Pyrrolidine Pathway Research Articles

Characterization of cotinine degradation in a newly isolated Gram-negative strain Pseudomonas sp. JH-2.

Cotinine, the primary metabolite of nicotine in the human body, is an emerging pollutant in aquatic environments. It causes environmental problems and is harmful to the health of humans and other mammals; however, the mechanisms of its biodegradation have been elucidated incompletely. In this study, a novel Gram-negative strain that could degrade and utilize cotinine as a sole carbon source was isolated from municipal wastewater samples, and its cotinine degradation characteristics and kinetics were determined. Pseudomonas sp. JH-2 was able to degrade 100mg/L (0.56 mM) of cotinine with high efficiency within 5 days at 30 ℃, pH 7.0, and 1% NaCl. Two intermediates, 6-hydroxycotinine and 6-hydroxy-3-succinoylpyridine (HSP), were identified by high-performance liquid chromatography and liquid chromatograph mass spectrometer. The draft whole genome sequence of strain JH-2 was obtained and analyzed to determine genomic structure and function. No homologs of proteins predicted in Nocardioides sp. JQ2195 and reported in nicotine degradation Pyrrolidine pathway were found in strain JH-2, suggesting new enzymes that responsible for cotinine catabolism. These findings provide meaningful insights into the biodegradation of cotinine by Gram-negative bacteria.

A multifunctional flavoprotein monooxygenase HspB for hydroxylation and C-C cleavage of 6-hydroxy-3-succinoyl-pyridine.

Flavoprotein monooxygenases catalyze reactions, including hydroxylation and epoxidation, involved in the catabolism, detoxification, and biosynthesis of natural substrates and industrial contaminants. Among them, the 6-hydroxy-3-succinoyl-pyridine (HSP) monooxygenase (HspB) from Pseudomonas putida S16 facilitates the hydroxylation and C-C bond cleavage of the pyridine ring in nicotine. However, the mechanism for biodegradation remains elusive. Here, we refined the crystal structure of HspB and elucidated the detailed mechanism behind the oxidative hydroxylation and C-C cleavage processes. Leveraging structural information about domains for binding the cofactor flavin adenine dinucleotide (FAD) and HSP substrate, we used molecular dynamics simulations and quantum/molecular mechanics calculations to demonstrate that the transfer of an oxygen atom from the reactive FAD peroxide species (C4a-hydroperoxyflavin) to the C3 atom in the HSP substrate constitutes a rate-limiting step, with a calculated reaction barrier of about 20 kcal/mol. Subsequently, the hydrogen atom was rebounded to the FAD cofactor, forming C4a-hydroxyflavin. The residue Cys218 then catalyzed the subsequent hydrolytic process of C-C cleavage. Our findings contribute to a deeper understanding of the versatile functions of flavoproteins in the natural transformation of pyridine and HspB in nicotine degradation.IMPORTANCEPseudomonas putida S16 plays a pivotal role in degrading nicotine, a toxic pyridine derivative that poses significant environmental challenges. This study highlights a key enzyme, HspB (6-hydroxy-3-succinoyl-pyridine monooxygenase), in breaking down nicotine through the pyrrolidine pathway. Utilizing dioxygen and a flavin adenine dinucleotide cofactor, HspB hydroxylates and cleaves the substrate's side chain. Structural analysis of the refined HspB crystal structure, combined with state-of-the-art computations, reveals its distinctive mechanism. The crucial function of Cys218 was never discovered in its homologous enzymes. Our findings not only deepen our understanding of bacterial nicotine degradation but also open avenues for applications in both environmental cleanup and pharmaceutical development.

Characterization on nicotine degradation and research on heavy metal resistance of a strain Pseudomonas sp. NBB

The remediation of polluted sites containing multiple contaminants like nicotine and heavy metals poses significant challenges, due to detrimental effects like cell death. In this study, we isolated a new strain Pseudomonas sp. NBB capable of efficiently degrading nicotine even in high level of heavy metals. It degraded nicotine through pyrrolidine pathway and displayed minimum inhibitory concentrations of 2 mM for barium, copper, and lead, and 5 mM for manganese. In the presence of 2 mM Ba2+ or Pb2+, 3 g L-1 nicotine could be completely degraded within 24 h. Moreover, under 0.5 mM Cu2+ or 5 mM Mn2+ stress, 24.13% and 72.56% of nicotine degradation were achieved in 60 h, respectively. Strain NBB tolerances metal stress by various strategies, including morphological changes, up-regulation of macromolecule transporters, cellular response to DNA damage, and down-regulation of ABC transporters. Notably, among the 153 up-regulated genes, cds_821 was identified as manganese exporter (MneA) after gene disruption and recovery experiments. This study presents a novel strain capable of efficiently degrading nicotine and displaying remarkable resistance to heavy metals. The findings of this research provide valuable insights into the potential application of nicotine bioremediation in heavy metal-contaminated areas.

Regulation Mechanism of Nicotine Catabolism in Sphingomonas melonis TY by a Dual Role Transcriptional Regulator NdpR.

A gene cluster ndp, responsible for nicotine degradation via a variant of the pyridine and pyrrolidine pathways, was previously identified in Sphingomonas melonis TY, but the regulation mechanism remains unknown. The gene ndpR within the cluster was predicted to encode a TetR family transcriptional regulator. Deletion of ndpR resulted in a notably shorter lag phase, higher maximum turbidity, and faster substrate degradation when cultivated in the presence of nicotine. Real-time quantitative PCR and promoter activity analysis in wild-type TY and TYΔndpR strains revealed that genes in the ndp cluster were negatively regulated by NdpR. However, complementation of ndpR to TYΔndpR did not restore transcription repression, but, instead, the complemented strain showed better growth than TYΔndpR. Promoter activity analysis indicates that NdpR also functions as an activator in the transcription regulation of ndpHFEGD. Further analysis through electrophoretic mobility shift assay and DNase I footprinting assay revealed that NdpR binds five DNA sequences within ndp and that NdpR has no autoregulation. These binding motifs overlap with the -35 or -10 box or are located distal upstream of the corresponding transcriptional start site. Multiple sequence alignment of these five NdpR-binding DNA sequences found a conserved motif, with two of the binding sequences being partially palindromic. 2,5-Dihydroxypyridine acted as a ligand of NdpR, preventing NdpR from binding to the promoter region of ndpASAL, ndpTB, and ndpHFEGD. This study revealed that NdpR binds to three promoters in the ndp cluster and is a dual-role transcriptional regulator in nicotine metabolism. IMPORTANCE Gene regulation is critical for microorganisms in the environment in which they may encounter various kinds of organic pollutants. Our study revealed that transcription of ndpASAL, ndpTB, and ndpHFEGD is negatively regulated by NdpR, and NdpR also exhibits a positive regulatory effect on PndpHFEGD. Furthermore, 2,5-dihydroxypyridine was identified as the effector molecular for NdpR and can both prevent the binding of free NdpR to the promoter and release NdpR from the promoters, which is different from previously reported NicR2. Additionally, NdpR was found to have both negative and positive transcription regulatory effects on the same target, PndpHFEGD, while only one binding site was identified, which is notably different from the previously reported TetR family regulators. Moreover, NdpR was revealed to be a global transcriptional regulator. This study provides new insight into the complex gene expression regulation of the TetR family.

Nicotine metabolism pathway in bacteria: mechanism, modification, and application.

Nicotine is a harmful pollutant mainly from the waste of tobacco factories. It is necessary to remove nicotine via high efficient strategies such as bioremediation. So far, an increasing number of nicotine degrading strains have been isolated. However, their degrading efficiency and tolerance to high content nicotine is still not high enough for application in real environment. Thus, the modification of nicotine metabolism pathway is obligated and requires comprehensive molecular insights into whole cell metabolism of nicotine degrading strains. Obviously, the development of multi-omics technology has accelerated the mechanism study on microbial degradation of nicotine and supplied more novel strategy of strains modification. So far, three pathways of nicotine degradation, pyridine pathway, pyrrolidine pathway, and the variant of pyridine and pyrrolidine pathway (VPP pathway), have been clearly identified in bacteria. Muti-omics analysis further revealed specific genome architecture, regulation mechanism, and specific genes or enzymes of three pathways, in different strains. Especially, muti-omics analysis revealed that functional modules coexisted in different genome loci and played additional roles on enhanced degradation efficiency in bacteria. Based on the above discovery, genomic editing strategy becomes more feasible to greatly improve bacterial degrading efficiency of nicotine.

Rid Enhances the 6-Hydroxypseudooxynicotine Dehydrogenase Reaction in Nicotine Degradation by Agrobacterium tumefaciens S33.

Agrobacterium tumefaciens S33 degrades nicotine through a hybrid of the pyridine and pyrrolidine pathways. The oxidation of 6-hydroxypseudooxynicotine to 6-hydroxy-3-succinoyl-semialdehyde-pyridine by 6-hydroxypseudooxynicotine dehydrogenase (Pno) is an important step in the breakdown of the N-heterocycle in this pathway. Although Pno has been characterized, the reaction is not fully understood; what is known is that it starts at a high speed followed by a rapid drop in the reaction rate, leading to the formation of a very small amount of product. In this study, we speculated that an unstable imine intermediate that is toxic with regard to the metabolism is produced in the reaction. We found that a Rid protein (designated Rid-NC) encoded by a gene in the nicotine-degrading gene cluster enhanced the reaction. Rid is a widely distributed family of small proteins with various functions, and some subfamilies have deaminase activity to eliminate the toxicity of the reactive intermediate, imine. Biochemical analyses showed that Rid-NC relieved the toxicity of the presumed imine intermediate produced in the Pno reaction and that, in the presence of Rid-NC, Pno maintained a high level of activity and the amount of the reaction product was increase by at least 5-fold. Disruption of the rid-NC gene led to slower growth of strain S33 on nicotine. The mechanism of Rid-NC-mediated detoxification of the imine intermediate was discussed. A phylogenetic analysis indicated that Rid-NC belongs to the rarely studied Rid6 subfamily. These results further our understanding of the biochemical mechanism of nicotine degradation and provide new insights into the function of the Rid6 subfamily proteins.IMPORTANCE Rid is a family of proteins that participate in metabolite damage repair and is widely distributed in different organisms. In this study, we found that Rid-NC, which belongs to the Rid6 subfamily, promoted the 6-hydroxypseudooxynicotine dehydrogenase (Pno) reaction in the hybrid of the pyridine and pyrrolidine pathways for nicotine degradation by Agrobacterium tumefaciens S33. Rid-NC hydrolyzed the presumed reactive imine intermediate produced in the reaction to remove its toxicity on Pno. The finding furthers our understanding of the metabolic process of the toxic N-heterocyclic aromatic compounds in microorganisms. This study demonstrated that the Rid family of proteins also functions in the metabolism of N-heterocyclic aromatic alkaloids, in addition to the amino acid metabolism, and that Rid6-subfamily proteins also have deaminase activity, similar to the RidA subfamily. The ability of reactive imines to damage a non-pyridoxal-5'-phosphate-dependent enzyme was reported. This study provides new insights into the function of the Rid family of proteins.

Physiology of a Hybrid Pathway for Nicotine Catabolism in Bacteria

Nicotine is a major N-heterocyclic aromatic alkaloid produced in tobacco plants and the main toxic chemical in tobacco waste. Due to its complex physiological effects and toxicity, it has become a concern both in terms of public health and the environment. A number of bacteria belonging to the genera Arthrobacter and Pseudomonas can degrade nicotine via the pyridine and pyrrollidine pathways. Recently, a novel hybrid of the pyridine and pyrrolidine pathways (also known as the VPP pathway) was found in the Rhizobiale group bacteria Agrobacterium tumefaciens S33, Shinella sp. HZN7 and Ochrobactrum sp. SJY1 as well as in other group bacteria. The special mosaic pathway has attracted much attention from microbiologists in terms of the study of their molecular and biochemical mechanisms. This will benefit the development of new biotechnologies in terms of the use of nicotine, the enzymes involved in its catabolism, and the microorganisms capable of degrading the alkaloid. In this pathway, some metabolites are hydroxylated in the pyridine ring or modified in the side chain with active groups, which can be used as precursors for the synthesis of some important compounds in the pharmaceutical and agricultural industries. Moreover, some enzymes may be used for industrial biocatalysis to transform pyridine derivatives into desired chemicals. Here, we review the molecular and biochemical basis of the hybrid nicotine-degrading pathway and discuss the electron transport in its oxidative degradation for energy conservation and bacterial growth.

Structural Insights into 6-Hydroxypseudooxynicotine Amine Oxidase from Pseudomonas geniculata N1, the Key Enzyme Involved in Nicotine Degradation.

Bacteria degrade nicotine mainly using pyridine and pyrrolidine pathways. Previously, we discovered a hybrid of the pyridine and pyrrolidine pathways (the VPP pathway) in Pseudomonas geniculata N1 and characterized its key enzyme, 6-hydroxypseudooxynicotine amine oxidase (HisD). It catalyzes oxidative deamination of 6-hydroxypseudooxynicotine to 6-hydroxy-3-succinoylsemialdehyde-pyridine, which is the crucial step connecting upstream and downstream portions of the VPP pathway. We determined the crystal structure of wild-type HisD to 2.6 Å. HisD is a monomer that contains a flavin mononucleotide, an iron-sulfur cluster, and ADP. On the basis of sequence alignment and structure comparison, a difference has been found among HisD, closely related trimethylamine dehydrogenase (TMADH), and histamine dehydrogenase (HADH). The flavin mononucleotide (FMN) cofactor is not covalently bound to any residue, and the FMN isoalloxazine ring is planar in HisD compared to TMADH or HADH, which forms a 6-S-cysteinyl flavin mononucleotide cofactor and has an FMN isoalloxazine ring in a "butterfly bend" conformation. Based on the structure, docking study, and site-directed mutagenesis, the residues Glu60, Tyr170, Asp262, and Trp263 may be involved in substrate binding. The expanded understanding of the substrate binding mode from this study may guide rational engineering of such enzymes for biodegradation of potential pollutants or for bioconversion to generate desired products.IMPORTANCE Nicotine is a major tobacco alkaloid in tobacco waste. Pyridine and pyrrolidine pathways are the two best-elucidated nicotine metabolic pathways; Pseudomonas geniculata N1 catabolizes nicotine via a hybrid between the pyridine and pyrrolidine pathways. The crucial enzyme, 6-hydroxypseudooxynicotine amine oxidase (HisD), links the upstream and downstream portions of the VPP pathway; however, there is little structural information about this important enzyme. In this study, we determined the crystal structure of HisD from Pseudomonas geniculata N1. Its basic insights about the structure may help us to guide the engineering of such enzymes for bioremediation and bioconversion applications.

6-Hydroxypseudooxynicotine Dehydrogenase Delivers Electrons to Electron Transfer Flavoprotein during Nicotine Degradation by Agrobacterium tumefaciens S33.

Agrobacterium tumefaciens S33 degrades nicotine via a novel hybrid of the pyridine and the pyrrolidine pathways. The hybrid pathway consists of at least six steps involved in oxidoreductive reactions before the N-heterocycle can be broken down. Collectively, the six steps allow electron transfer from nicotine and its intermediates to the final acceptor O2 via the electron transport chain (ETC). 6-Hydroxypseudooxynicotine oxidase, renamed 6-hydroxypseudooxynicotine dehydrogenase in this study, has been characterized as catalyzing the fourth step using the artificial electron acceptor 2,6-dichlorophenolindophenol. Here, we used biochemical, genetic, and liquid chromatography-mass spectrometry (LC-MS) analyses to determine that 6-hydroxypseudooxynicotine dehydrogenase utilizes the electron transfer flavoprotein (EtfAB) as the physiological electron acceptor to catalyze the dehydrogenation of pseudooxynicotine, an analogue of the true substrate 6-hydroxypseudooxynicotine, in vivo, into 3-succinoyl-semialdehyde-pyridine. NAD(P)+, O2, and ferredoxin could not function as electron acceptors. The oxygen atom in the aldehyde group of the product 3-succinoyl-semialdehyde-pyridine was verified to be derived from H2O. Disruption of the etfAB genes in the nicotine-degrading gene cluster decreased the growth rate of A. tumefaciens S33 on nicotine but not on 6-hydroxy-3-succinoylpyridine, an intermediate downstream of the hybrid pathway, indicating the requirement of EtfAB for efficient nicotine degradation. The electrons were found to be further transferred from the reduced EtfAB to coenzyme Q by the catalysis of electron transfer flavoprotein:ubiquinone oxidoreductase. These results aid in an in-depth understanding of the electron transfer process and energy metabolism involved in the nicotine oxidation and provide novel insights into nicotine catabolism in bacteria.IMPORTANCE Nicotine has been studied as a model for toxic N-heterocyclic aromatic compounds. Microorganisms can catabolize nicotine via various pathways and conserve energy from its oxidation. Although several oxidoreductases have been characterized to participate in nicotine degradation, the electron transfer involved in these processes is poorly understood. In this study, we found that 6-hydroxypseudooxynicotine dehydrogenase, a key enzyme in the hybrid pyridine and pyrrolidine pathway for nicotine degradation in Agrobacterium tumefaciens S33, utilizes EtfAB as a physiological electron acceptor. Catalyzed by the membrane-associated electron transfer flavoprotein:ubiquinone oxidoreductase, the electrons are transferred from the reduced EtfAB to coenzyme Q, which then could enter into the classic ETC. Thus, the route for electron transport from the substrate to O2 could be constructed, by which ATP can be further sythesized via chemiosmosis to support the baterial growth. These findings provide new knowledge regarding the catabolism of N-heterocyclic aromatic compounds in microorganisms.

Characterization of the ModABC Molybdate Transport System of Pseudomonas putida in Nicotine Degradation.

Pseudomonas putida J5 is an efficient nicotine-degrading bacterial strain that catabolizes nicotine through the pyrrolidine pathway. In our previous study, we used Tn5 transposon mutagenesis to investigate nicotine metabolism-associated genes, and 18 nicotine degradation-deficient mutants were isolated from 16,324 Tn5-transformants. Three of the mutants were Tn5 inserts into the modABC gene cluster that encoded an ABC-type, high-affinity, molybdate transporter. In-frame deletion of the modABC genes abolished the nicotine-degrading ability of strain J5, and complementation with modABC either from P. putida or Arthrobacter oxidans restored the degrading activity of the mutant to wild-type level. Nicotine degradation of J5 was inhibited markedly by addition of tungstate, a specific antagonist of molybdate. Molybdate at a non-physiologically high concentration (100 μM) fully restored nicotine-degrading activity and recovered growth of the modABC mutant in a nicotine minimal medium. Transcriptional analysis revealed that the expression of modABC was up-regulated at low molybdate concentrations and down-regulated at high moybdate concentrations, which indicated that at least one other system was able to transport molybdate, but with lower affinity. These results suggested that the molybdate transport system was essential to nicotine metabolism in P. putida J5.

The enzymes of microbial nicotine metabolism

Because of nicotine’s toxicity and the high levels found in tobacco and in the waste from tobacco processing, there is a great deal of interest in identifying bacteria capable of degrading it. A number of microbial pathways have been identified for nicotine degradation. The first and best-understood is the pyridine pathway, best characterized for Arthrobacter nicotinovorans, in which the first reaction is hydroxylation of the pyridine ring. The pyrrolidine pathway, which begins with oxidation of a carbon–nitrogen bond in the pyrrolidine ring, was subsequently characterized in a number of pseudomonads. Most recently, a hybrid pathway has been described, which incorporates the early steps in the pyridine pathway and ends with steps in the pyrrolidine pathway. This review summarizes the present status of our understanding of these pathways, focusing on what is known about the individual enzymes involved.

Characterization of Nicotine Catabolism through a Novel Pyrrolidine Pathway in Pseudomonas sp. S-1.

Nicotine is a major toxic alkaloid in wastes generated from tobacco production and cigarette manufacturing. In the present work, a nicotine-degrading bacterial strain was isolated from tobacco powdery waste. The isolate was identified as Pseudomonas sp. S-1 based on morphology, physiology, and 16S rRNA gene sequence. Suitable conditions of isolate S-1 for nicotine degradation were pH 7.0 and 30 °C. Catabolic intermediates of nicotine were isolated with preparative-HPLC and characterized with LC-HRMS and NMR. The catabolic pathways of nicotine were involved in dehydrogenation, oxidation, hydrolysis, and hydroxylation. Interestingly, nicotine catabolism in strain S-1 undergoes a new pyrrolidine pathway that differs from the other three catabolic pathways in bacterial species. This work sheds light on catabolic diversity of nicotine and heteroaromatics.

Green route to synthesis of valuable chemical 6-hydroxynicotine from nicotine in tobacco wastes using genetically engineered Agrobacterium tumefaciens S33

BackgroundTobacco is widely planted as an important nonfood economic crop throughout the world, and large amounts of tobacco wastes are generated during the tobacco manufacturing process. Tobacco and its wastes contain high nicotine content. This issue has become a major concern for health and environments due to its toxicity and complex physiological effects. The microbial transformation of nicotine into valuable functionalized pyridine compounds is a promising way to utilize tobacco and its wastes as a potential biomass resource. Agrobacterium tumefaciens S33 is able to degrade nicotine via a novel hybrid of the pyridine and pyrrolidine pathways, in which several intermediates, such as 6-hydroxynicotine, can be used as renewable precursors to synthesize drugs and insecticides. This provides an opportunity to produce valuable chemical 6-hydroxynicotine from nicotine via biocatalysis using strain S33.ResultsTo accumulate the intermediate 6-hydroxynicotine, we firstly identified the key enzyme decomposing 6-hydroxynicotine, named 6-hydroxynicotine oxidase, and then disrupted its encoding gene in A. tumefaciens S33. With the whole cells of the mutant as a biocatalyst, we tested the possibility to produce 6-hydroxynicotine from the nicotine of tobacco and its wastes and optimized the reaction conditions. At 30 °C and pH 7.0, nicotine could be efficiently transformed into 6-hydroxynicotine by the whole cells cultivated with glucose/ammonium/6-hydroxy-3-succinoylpyridine medium. The molar conversion and the specific catalytic rate reached approximately 98% and 1.01 g 6-hydroxynicotine h−1 g−1 dry cells, respectively. The product could be purified easily by dichloromethane extraction with a recovery of 76.8%, and was further confirmed by UV spectroscopy, mass spectroscopy, and NMR analysis.ConclusionsWe successfully developed a novel biocatalytic route to 6-hydroxynicotine from nicotine by blocking the nicotine catabolic pathway via gene disruption, which provides an alternative green strategy to utilize tobacco and its wastes as a biomass resource by converting nicotine into valuable hydroxylated-pyridine compounds.

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.

Genomic and transcriptomic analyses of Agrobacterium tumefaciens S33 reveal the molecular mechanism of a novel hybrid nicotine-degrading pathway

Agrobacterium tumefaciens S33 is able to degrade nicotine via a novel hybrid of the pyridine and pyrrolidine pathways. It can be utilized to remove nicotine from tobacco wastes and transform nicotine into important functionalized pyridine precursors for some valuable drugs and insecticides. However, the molecular mechanism of the hybrid pathway is still not completely clear. Here we report the genome analysis of strain S33 and its transcriptomes grown in glucose-ammonium medium and nicotine medium. The complete gene cluster involved in nicotine catabolism was found to be located on a genomic island composed of genes functionally similar but not in sequences to those of the pyridine and pyrrolidine pathways, as well as genes encoding plasmid partitioning and replication initiation proteins, conjugal transfer proteins and transposases. This suggests that the evolution of this hybrid pathway is not a simple fusion of the genes involved in the two pathways, but the result of a complicated lateral gene transfer. In addition, other genes potentially involved in the hybrid pathway could include those responsible for substrate sensing and transport, transcription regulation and electron transfer during nicotine degradation. This study provides new insights into the molecular mechanism of the novel hybrid pathway for nicotine degradation.

A model for evolution and regulation of nicotine biosynthesis regulon in tobacco

ABSTRACTIn tobacco, the defense alkaloid nicotine is produced in roots and accumulates mainly in leaves. Signaling mediated by jasmonates (JAs) induces the formation of nicotine via a series of structural genes that constitute a regulon and are coordinated by JA-responsive transcription factors of the ethylene response factor (ERF) family. Early steps in the pyrrolidine and pyridine biosynthesis pathways likely arose through duplication of the polyamine and nicotinamide adenine dinucleotide (NAD) biosynthetic pathways, respectively, followed by recruitment of duplicated primary metabolic genes into the nicotine biosynthesis regulon. Transcriptional regulation of nicotine biosynthesis by ERF and cooperatively-acting MYC2 transcription factors is implied by the frequency of cognate cis-regulatory elements for these factors in the promoter regions of the downstream structural genes. Indeed, a mutant tobacco with low nicotine content was found to have a large chromosomal deletion in a cluster of closely related ERF genes at the nicotine-controlling NICOTINE2 (NIC2) locus.

Characterization of a Novel Nicotine Degradation Gene Cluster ndp in Sphingomonas melonis TY and Its Evolutionary Analysis.

Sphingomonas melonis TY utilizes nicotine as a sole source of carbon, nitrogen, and energy through a variant of the pyridine and pyrrolidine pathways (VPP). A 31-kb novel nicotine-degrading gene cluster, ndp, in strain TY exhibited a different genetic organization with the vpp cluster in strains Ochrobactrum rhizosphaerae SJY1 and Agrobacterium tumefaciens S33. Genes in vpp were separated by a 20-kb interval sequence, while genes in ndp were localized together. Half of the homolog genes were in different locus in ndp and vpp. Moreover, there was a gene encoding putative transporter of nicotine or other critical metabolite in ndp. Among the putative nicotine-degrading related genes, the nicotine hydroxylase, 6-hydroxy-L-nicotine oxidase, 6-hydroxypseudooxynicotine oxidase, and 6-hydroxy-3-succinyl-pyridine monooxygenase responsible for catalyzing the transformation of nicotine to 2, 5-dihydropyridine in the initial four steps of the VPP were characterized. Hydroxylation at C6 of the pyridine ring and dehydrogenation at the C2–C3 bond of the pyrrolidine ring were the key common reactions in the VPP, pyrrolidine and pyridine pathways. Besides, VPP and pyrrolidine pathway shared the same latter part of metabolic pathway. After analysis of metabolic genes in the pyridine, pyrrolidine, and VPP pathways, we found that both the evolutionary features and metabolic mechanisms of the VPP were more similar to the pyrrolidine pathway. The linked ndpHFEG genes shared by the VPP and pyrrolidine pathways indicated that these two pathways might share the same origin, but variants were observed in some bacteria. And we speculated that the pyridine pathway was distributed in Gram-positive bacteria and the VPP and pyrrolidine pathways were distributed in Gram-negative bacteria by using comprehensive homologs searching and phylogenetic tree construction.

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.

The Complete Genome Sequence of the Nicotine-Degrading Bacterium Shinella sp. HZN7

Nicotine is a natural alkaloid that is very toxic to humans. To eliminate the harmful effects of nicotine in the environment, biological methods employing microbes to degrade nicotine are required (Brandsch, 2006; Liu et al., 2015). Shinella sp. HZN7 can degrade nicotine efficiently via the variant of a pyridine and pyrrolidine pathways (VPP; Ma et al., 2013; Qiu et al., 2014, 2015). The main intermediates in this pathway include 6-hydroxy-nicotine, 6-hydroxy-N-methylmyosmine, 6-hydroxypseudooxynicotine, 6-hydroxy-3-succinoyl-pyridine, and 2,5-dihydroxypyridine. This strain is the first nicotine-degrading bacterium to be isolated from the genus Shinella. The genus Shinella was established in 2006 within the “Rhizobiaceae group” of the Alphaproteobacteria (An et al., 2006). Six species were assigned to this genus namely S. daejeonensis, S. fusca, S. granuli, S. kummerowiae, S. yambaruensis, S. zoogloeoides. However, most strains in this genus have not been identified at the species level. Shinella spp. have been isolated from various environmental samples, such as active sludge, zooplankton gut, soils, and water. They also exhibit a range of functional diversity, such as nitrogen fixation (Lin et al., 2008), assimilation of phosphite (Poehlein et al., 2016), and degradation of the toxic pollutants, 4-aminobenzenesulfonate (Biala et al., 2014), chlorothalonil (Liang et al., 2011), and pyridine (Bai et al., 2009). Until now, only three Shinella draft genomes have been deposited in GenBank (http://www.ncbi.nlm.nih.gov/genome/ genomes/32494). To further understand the molecular mechanism of nicotine degradation and advance the potential biotechnological applications of Shinella strains, we present the first complete genome sequence of Shinella sp. HZN7 and its features.

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.