Nicotine Dehydrogenase Research Articles

Bacillus sp. YC7 from intestines of Lasioderma serricorne degrades nicotine due to nicotine dehydrogenase

A large number of nicotine-containing wastes produced during the tobacco manufacturing process are seriously harmful to the environment and human health. The degradation and transformation of nicotine-containing environmental contaminants to harmless substances has become an urgent requirement. Lasioderma serricorne can grow and reproduce in nicotine-rich sources, and their intestinal microbiota show promising potential to degrade and utilize nicotine. The purpose of this study is to screen and identify nicotine-degrading bacteria from the intestines of L. serricorne and explore their degradation characteristics. A dominant strain, YC7, with significant nicotine degradation capabilities was isolated from the intestines of L. serricorne. The strain was identified as Bacillus using a polyphasic approach. The test results showed it can produce multiple enzymes that include β-glucosidase, cellulase, proteases, and amylases. The nicotine-degrading bacteria were functionally annotated using databases. Nicotine dehydrogenase (NDH) was found by combining an activity tracking test and protein mass spectrometry analysis. The YC-7 NDH in the pathway was molecularly docked and functionally verified via the gene knockdown method. The binding ability of nicotine to nicotine-degrading enzymes was investigated using molecular docking. A high-efficiency nicotine-degrading bacteria, YC-7, was isolated and screened from tobacco, and the gene functions related to degradation were verified. This investigation provides a new hypothesis for screening nicotine-degrading bacteria and increases our knowledge of potential nicotine-degrading microbial sources.

Function Enhancement of a Metabolic Module via Endogenous Promoter Replacement for Pseudomonas sp. JY-Q to Degrade Nicotine in Tobacco Waste Treatment.

Nicotine-degrading Pseudomonas sp. JY-Q is a preferred strain utilized in reconstituted tobacco process for tobacco waste treatment. However, its efficiency of nicotine metabolism still requires to be improved via genomic technology such as promoter engineering based on genomic information. Concerning upstream module of nicotine metabolic pathway, we found that two homologous genes of nicotine dehydrogenase (nicA2 and nox) coexisted in strain JY-Q. However, the transcriptional amount of nox was 20-fold higher than that of nicA2. Thus, the nicA2 expression required improvement. Combinatorial displacement was accomplished for two predicted endogenous promoters, named as PnicA2 and Pnox for nicA2 and nox, respectively. The mutant with Pnox as the promoters for both nicA2 and nox exhibited the best nicotine metabolic capacity which increased by 66% compared to the wild type. These results suggested that endogenous promoter replacement is also feasible for function improvementof metabolic modules and strain enhancement of biodegradation capacity to meet real environment demand.

Periplasmic Nicotine Dehydrogenase NdhAB Utilizes Pseudoazurin as Its Physiological Electron Acceptor in Agrobacterium tumefaciens S33.

Agrobacterium tumefaciens S33 can grow with nicotine as the sole source of carbon, nitrogen, and energy via a novel hybrid of the pyridine pathway and the pyrrolidine pathway. Characterization of the enzymes involved in the hybrid pathway is important for understanding its biochemical mechanism. Here, we report that the molybdenum-containing nicotine dehydrogenase (NdhAB), which catalyzes the initial step of nicotine degradation, is located in the periplasm of strain S33, while the 6-hydroxynicotine oxidase and 6-hydroxypseudooxynicoine oxidase are in the cytoplasm. This is consistent with the fact that NdhA has a Tat signal peptide. Interestingly, an open reading frame (ORF) adjacent to the ndhAB gene was verified to encode a copper-containing electron carrier, pseudoazurin (Paz), which has a signal peptide typical of bacterial Paz proteins. Both were transported into the periplasm after being produced in the cytoplasm. We purified NdhAB from the periplasmic fraction of strain S33 and found that with Paz as the physiological electron acceptor, NdhAB catalyzed the hydroxylation of nicotine at a specific rate of 110.52 ± 8.09 μmol · min-1 · mg of protein-1, where the oxygen atom in the hydroxyl group of the product 6-hydroxynicotine was derived from H2O. The apparent Km values for nicotine and Paz were 1.64 ± 0.07 μM and 3.61 ± 0.23 μM, respectively. NAD(P)+, O2, and ferredoxin could not serve as electron acceptors. Disruption of the paz gene disabled the strain for nicotine degradation, indicating that Paz is required for nicotine catabolism in the strain. These findings help our understanding of electron transfer during nicotine degradation in bacteria.IMPORTANCE Nicotine is a toxic and addictive N-heterocyclic aromatic alkaloid produced in tobacco. Its catabolism in organisms and degradation in tobacco wastes have become major concerns for human health and the environment. Bacteria usually decompose nicotine using the classical strategy of hydroxylating the pyridine ring with the help of activated oxygen by nicotine dehydrogenase, which binds one molybdopterin, two [2Fe2S] clusters, and usually one flavin adenine dinucleotide (FAD) as well. However, the physiological electron acceptor for the reaction is still unknown. In this study, we found that the two-component nicotine dehydrogenase from Agrobacterium tumefaciens S33, naturally lacking an FAD-binding domain, is located in the periplasmic space and uses a copper-containing electron carrier, pseudoazurin, as its physiological electron acceptor. We report here the role of pseudoazurin in a reaction catalyzed by a molybdopterin-containing hydroxylase occurring in the periplasmic space. These results provide new biochemical knowledge on microbial degradation of N-heterocyclic aromatic compounds.

Two Novel Sets of Genes Essential for Nicotine Degradation by Sphingomonas melonis TY.

Nicotine is a type of environmental pollutant present in the tobacco waste that is generated during tobacco manufacturing. Sphingomonas melonis TY can utilize nicotine as a sole source of carbon, nitrogen and energy via a variant of the pyridine and pyrrolidine pathway (the VPP pathway). In this study, we report the identification of two novel sets of genes, ndrA1A2A3, and ndrB1B2B3B4, which are crucial for nicotine degradation by strain TY. ndrA1A2A3 and ndrB1B2B3B4 exhibit similarity with both nicotine dehydrogenase ndh from Arthrobacter nicotinovorans and nicotine hydroxylase vppA from Ochrobactrum sp. SJY1. The transcriptional levels of ndrA1A2A3 and ndrB1B2B3B4 in strain TY were significantly upregulated in the presence of nicotine. Furthermore, ndrA1 or ndrB2 knockout resulted in a loss of the ability to degrade nicotine, whereas gene complementation restored the capacity of each mutant to utilize nicotine for growth. Biodegradation assays indicated that the mutant strains retained the ability to degrade the first intermediate in the pathway, 6-hydroxynicotine (6 HN). However, heterologous expression of ndrA1A2A3 and ndrB1B2B3B4 did not confer nicotine dehydrogenase activity to E. coli DH5α, Pseudomonas putida KT2440 or Sphingomonas aquatilis. These results provide information on the VPP pathway of nicotine degradation in S. melonis TY, and we conclude that these two sets of genes have essential functions in the conversion of nicotine to 6 HN in strain TY.

Biodegradation and metabolic pathway of nicotine in Rhodococcus sp. Y22.

Nicotine in tobacco is harmful to health and the environment, so there is an environmental requirement to remove nicotine from tobacco and tobacco wastes. In this study, the biotransformation of nicotine by Rhodococcus sp. Y22 was investigated, and three metabolites (NIC1, NIC4 and NIC5) were isolated by column separation, preparative TLC and solid plate's method, respectively. NIC1 was identified as 6-hydoxynicotine based on the results of NMR, MS, HPLC-UV and HRESIMS analysis; NIC4 was a novel compound and identified as 5-(3-methyl-[1,3]oxazinan-2-ylidene)-5H-pyridin-2-one based on the results of NMR, MS and UV analysis; NIC5 was identified as nicotine blue based on the results of NMR and MS analysis. Meanwhile, two metabolites NIC2 and NIC3 were identified as 6-hydroxy-N-methylmyosmine and 6-hydroxypseudooxynicotine by HRESIMS analysis, respectively. According to these metabolites, the possible pathway of nicotine degradation by Rhodococcus sp. Y22 was proposed. The nicotine can be transformed to nicotine blue through two pathways (A and B), and 6-hydroxy-N-methylmyosmine is the key compound, which can be converted to 6-hydroxypseudooxynicotine (pathway A) and 5-(3-methyl-[1,3]oxazinan-2-ylidene)-5H-pyridin-2-one (pathway B), respectively. Moreover, the encoding gene of nicotine dehydrogenase, ndh, was amplified from Rhodococcus sp. Y22, and its transcriptional level could be up-regulated obviously under nicotine induction. Our studies reported the key metabolites and possible biotransformation pathway of nicotine in Rhodococcus sp. Y22, and provided new insights into the microbial metabolism of nicotine.

Conversion of nornicotine to 6-hydroxy-nornicotine and 6-hydroxy-myosmine by Shinella sp. strain HZN7.

Nornicotine is a natural alkaloid produced by plants in the genus Nicotiana and is structurally related to nicotine. Importantly, nornicotine is the direct precursor of tobacco-specific nitrosamine N'-nitrosonornicotine, which is a highly potent human carcinogen. Microbial detoxification and degradation of nicotine have been well characterized; however, until now, there has been no information on the molecular mechanism of nornicotine degradation. In this study, we demonstrate the transformation of nornicotine by the nicotine-degrading strain Shinella sp. HZN7. Three transformation products were identified as 6-hydroxy-nornicotine, 6-hydroxy-myosmine, and 6-hydroxy-pseudooxy-nornicotine by UV spectroscopy, high-resolution mass spectrometry, nuclear magnetic resonance, and Fourier transform-infrared spectroscopy analyses. The two-component nicotine dehydrogenase genes nctA1 and nctA2 were cloned, and their product, NctA, was confirmed to be responsible for the conversion of nornicotine into 6-hydroxy-nornicotine as well as nicotine into 6-hydroxy-nicotine. The 6-hydroxy-nicotine oxidase, NctB, catalyzed the oxidation of 6-hydroxy-nornicotine to 6-hydroxy-myosmine, and it spontaneously hydrolyzed into 6-hydroxy-pseudooxy-nornicotine. However, 6-hydroxy-pseudooxy-nornicotine could not be further degraded by strain HZN7. This study demonstrated that nornicotine is partially transformed by strain HZN7 via nicotine degradation pathway.

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

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 ∼26 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.

Identification of nicotine biotransformation intermediates by Agrobacterium tumefaciens strain S33 suggests a novel nicotine degradation pathway

Nicotine, a major alkaloid in tobacco plants and the main toxic chemical in tobacco wastes, can be transformed by bacteria into hydroxylated-pyridine intermediates, which are important precursors for the chemical synthesis of valuable drugs and insecticides. Such biotransformation could be a useful approach to utilize tobacco and its wastes. In this study, we explored nicotine degradation by a recently isolated Agrobacterium tumefaciens S33 by identifying the intermediates during its growth on nicotine and during transformation of nicotine with its resting cells. Five hydroxylated-pyridine intermediates were detected through multiple approaches, including GC-HR-MS, HPLC, and ESI-Q-TOF MS analyses. Surprisingly, these identified intermediates suggest that strain S33 employs a novel pathway that is different from the two characterized pathways described in Arthrobacter and Pseudomonas. Based on these findings, we propose that strain S33 is able to transform nicotine to 6-hydroxy-pseudooxynicotine first via the pyridine pathway through 6-hydroxy-L-nicotine and 6-hydroxy-N-methylmyosmine, and then, it turns to the pyrrolidine pathway with the formation of 6-hydroxy-3-succinoylpyridine and 2,5-dihydroxypyridine. The activities of the key enzymes, nicotine dehydrogenase, 6-hydroxy-L-nicotine oxidase, and 6-hydroxy-3-succinoylpyridine hydroxylase, were demonstrated in the cell extract of strain S33 and by partially enriched enzymes. Moreover, the cell extract could transform 6-hydroxy-pseudooxynicotine into 6-hydroxy-3-succinoylpyridine by coupling with 6-hydroxy-L-nicotine oxidation reaction by 6-hydroxy-L-nicotine oxidase. These results indicated that strain S33 can transform nicotine into renewable hydroxylated-pyridine intermediates by the special pathway, in which at least three intermediates, 6-hydroxy-L-nicotine, 6-hydroxy-3-succinoylpyridine, and 2,5-dihydroxypyridine, have potential to be further chemically modified into useful compounds.

Uptake of l-nicotine and of 6-hydroxy-l-nicotine by Arthrobacter nicotinovorans and by Escherichia coli is mediated by facilitated diffusion and not by passive diffusion or active transport

The mechanism by which l-nicotine is taken up by bacteria that are able to grow on it is unknown. Nicotine degradation by Arthrobacter nicotinovorans, a Gram-positive soil bacterium, is linked to the presence of the catabolic megaplasmid pAO1. l-[(14)C]Nicotine uptake assays with A. nicotinovorans showed transport of nicotine across the cell membrane to be energy-independent and saturable with a K(m) of 6.2+/-0.1 microM and a V(max) of 0.70+/-0.08 micromol min(-1) (mg protein)(-1). This is in accord with a mechanism of facilitated diffusion, driven by the nicotine concentration gradient. Nicotine uptake was coupled to its intracellular degradation, and an A. nicotinovorans strain unable to degrade nicotine (pAO1(-)) showed no nicotine import. However, when the nicotine dehydrogenase genes were expressed in this strain, import of l-[(14)C]nicotine took place. A. nicotinovorans pAO1(-) and Escherichia coli were also unable to import 6-hydroxy-l-nicotine, but expression of the 6-hydroxy-l-nicotine oxidase gene allowed both bacteria to take up this compound. l-Nicotine uptake was inhibited by d-nicotine, 6-hydroxy-l-nicotine and 2-amino-l-nicotine, which may indicate transport of these nicotine derivatives by a common permease. Attempts to correlate nicotine uptake with pAO1 genes possessing similarity to amino acid transporters failed. In contrast to the situation at the blood-brain barrier, nicotine transport across the cell membrane by these bacteria was not by passive diffusion or active transport but by facilitated diffusion.

Gene structures and properties of enzymes of the plasmid-encoded nicotine catabolism of Arthrobacter nicotinovorans

Arthrobacter nicotinovorans is a Gram-positive aerobic soil bacterium able to grow on nicotine as its sole source of carbon and nitrogen. The initial steps of nicotine catabolism are catalyzed by nicotine dehydrogenase, the l- and d-specific 6-hydroxynicotine oxidases, and ketone dehydrogenase. The genes encoding these enzymes reside on a 160 kb plasmid, pAO1. The cccDNA of this plasmid was isolated in high purity and reasonable yield. It served as template material for the construction of a λ-phage DNA library of the plasmid. The genes coding for 6-hydroxy-l-nicotine oxidase and for the subunits of the heterotrimeric ketone dehydrogenase were identified, subcloned and sequenced. The 6-hlno gene was identified as a 1278 bp open reading frame; its regulatory elements were also recognized. The derived primary structure of the monomer of apo-6-hydroxy-l-nicotine oxidase (46,264.5 Da) agrees with the data obtained by partial amino acid sequencing. 6-Hydroxy-l-nicotine oxidase and 6-hydroxy-d-nicotine oxidase were expressed in Escherichia coli and obtained in a state of high purity and crystallized.Ketone dehydrogenase (KDH) was found to be a heterotrimer with subunits of molecular mass 89,021.71, 26,778.65 and 17,638.88. The genes of KDH-A and KDH-B are juxtaposed; the A of the stop codon of KDH-A is used in the start codon of KDH-B, eliciting a frame shift. KDH-C is separated from KDH-A by 281 bp.

IS1473, a Putative Insertion Sequence Identified in the Plasmid pAO1 fromArthrobacter nicotinovorans:Isolation, Characterization, and Distribution amongArthrobacterSpecies

A putative insertion sequence (IS1473) has been cloned and sequenced. The 1087-bp element was found between themoaAand thendhAgenes in the upstream region of the nicotine dehydrogenase (ndh) operon in the 160-kb pAO1 plasmid ofArthrobacter nicotinovorans.It is flanked by an imperfect repeat of 33 bp and carries two overlapping open reading frames which, by programmed −1 translational frameshifting, may produce a transposase of 36.735 Da with a pI= 10.18. The deduced protein is similar to the transposases IS481 and IS1002 fromBordetellaand IS476 fromXanthomonas campestris,all members of the IS3 family. The putative insertion element was found as a single copy in the pAO1 plasmid and absent on the chromosome of theA. nicotinovoransgenome. Similar sequences were detected by hybridization on total DNA fromArthrobacter globiformis, Arthrobacter ramosus,andArthrobacter ureafaciens.

A pAO1-encoded molybdopterin cofactor gene (moaA) of Arthrobacter nicotinovorans: characterization and site-directed mutagenesis of the encoded protein.

A gene homologous to moaA, the gene responsible for the expression of a protein involved in an early step in the synthesis of the molybdopterin cofactor of Escherichia coli, was found to be located 2.7-kb upstream of the nicotine dehydrogenase (ndh) operon on the catabolic plasmid pAO1 of Arthrobacter nicotinovorans. The MoaA protein, containing 354 amino acids, migrated on an SDS-polyacrylamide gel with an apparent molecular weight of 40,000, in good agreement with the predicted molecular weight of 38,880. The pAO1-encoded moaA gene from A. nicotinovorans was expressed in E. coli as an active protein that functionally complemented moaA mutants. Its deduced amino acid sequence shows 43% identity to the E. coli MoaA, 44% to the NarAB gene product from Bacillus subtilis, and 42% to the gene product of two contiguous ORFs from Methanobacterium formicicum. N-terminal sequences, including the motif CxxxCxYC, are conserved among the MoaA and NarAB proteins. This motif is also present in proteins involved in PQQ cofactor synthesis in almost all the NifB proteins reported so far and in the fixZ gene product from Rhizobium leguminosarum. Mutagenesis of any of these three conserved cysteine residues to serine abolished the biological activity of MoaA, while substitution of the tyrosine by either serine, phenylalanine, or alanine did not alter the capacity of the protein to complement the moaA mutation in E. coli. A second Cys-rich domain with the motif FCxxC(13x)C is found close to the C-terminus of MoaA and NarAB proteins. These two Cys-rich sequences may be involved in the coordination of a metal ions. The pAO1 copy of moaA may not be unique in the A. nicotinovorans genome since the molybdopterin cofactor oxidation products were detected in cell extracts from a plasmidless strain.

Riboflavin-dependent expression of flavoenzymes of the nicotine regulon of Arthrobacter oxidans

In cells of an Arthrobacter oxidans riboflavin-dependent mutant the specific activity of the DL-nicotine-inducible nicregulon enzymes nicotine dehydrogenase (NDH, EC 1.5.99.4), 6-hydroxy-L-nicotine oxidase (6-HLNO, EC 1.5.3.5) and 6-hydroxy-D-nicotine oxidase (6-HDNO, EC 1.5.3.6) was shown to be dependent on the supply of the vitamin in the growth medium. Experiments designed to identify at which level riboflavin directs the biosynthesis of these flavoenzymes revealed that the steady-state levels of enzyme protein analysed on Western blots correlated directly with riboflavin supply from the minimal concentration of 0.5 microns-riboflavin required for growth up to 8 microns-riboflavin. Mutant cells grown at the higher riboflavin concentration showed on dot-blots increased levels of RNA which hybridized to 32P-labelled probes derived from the nic-regulon genes. When cells grown at 2 microns-riboflavin were shifted to 8 microns-riboflavin, 6-HDNO expression increased as indicated by elevated enzyme and RNA levels. When the rates of synthesis of the 6-HDNO and 6-HLNO polypeptides after DL-nicotine induction was analysed in cells grown at 0.5 microns and 8 microns-riboflavin, only cells grown at the higher riboflavin concentration showed on Western blots an accumulation of the polypeptides. No 6-HDNO or 6-HLNO polypeptide was identified in cell extracts from cells grown on 0.5 microns-riboflavin. Pulse-chase experiments with [35S]methionine showed that 6-HDNO- and 6-HLNO synthesis was prevented in cells grown at the low riboflavin concentration. The absence of detectable enzyme levels seemed not to be caused by proteolytic breakdown. Incubation in vitro of apo-6HDNO with low- or high-riboflavin-grown-cell extracts showed no increased proteolytic activity in 0.5 microns-riboflavin-grown cells. From these results it is concluded that riboflavin supply co-regulates the expression of the nicregulon genes at the level of transcription and/or mRNA turnover.

Nicotine dehydrogenase from Arthrobacter oxidans: A molybdenum-containing hydroxylase

The nicotine dehydrogenase from Arthrobacter oxidans was purified 40-fold to homogeneity with 26% recovery. SDS-polyacrylamide gel electrophoresis of the enzyme revealed three protein bands corresponding to Mr of 82 000, 30 000 and 15 000. The Mr of the native enzyme was calculated to be 12 0000 by gel chromatography. The enzyme contained about 1 FAD, 1 molybdenum, 4 iron and 2 labile sulfur.

Nicotine dehydrogenase fromArthrobacter oxidans: A molybdenum-containing hydroxylase

The nicotine dehydrogenase from Arthrobacter oxidans was purified 40-fold to homogeneity with 26% recovery. SDS-polyacrylamide gel electrophoresis of the enzyme revealed three protein bands corresponding to Mr of 82 000, 30 000 and 15 000. The Mr of the native enzyme was calculated to be 12 0000 by gel chromatography. The enzyme contained about 1 FAD, 1 molybdenum, 4 iron and 2 labile sulfur.