated N7-M9 and N7-W3, that fit the criteria described in Materials and Methods were identified. These mutants could degrade 1.0 mM nicotine into 6HN in 2 h without any further degradation. After quantification analysis by HPLC, the mole ratio of nicotine to 6HN was approximately 1:1, indicating a 100% transformation rate.

Cloning, sequencing, and analysis of interrupted DNA.

The transposon-interrupted DNA in the mutants was amplified using the SEFA-PCR method. A 3,777-bp DNA fragment and a 4,266-bp DNA fragment were cloned and assembled from N7-M9 and N7-W3, respectively. In the 3,777-bp fragment obtained from N7-M9, two complete ORFs were found. One ORF, designated nctB, encoded a protein of 437 amino acids with a predicted molecular mass of 48.736 kDa. The G+C content was 53.35%. The transposon was inserted into this gene between 957 and 958 bp from its start codon (see Fig. S1 in the supplemental material). The insertion event was further confirmed by PCR using primers nctBFw and nctBRv (see Fig. S2 in the supplemental material). Compared with the known enzymes available from the NCBI Protein Database, the predicted protein showed the highest similarity with several flavin-containing amine oxidases (pfam01593), such as nicotine oxidase (NOX; GenBank accession number AGH68979) from Pseudomonas sp. HZN6 (43% identity) (21), pseudooxynicotine amine oxidase (PNAO; GenBank accession number AFD54463) from Pseudomonas sp. HZN6 (31% identity) (20), monoamine oxidase (Mao; PDB accession number 2VVM) from Aspergillus niger (27% identity) (27), 6-hydroxy-l-nicotine oxidase (6HLNO; PDB accession number 3K7M) from A. nicotinovorans (26% identity) (28, 29), putrescine oxidase (PutO; PDB accession number 2YG5) from Rhodococcus erythropolis (24% identity) (30), and cyclohexylamine oxidase (CHAO; PDB accession number 4I58) from Brevibacterium oxydans IH-35A (24% identity) (31). Multiple-sequence alignments of NctB and these proteins revealed the consensus sequence of the FAD-binding motif (GXGXXG) at amino acids 12 to 17 in the N-terminal sequence (Fig. 2). In addition, phylogenetic analysis of NctB with these reported amine oxidases showed that NctB formed a branch with nicotine degradation-related enzymes, e.g., NAO, PNAO, and 6HLNO (see Fig. S3 in the supplemental material). The second ORF, designated tnp1, encoded a protein of 455 amino acids that exhibited 92% and 89% amino acid sequence identity with the transposase IS66 family proteins from Rhizobium leguminosarum WSM1325 (YP_002973085) and Sinorhizobium meliloti GR4 (YP_007194654), respectively.

FIG 2.

FIG 2

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Primary sequence alignment of NctB and related amine oxidases. Three conserved regions were selected. The conserved FAD-binding motif GXGXXG is indicated. NOX, nicotine oxidase from Pseudomonas sp. HZN6; PNAO, pseudooxynicotine amine oxidase from Pseudomonas sp. HZN6; 6HLNO, 6-hydroxy-l-nicotine oxidase from Arthrobacter nicotinovorans; Mao, monoamine oxidase from Aspergillus niger; PutO, putrescine oxidase from Rhodococcus erythropolis; CHAO, cyclohexylamine oxidase from Brevibacterium oxydans IH-35A.

In the 4,266-bp DNA fragment obtained from N7-W3, one complete ORF (bp 1934 to 3034), designated tnp2, was found. The transposon was inserted into this gene between 138 and 139 bp from its start codon. The insertion event was further confirmed by PCR using primers tnp2Fw and tnp2Rv (see Fig. S4 in the supplemental material). This ORF encoded a 366-amino-acid protein with a predicted molecular mass of 41.289 kDa. The predicted protein shared 63% and 56% amino acid sequence identity with the transposase IS110 family proteins from Sinorhizobium fredii HH103 (GenBank accession number YP_006575384) and Methylobacterium extorquens CM4 (GenBank accession number YP_002422113), respectively. No other ORFs (>500 bp) were identified in this fragment.

Expression and purification of NctB.

Recombinant NctB was overexpressed in E. coli BL21(DE3) as a C-terminal His6-tagged fusion protein and was purified from the crude extract using Ni-nitrilotriacetic acid affinity chromatography. A single band at an apparent molecular mass of 50 kDa was detected by SDS-PAGE, and this band corresponded to the molecular mass of His6-tagged NctB. The purified NctB was yellow, indicating that it is a flavoprotein. After the samples were boiled for 10 min and centrifuged, the sample formed a white protein pellet and a yellow supernatant, indicating that the flavin cofactor was noncovalently bound to the protein.

The spectrum of the His6-tagged NctB showed the characteristics of a typical flavoprotein. The visible and UV absorption spectra of the purified enzyme exhibited absorption maxima centered at 275, 384, and 460 nm and had an absorption minimum at 415 nm (Fig. 3). The visible spectrum of the flavin released from the enzyme after boiling was identical to that of FAD. HPLC analysis also revealed that the flavin was FAD; no flavin mononucleotide was detected. The FAD content was determined to be 1.0 ± 0.1 mol of FAD per mole subunit. NctB was considered to be a homodimer, as determined by gel filtration chromatography.

FIG 3.

FIG 3

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Visible and UV absorption spectra of purified NctB (continuous black line) and the FAD released from NctB (dotted line). (Inset) Expanded scale of the absorption in the 314- to 600-nm range.

Functional identification of NctB.

6HN (absorption maxima, 234 and 297 nm) was rapidly degraded by purified NctB, as shown in Fig. 4A, and 6HPON (absorption maximum, 291 nm) was formed. An isosbestic point of 244 nm was also observed. The consumption of 6HN and the accumulation of 6HPON were confirmed and quantified using UV and HPLC analysis. Furthermore, the formation of H2O2 was determined using a kit. The results showed that the ratio of the complete conversion of 6HN (56.2 μM) to 6HPON (51.5 ± 3.0 μM) and H2O2 (50.5 ± 5.1 μM) was close to 1:1:1.

FIG 4.

FIG 4

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Spectrophotometric changes during the transformation of 6HN (A) and nicotine (B) by purified NctB. The reaction was initiated by the addition of 6HN or nicotine, and the spectra were recorded every 2 min after the addition of the substrates. Arrows, directions of the spectral changes; blue and red, substrates and products, respectively.

Similarly, the rapid transformation of nicotine (absorption maximum, 260 nm) by recombinant NctB resulted in the formation of a product with absorption maxima of 241 and 267 nm (Fig. 4B), which was clearly different from the absorption maximum of nicotine. HPLC and LC-MS analyses showed that this product was identical to authentic PN. Furthermore, H2O2 was also detected and quantified. In a time course assay of the oxidation reaction, the level of nicotine consumption (123 μM) was equivalent to the level of accumulation of PN (115 ± 7 μM) and H2O2 (119 ± 6.2 μM), indicating that the ratio of the complete conversion of nicotine to PN and H2O2 was also close to 1:1:1.

Additionally, the two reactions described above were performed under anaerobic conditions, and little or no H2O2 was detected, suggesting that the reactions were oxygen dependent.

Biochemical characterization of recombinant NctB.

The kinetic constants of 6HN conversion by NctB showed an apparent Km of 0.019 ± 0.002 mM, a kcat of 7.3 ± 0.6 s−1, and a catalytic efficiency (kcat/Km) of 379 ± 57 s−1 mM−1; for nicotine, an apparent Km of 2.03 ± 0.094 mM, kcat of 0.396 ± 0.033 s−1, and catalytic efficiency (kcat/Km) of 0.195 ± 0.025 s−1 mM−1 were observed. The Km value for nicotine was 106-fold higher than that for 6HN. The catalytic efficiency for 6HN was 1,943-fold higher than that for nicotine. Therefore, 6HN was predicted to be the preferred substrate of NctB in vivo.

NctB was active over a broad range of pHs (pH 6.0 to 9.0) (Fig. 5A). The optimum pH was 7.0 in 50 mM PBS. The enzyme was stable between pH 6.0 and 9.8, and it retained more than 70% of its original activity after preincubation over that pH range for 1 h. The optimal temperature for NctB activity was 40°C. The enzyme was stable at temperatures lower than 50°C (Fig. 5B) and was fairly stable up to 50°C. It retained approximately 85% of its activity when incubated at 50°C for 1 h, and it retained 30% of its residual activity at 60°C. At 4°C, approximately 100% of the NctB activity was retained after 48 h, and approximately 50% activity remained after 7 days.

FIG 5.

FIG 5

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(A) Effect of pH on the enzyme stability (continuous black line) and activity (dotted line) of the recombinant NctB. ■, 50 mM citric acid-sodium citrate, pH 4, 5, and 6; ●, 50 mM KH2PO4-K2HPO4 buffer, pH 6, 7, and 8; ▲, 50 mM glycine-NaOH, pH 8.6, 9.0, and 9.8. (B) Effect of temperature on the enzyme stability (continuous black line) and activity (dotted line) of the recombinant NctB. The temperature was set at 4, 20, 30, 40, 50, and 60°C. (C) Effect of metal ions, EDTA, and SDS on the enzyme activity of the recombinant NctB. CK, control reaction carried out in the absence of metal ion; Al, Al3+; Ag, Ag+; Ca, Ca2+; Co, Co2+; Cu, Cu2+; Hg, Hg2+; Mg, Mg2+; Mn, Mn2+; Zn, Zn2+.

The effects of metal ions (1 mM) on NctB activity were also investigated (Fig. 5C). The enzyme did not require Al3+, Ca2+, Mg2+, or Mn2+ for its activity and was partially inhibited by Co2+ or Zn2+. NctB was significantly inhibited (more than 80%) by Ag+, Cu2+, and Hg2+. Incubation of NctB with 1 mM EDTA or SDS for 1 h completely inhibited its enzymatic activity.

Degradation of nicotine and its intermediates by the recombinant strains.

The degradation results for nicotine and other intermediates, including 6HN, 6HMM, 6HPON, HSP, 2, 5-DHP, and PN, degraded by HZN7, as well as its derivative mutants, are shown in Table 2.

TABLE 2.

Accumulation of different intermediates by Shinella sp. HZN7, the derivative mutants, or purified NctB

Substrate Intermediate accumulateda
HZN7 N7-M9 N7-M9-Com N7-ΔnctB N7-ΔnctB-Com N7-W3 N7-W3-tnp2 N7-Δtnp2 N7-W3-nctB NctB
(S)-Nicotine — (S)-6HN — (S)-6HN — (S)-6HN (S)-6HN — 6HPON PN
(R)-Nicotine (R)-6HN (R)-6HN (R)-6HN (R)-6HN (R)-6HN (R)-6HN (R)-6HN (R)-6HN (R)-6HN (R)-Nicotine
(S)-6HN — (S)-6HN — (S)-6HN — (S)-6HN (S)-6HN — 6HPON 6HPON
(R)-6HN (R)-6HN (R)-6HN (R)-6HN (R)-6HN (R)-6HN (R)-6HN (R)-6HN (R)-6HN (R)-6HN (R)-6HN
6HPON — — — — — 6HPON 6HPON — 6HPON ND
HSP — — — — — HSP HSP — HSP ND
2,5-DHP — — — — — 2,5-DHP 2,5-DHP — 2,5-DHP ND
PN — — — — — 6HPON 6HPON — 6HPON ND
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a

The intermediate indicates the final product degraded by strains that were monitored for 72 h. —, no accumulation of the intermediate was detected after 2 h of degradation; ND, not detected.

Mutants N7-M9 and N7-W3 could convert nicotine into 6HN without any further degradation. The ability of the enzyme to degrade 6HN was abolished in the nctB-disrupted recombinant N7-ΔnctB. The capacity to mineralize nicotine or 6HN was recovered in the complemented strains, N7-M9-Com and N7-ΔnctB-Com, and their mineralization capacity was no different from that of wild-type strain HZN7. Additionally, the N7-M9, N7-M9-Com, N7-ΔnctB, and N7-ΔnctB-Com strains could degrade the other four intermediates in the pathway, 6HMM, 6HPON, HSP, and 2,5-DHP. These results suggest that the disruption of the nctB gene did not interfere with the degradation of the intermediates downstream of 6HN.

In contrast to N7-M9, the N7-W3 mutant, in which the tnp2 gene was disrupted, exhibited some different degradation characteristics. Similar to the case for 6HN, mutant N7-W3 could not degrade 6HMM, 6HPON, HSP, or 2,5-DHP. After this mutant was complemented with the tnp2 gene, the recombinant N7-W3-tnp2 still could not degrade 6HN, 6HMM, 6HPON, HSP, or 2,5-DHP. The recombinant N7-W3-nctB (N7-W3 containing the nctB gene) could convert nicotine or 6HN into 6HPON without any further degradation. Interestingly, tnp2-disrupted mutant N7-Δtnp2 could degrade nicotine and all five other intermediates and showed no difference in degradation capacity from the HZN7 strain. These results might suggest that, except for the gene for the enzyme needed in the first step, the degradation-related genes were lost after transposon mutagenesis. This hypothesis was partially confirmed by PCR of the nctB gene using the primers nctBFw and nctBRv (see Table S1 in the supplemental material). The expected 1.68-kb fragment was not obtained from the N7-W3 mutant (see Fig. S4 in the supplemental material).

A previous study showed that strain HZN7 could degrade PN using the following pathway: PN, 6HMM, 6HPON, HSP, and 2,5-DHP (23). The N7-M9, N7-M9-Com, N7-ΔnctB, and N7-ΔnctB-Com mutants could also mineralize PN. Additionally, the N7-W3, N7-W3-tnp2, and N7-W3-nctB mutants could only hydroxylate PN into 6HPON without any further degradation, but recombinant N7-Δtnp2 could still completely degrade PN.

(R)-Nicotine and (R)-6HN degradation.

After degradation of (R)-nicotine by strain A. nicotinovorans pAO1, (R)-6HN was collected by semipreparative HPLC, and the results showed that its retention time was identical to that of (S)-6HN. LC-MS results showed that this compound had a molecular ion peak at 179.1[M + H]+ m/z, and its fragment ions peaked at 148.1[M + H]+ m/z. Thus, we confirmed that this compound was (R)-6HN.

The ability to degrade (R)-nicotine and (R)-6HN was monitored in strain HZN7 and its derivative mutants. These results showed that all of these strains could degrade (R)-nicotine into (R)-6HN but could not convert (R)-6HN into any other compound. Additionally, purified NctB showed no activity toward (R)-nicotine or (R)-6HN (Table 2).

DISCUSSION

In this study, we cloned, expressed, and characterized the novel, FAD-containing oxidase NctB from the newly isolated nicotine-degrading strain Shinella sp. HZN7. The deduced amino acid sequences showed relatively low homogenetic identities (approximately 30%) with known amino oxidases. Although the conserved amino oxidase FAD-binding motif GXXGXG was found at the N-terminal end, the other regions of the protein shared little identity with these known proteins (Fig. 2). Previous studies reported that some nicotine-degrading enzymes exhibit substrate specificity. For example, the activity of 6HLNO from A. nicotinovorans pAO1 is specific for 6HN, resulting in the production of 6HPON (29). NicA from P. putida S16 is specific for nicotine, resulting in SP (16). NOX from Pseudomonas sp. HZN6 can only convert nicotine into PN (21). In contrast to the proteins described above, NctB could mediate the dehydrogenation of 6HN and nicotine, yielding the products 6HMM and NMM, which were spontaneously hydrolyzed into 6HPON and PN, respectively (Fig. 1). Although the enzymatic affinity and catalytic efficiency of NctB toward nicotine were relatively lower than those of 6HN, this oxidase is different from any other reported nicotine-degrading related enzymes. The nctB-disrupted mutant strains (N7-M9 and N7-ΔnctB) lost the ability to degrade 6HN, and their complementary strains (N7-M9-Com and N7-ΔnctB-Com) restored this ability. These results reveal that NctB is a novel enzyme responsible for 6HN degradation in Shinella sp. HZN7.

In our previous study, strain HZN7 was determined to employ a variant of the pyridine and pyrrolidine pathways: nicotine, 6HN, 6HMM, 6HPON, HSP, and 2,5-DHP (Fig. 1) (23). The first three intermediates, 6HN, 6HMM, and 6HPON, belong to the pyridine pathway used by A. nicotinovorans pAO1 (8), while the last two intermediates, HSP and 2,5-DHP, are involved in the pyrrolidine pathway utilized by P. putida S16 (32) and Pseudomonas sp. HZN6 (20). Additionally, strain HZN7 can completely degrade PN via the pathway PN, 6HPON, HSP, and 2,5-DHP (Fig. 1) (23). It appears as though only one conversion, from nicotine to PN, resulted in a novel variant degradation pathway: nicotine, NMM, PN, 6HPON, HSP, and 2,5-DHP. Interestingly, in this study, the purified NctB was able to convert nicotine into PN in vitro, thereby seemingly closing the gap between nicotine and PN. However, neither NMM nor PN was detected in the samples degraded by strain HZN7 or its derivative strains. Moreover, the Km value of NctB for nicotine was 105-fold higher than that for 6HN, whereas the catalytic efficiency for 6HN was 1,941-fold higher than that for nicotine. The two kinetic constants indicate that 6HN is the preferred substrate of NctB. Therefore, the latter variant nicotine utilization pathway seemingly does not exist at the strain level.

The 6HN degradation-deficient mutant N7-W3, which has a disrupted tnp2 gene, exhibited characteristics different from those of the N7-M9 mutant. We believe that mutant N7-W3 most likely lost the nicotine degradation genes from the genome during transposon mutagenesis, except for the gene(s) responsible for the first step of the pathway. This was supported by the facts that (i) mutant M7-W3 could only hydrolyze nicotine to 6HN, but it could not degrade any of the intermediates after 6HN in the pathway, (ii) the tnp2-disrupted mutant N7-Δtnp2 could still degrade nicotine and the other five intermediates, and (iii) the nctB gene fragment could not be amplified from the genomic DNA of N7-W3, but it could be obtained from N7-Δtnp2 (see Fig. S2 and S4 in the supplemental material). The tnp2 gene encodes a putative transposase that shares low identities (approximately 50 to 60%) with members of the IS110 family of proteins. This type of transposase may be responsible for the horizontal gene transfer (33, 34). The disruption of the tnp2 gene by transposon mutagenesis might result in the loss of the nicotine-degrading gene cluster. However, the other tnp2-disrupted mutant, N7-Δtnp2, which was constructed using a two-step homologous recombination method, did not show the same characteristics as N7-W3 but showed no differences from wild-type strain HZN7. The differences between N7-W3 and N7-Δtnp2 suggest that the generation of mutant N7-W3 by random transposon mutagenesis and its loss of genetic information were accidental events.

Previous studies have focused on the biodegradation of (S)-nicotine, whereas (R)-nicotine degradation has been poorly studied. It has been reported that nicotine dehydrogenase (NDH) from A. nicotinovorans pAO1 exhibits no stereoselectivity and is active against both enantiomers of nicotine, resulting in 6HN (8, 35). (R)-6HN and (S)-6HN are then specifically degraded by 6HLNO and 6-hydroxy-d-nicotine oxidase (36), respectively. Strain Pseudomonas sp. HZN6 can degrade both the R and S enantiomers of nicotine into PN (21), whereas strain P. putida NRRL B-8061 can degrade only (S)-nicotine (37). It is still not clear whether (R)-nicotine can be degraded by strain P. putida S16 (14) or A. tumefaciens S33 (22). In this study, the degradation profiles of nicotine and 6HN were further investigated at the enantiomeric level. First, similar to strain A. nicotinovorans pAO1, strain HNZ7 could convert (R)- and (S)-nicotine into (R)- and (S)-6HN, respectively, which retained the configuration. Second, (S)-6HN could be completely degraded, but (R)-6HN could not be degraded anymore; this is different from the findings for other strains reported previously. Third, purified NctB showed no activity toward (R)-nicotine or (R)-6HN. These results suggest that strain HZN7 does not contain an enzyme that can deal with (R)-6HN. These results also reveal that different strains exhibit different degradation characteristics, resulting in a functional diversity in nicotine catabolism.

Taken together, this study identified a novel (S)-6HN oxidase, NctB, involved in nicotine degradation in Shinella sp. strain HNZ7. Purified NctB could oxidize (S)-6HN and (S)-nicotine into 6HPON and PN, respectively, which indicates a potential shunt in the nicotine degradation pathway. The data presented in this study provide us with the first insight into the nicotine catabolic mechanism at the enzymatic level in the new variant of the pyridine and pyrrolidine pathways. Further studies are needed to identify the genes or enzymes involved in the rest of the steps of nicotine degradation in Shinella sp. HNZ7.

Supplementary Material

Supplemental material
supp_80_18_5552__index.html (1.1KB, html)

ACKNOWLEDGMENTS

We are grateful to Roderich Brandsch (Institute of Biochemistry and Molecular Biology, University of Freiburg, Freiburg, Germany) for his kind provision of strain A. nicotinovorans pAO1.

This work was funded in the part by the National Natural Science Foundation of China (no. 21177112 and 21320102007) and the Ph.D. Programs Foundation of the Ministry of Education of China (no. 20120101110132).

Footnotes

Published ahead of print 7 July 2014

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.01312-14.

REFERENCES

  • 1.Benowitz NL. 2010. Nicotine addiction. N. Engl. J. Med. 362:2295–2303. 10.1056/NEJMra0809890 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Briški F, Horgas N, Vuković M, Gomzi Z. 2003. Aerobic composting of tobacco industry solid waste—simulation of the process. Clean Technol. Environ. Policy 5:295–301. 10.1007/s10098-003-0218-7 [DOI] [Google Scholar]
  • 3.Nielsen SS, Franklin GM, Longstreth W, Swanson PD, Checkoway H. 2013. Nicotine from edible Solanaceae and risk of Parkinson disease. Ann. Neurol. 74:472–477. 10.1002/ana.23884 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Jiang HJ, Ma Y, Qiu GJ, Wu FL, Chen SL. 2011. Biodegradation of nicotine by a novel strain Shinella sp. HZN1 isolated from activated sludge. J. Environ. Sci. Health B 46:703–708 [DOI] [PubMed] [Google Scholar]
  • 5.Raman G, Mohan K, Manohar V, Sakthivel N. 2014. Biodegradation of nicotine by a novel nicotine-degrading bacterium, Pseudomonas plecoglossicida TND35 and its new biotransformation intermediates. Biodegradation 25:95–107. 10.1007/s10532-013-9643-4 [DOI] [PubMed] [Google Scholar]
  • 6.Wang SN, Xu P, Tang HZ, Meng J, Liu XL, Ma CQ. 2005. “Green” route to 6-hydroxy-3-succinoyl-pyridine from (S)-nicotine of tobacco waste by whole cells of a Pseudomonas sp. Environ. Sci. Technol. 39:6877–6880. 10.1021/es0500759 [DOI] [PubMed] [Google Scholar]
  • 7.Schmid A, Dordick J, Hauer B, Kiener A, Wubbolts M, Witholt B. 2001. Industrial biocatalysis today and tomorrow. Nature 409:258–268. 10.1038/35051736 [DOI] [PubMed] [Google Scholar]
  • 8.Brandsch R. 2006. Microbiology and biochemistry of nicotine degradation. Appl. Microbiol. Biotechnol. 69:493–498. 10.1007/s00253-005-0226-0 [DOI] [PubMed] [Google Scholar]
  • 9.Chiribau CB, Mihasan M, Ganas P, Igloi GL, Artenie V, Brandsch R. 2006. Final steps in the catabolism of nicotine—deamination versus demethylation of gamma-N-methylaminobutyrate. FEBS J. 273:1528–1536. 10.1111/j.1742-4658.2006.05173.x [DOI] [PubMed] [Google Scholar]
  • 10.Baitsch D, Sandu C, Brandsch R, Igloi GL. 2001. Gene cluster on pAO1 of Arthrobacter nicotinovorans involved in degradation of the plant alkaloid nicotine: cloning, purification, and characterization of 2,6-dihydroxypyridine 3-hydroxylase. J. Bacteriol. 183:5262–5267. 10.1128/JB.183.18.5262-5267.2001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Igloi GL, Brandsch R. 2003. Sequence of the 165-kilobase catabolic plasmid pAO1 from Arthrobacter nicotinovorans and identification of a pAO1-dependent nicotine uptake system. J. Bacteriol. 185:1976–1986. 10.1128/JB.185.6.1976-1986.2003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Ganas P, Sachelaru P, Mihasan M, Igloi GL, Brandsch R. 2008. Two closely related pathways of nicotine catabolism in Arthrobacter nicotinovorans and Nocardioides sp. strain JS614. Arch. Microbiol. 189:511–517. 10.1007/s00203-007-0340-8 [DOI] [PubMed] [Google Scholar]
  • 13.Cobzaru C, Ganas P, Mihasan M, Schleberger P, Brandsch R. 2011. Homologous gene clusters of nicotine catabolism, including a new ω-amidase for α-ketoglutaramate, in species of three genera of Gram-positive bacteria. Res. Microbiol. 162:285–291. 10.1016/j.resmic.2011.01.001 [DOI] [PubMed] [Google Scholar]
  • 14.Tang H, Yao Y, Wang L, Yu H, Ren Y, Wu G, Xu P. 2012. Genomic analysis of Pseudomonas putida: genes in a genome island are crucial for nicotine degradation. Sci. Rep. 2:377. 10.1038/srep00377 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Tang H, Yao Y, Zhang D, Meng X, Wang L, Yu H, Ma L, Xu P. 2011. A novel NADH-dependent and FAD-containing hydroxylase is crucial for nicotine degradation by Pseudomonas putida. J. Biol. Chem. 286:39179–39187. 10.1074/jbc.M111.283929 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Tang HZ, Wang LJ, Meng XZ, Ma LY, Wang SN, He XF, Wu G, Xu P. 2009. Novel nicotine oxidoreductase-encoding gene involved in nicotine degradation by Pseudomonas putida strain S16. Appl. Environ. Microbiol. 75:772–778. 10.1128/AEM.02300-08 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Tang HZ, Wang SN, Ma LY, Meng XZ, Deng ZX, Zhang D, Ma CQ, Xu P. 2008. A novel gene, encoding 6-hydroxy-3-succinoylpyridine hydroxylase, involved in nicotine degradation by Pseudomonas putida strain S16. Appl. Environ. Microbiol. 74:1567–1574. 10.1128/AEM.02529-07 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Tang H, Wang L, Wang W, Yu H, Zhang K, Yao Y, Xu P. 2013. Systematic unraveling of the unsolved pathway of nicotine degradation in Pseudomonas. PLoS Genet. 9:e1003923. 10.1371/journal.pgen.1003923 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Qiu J, Ma Y, Chen L, Wu L, Wen Y, Liu W. 2011. A sirA-like gene, sirA2, is essential for 3-succinoyl-pyridine metabolism in the newly isolated nicotine-degrading Pseudomonas sp. HZN6 strain. Appl. Microbiol. Biotechnol. 92:1023–1032. 10.1007/s00253-011-3353-9 [DOI] [PubMed] [Google Scholar]
  • 20.Qiu J, Ma Y, Wen Y, Chen L, Wu L, Liu W. 2012. Functional identification of two novel genes from Pseudomonas sp. strain HZN6 involved in the catabolism of nicotine. Appl. Environ. Microbiol. 78:2154–2160. 10.1128/AEM.07025-11 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Qiu J, Ma Y, Zhang J, Wen Y, Liu W. 2013. Cloning of a novel nicotine oxidase gene from Pseudomonas sp. strain HZN6 whose product nonenantioselectively degrades nicotine to pseudooxynicotine. Appl. Environ. Microbiol. 79:2164–2171. 10.1128/AEM.03824-12 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Wang SN, Huang H, Xie K, Xu P. 2012. Identification of nicotine biotransformation intermediates by Agrobacterium tumefaciens strain S33 suggests a novel nicotine degradation pathway. Appl. Microbiol. Biotechnol. 95:1567–1578. 10.1007/s00253-012-4007-2 [DOI] [PubMed] [Google Scholar]
  • 23.Ma Y, Wei Y, Qiu J, Wen R, Hong J, Liu W. 2014. Isolation, transposon mutagenesis, and characterization of the novel nicotine-degrading strain Shinella sp. HZN7. Appl. Microbiol. Biotechnol. 98:2625–2636. 10.1007/s00253-013-5207-0 [DOI] [PubMed] [Google Scholar]
  • 24.Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY [Google Scholar]
  • 25.Wang SM, He J, Cui ZL, Li SP. 2007. Self-formed adaptor PCR: a simple and efficient method for chromosome walking. Appl. Environ. Microbiol. 73:5048–5051. 10.1128/AEM.02973-06 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254. 10.1016/0003-2697(76)90527-3 [DOI] [PubMed] [Google Scholar]
  • 27.Atkin KE, Reiss R, Koehler V, Bailey KR, Hart S, Turkenburg JP, Turner NJ, Brzozowski AM, Grogan G. 2008. The structure of monoamine oxidase from Aspergillus niger provides a molecular context for improvements in activity obtained by directed evolution. J. Mol. Biol. 384:1218–1231. 10.1016/j.jmb.2008.09.090 [DOI] [PubMed] [Google Scholar]
  • 28.Kachalova GS, Bourenkov GP, Mengesdorf T, Schenk S, Maun HR, Burghammer M, Riekel C, Decker K, Bartunik HD. 2010. Crystal structure analysis of free and substrate-bound 6-hydroxy-l-nicotine oxidase from Arthrobacter nicotinovorans. J. Mol. Biol. 396:785–799. 10.1016/j.jmb.2009.12.009 [DOI] [PubMed] [Google Scholar]
  • 29.Kachalova GS, Decker K, Holt A, Bartunik HD. 2011. Crystallographic snapshots of the complete reaction cycle of nicotine degradation by an amine oxidase of the monoamine oxidase (MAO) family. Proc. Natl. Acad. Sci. U. S. A. 108:4800–4805. 10.1073/pnas.1016684108 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Kopacz MM, Rovida S, van Duijn E, Fraaije MW, Mattevi A. 2011. Structure-based redesign of cofactor binding in putrescine oxidase. Biochemistry 50:4209–4217. 10.1021/bi200372u [DOI] [PubMed] [Google Scholar]
  • 31.Mirza IA, Burk DL, Xiong B, Iwaki H, Hasegawa Y, Grosse S, Lau PC, Berghuis AM. 2013. Structural analysis of a novel cyclohexylamine oxidase from Brevibacterium oxydans IH-35A. PLoS One 8:e60072. 10.1371/journal.pone.0060072 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Wang SN, Liu Z, Tang HZ, Meng J, Xu P. 2007. Characterization of environmentally friendly nicotine degradation by Pseudomonas putida biotype A strain S16. Microbiology 153:1556–1565. 10.1099/mic.0.2006/005223-0 [DOI] [PubMed] [Google Scholar]
  • 33.Choi S, Ohta S, Ohtsubo E. 2003. A novel IS element, IS621, of the IS110/IS492 family transposes to a specific site in repetitive extragenic palindromic sequences in Escherichia coli. J. Bacteriol. 185:4891–4900. 10.1128/JB.185.16.4891-4900.2003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Mahillon J, Chandler M. 1998. Insertion sequences. Microbiol. Mol. Biol. Rev. 62:725–774 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Grether-Beck S, Igloi GL, Pust S, Schilz E, Decker K, Brandsch R. 1994. Structural analysis and molybdenum-dependent expression of the pAO1-encoded nicotine dehydrogenase genes of Arthrobacter nicotinovorans. Mol. Microbiol. 13:929–936. 10.1111/j.1365-2958.1994.tb00484.x [DOI] [PubMed] [Google Scholar]
  • 36.Koetter JW, Schulz GE. 2005. Crystal structure of 6-hydroxy-d-nicotine oxidase from Arthrobacter nicotinovorans. J. Mol. Biol. 352:418–428. 10.1016/j.jmb.2005.07.041 [DOI] [PubMed] [Google Scholar]
  • 37.DeTraglia MC, Tometsko AM. 1980. Separation of d-(+)-nicotine from a racemic mixture by stereospecific degradation of the l-(−) isomer with Pseudomonas putida. Appl. Environ. Microbiol. 39:1067–1069 [DOI] [PMC free article] [PubMed] [Google Scholar]

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