Structural, Mechanistic, and Functional Insights into an Arthrobacter nicotinovorans Molybdenum Hydroxylase Involved in Nicotine Degradation

Arthrobacter nicotinovorans decomposes nicotine through the pyridine pathway. 6-hydroxypseudooxynicotine 2-oxidoreductase (also named ketone dehydrogenase, Kdh) is an important enzyme in nicotine degradation pathway of A. nicotinovorans, and is responsible for the second hydroxylation of nicotine. Kdh belongs to the molybdenum hydroxylase family, and catalyzes the oxidation of 6-hydroxy-pseudooxynicotine (6-HPON) to 2,6-dihydroxy-pseudooxynicotine (2,6-DHPON). We determined the crystal structure of the Kdh holoenzyme from A. nicotinovorans, with its three subunits KdhL, KdhM, and KdhS, and their associated cofactors molybdopterin cytosine dinucleotide (MCD), two iron-sulfur clusters (Fe2S2), and flavin adenine dinucleotide (FAD), respectively. In addition, we obtained a structural model of the substrate 6-HPON-bound Kdh through molecular docking, and performed molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations to unveil the catalytic mechanism of Kdh. The residues Glu345, Try551, and Glu748 of KdhL were found to participate in substrate binding, and Phe269 and Arg383 of KdhL were found to contribute to stabilize the MCD conformation. Furthermore, site-directed mutagenesis and enzymatic activity assays were performed to support our structural and computational results, which also revealed a trend of increasing catalytic efficiency with the increase in the buffer pH. Lastly, our electrochemical results demonstrated electron transfer among the various cofactors of Kdh. Therefore, our work provides a comprehensive structural, mechanistic, and functional study on the molybdenum hydroxylase Kdh in the nicotine degradation pathway of A. nicotinovorans.

Dysregulated Metabolites Serve as Novel Biomarkers for Metabolic Diseases Caused by E-Cigarette Vaping and Cigarette Smoking

Metabolites are essential intermediate products in metabolism, and metabolism dysregulation indicates different types of diseases. Previous studies have shown that cigarette smoke dysregulated metabolites; however, limited information is available with electronic cigarette (e-cig) vaping. We hypothesized that e-cig vaping and cigarette smoking alters systemic metabolites, and we propose to understand the specific metabolic signature between e-cig users and cigarette smokers. Plasma from non-smoker controls, cigarette smokers, and e-cig users was collected, and metabolites were identified by UPLC–MS (ultra-performance liquid chromatography mass spectrometer). Nicotine degradation was activated by e-cig vaping and cigarette smoking with increased concentrations of cotinine, cotinine N-oxide, (S)-nicotine, and (R)-6-hydroxynicotine. Additionally, we found significantly decreased concentrations in metabolites associated with tricarboxylic acid (TCA) cycle pathways in e-cig users versus cigarette smokers, such as d-glucose, (2R,3S)-2,3-dimethylmalate, (R)-2-hydroxyglutarate, O-phosphoethanolamine, malathion, d-threo-isocitrate, malic acid, and 4-acetamidobutanoic acid. Cigarette smoking significant upregulated sphingolipid metabolites, such as D-sphingosine, ceramide, N-(octadecanoyl)-sphing-4-enine, N-(9Z-octadecenoyl)-sphing-4-enine, and N-[(13Z)-docosenoyl]-sphingosine, versus e-cig vaping. Overall, e-cig vaping dysregulated TCA cycle-related metabolites while cigarette smoking altered sphingolipid metabolites. Both e-cig and cigarette smoke increased nicotinic metabolites. Therefore, specific metabolic signatures altered by e-cig vaping and cigarette smoking could serve as potential systemic biomarkers for early pathogenesis of cardiopulmonary diseases.

Dysregulated Metabolites Serve as Novel Biomarkers for Metabolic Diseases Caused by Vaping and Cigarette Smoking

Metabolites are essential intermediate products in metabolism, and metabolism dysregulation indicates different types of diseases. Previous studies have shown that cigarette smoke dysregulated metabolites; however, limited information is available with electronic cigarette (E-cig) vaping. We hypothesized that E-cig vaping and cigarette smoking altered systemic metabolites, and we propose to understand the specific metabolic signature between E-cig users and cigarette smokers. Plasma from non-smoker controls, cigarette smokers, and e-cig users were collected, and metabolites were identified by UPLC–MS (Ultraperformance liquid chromatography-mass spectrometer). Nicotine degradation was activated by e-cig vaping and cigarette smoking with increased concentrations of cotinine, cotinine N-oxide, (S)-nicotine, and (R)-6-hydroxynicotine. Additionly, we found significant decreased concentrations in metabolites associated with tricarboxylic acid (TCA) cycle pathways in e-cig users verses cigarette smokers, such as: D-glucose, (2R,3S)-2,3-dimethylmalate, (R)-2-hydroxyglutarate, O-phosphoethanolamine, malathion, D-threo-isocitrate, malic acid, and 4-acetamidobutanoic acid. Cigarette smoking significant up-regulated sphingolipid metabolites, such as D-sphingosine, ceramide, N-(octadecanoyl)-sphing-4-enine, N-(9Z-octadecenoyl)-sphing-4-enine, and N-[(13Z)-docosenoyl]sphingosine, verses e-cig vaping. Overall, e-cig vaping dysregulated TCA cycle realted metabolites while cigarette smoking altered sphingolipid metabolites. Both e-cig and cigarette smoke increased nicotinic metabolites. Therefore, specific metabolic signature altered by e-cig vaping and cigarette smoking could serve as potential systemic biomarkers for early cardiopulmonary diseases.

Type VI secretion system facilitates fitness, homeostasis, and competitive advantages for environmental adaptability and efficient nicotine biodegradation

Gram-negative bacteria employ secretion systems to translocate proteinaceous effectors from the cytoplasm to the extracellular milieu, thus interacting with the surrounding environment or micro-niche. It is known that bacteria can benefit from type VI secretion system (T6SS) by transporting ions to combat reactive oxygen species (ROS). Here, we report that T6SS activities conferred bacterial tolerance to nicotine-induced oxidative stress in Pseudomonas sp. strain JY-Q, a highly active nicotine degradation strain isolated from tobacco waste extract. AA098_13375 was identified to encode a dual-functional effector with anti-microbial and anti-ROS activities. Wild type strain JY-Q grew better than AA098_13375 deletion mutant in nicotine-containing medium by antagonizing increased intracellular ROS levels. It was, therefore, tentatively designated as TseN (Type VI Secretion system Effector for Nicotine tolerance), of which homologs were observed to be broadly ubiquitous in Pseudomonas species. TseN was identified as a Tse6-like bacteriostatic toxin via monitoring intracellular NAD+. TseN presented potential antagonism against ROS to fine tune the heavy traffic of nicotine metabolism in strain JY-Q. It is feasible that the dynamic tuning of NAD+ driven by TseN could satisfy demands from nicotine degradation with less cytotoxicity. In this scenario, T6SS involves a fascinating accommodation cascade that prompts constitutive biotransformation of N-heterocyclic aromatics by improving bacterial robustness/growth. In summary, T6SS in JY-Q mediated resistance to oxidative stress and promoted bacterial fitness via a contact-independent growth competitive advantage besides the well-studied T6SS-dependent antimicrobial activities. IMPORTANCE Mixtures of various pollutants and co-existence of numerous species of organisms are usually found in the adverse environments. Scientific community concerning biodegradation of nitrogen-heterocyclic contaminants commonly focused on screening functional enzymes transforming pollutants into intermediates of attenuated toxicity or for primary metabolism. Here we identified dual roles of T6SS effector TseN in Pseudomonas sp. JY-Q capable of degrading nicotine. T6SS could deliver TseN to kill competitors, and provide growth advantage by contact-independent pattern. TseN could monitor intracellular NAD+ level by its hydrolase activity, conferring cytotoxicity on competitive rivals but metabolic homeostasis on JY-Q. Moreover, JY-Q could be protected from TseN toxicity by its immunity protein TsiN. In conclusion, we found that TseN with cytotoxicity to bacterial competitors facilitated nicotine tolerance of JY-Q. We therefore revealed a working model between T6SS and nicotine metabolism. This finding indicates that multiple diversified weapons have been evolved by bacteria for their growth and robustness.

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, i.e., 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 is produced in the reaction, which may be toxic to the metabolism. We found that a Rid protein (designated as 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 presumable imine intermediate produced in the Pno reaction, and that, in the presence of Rid-NC, Pno maintained a high activity and the amount of the reaction product was increase by at least 5-fold. Disruption of the rid-NC gene caused a 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 presumable 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 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.

Additional role of nicotinic acid hydroxylase for the transformation of 3-succinoyl-pyridine by Pseudomonas sp. JY-Q

Nicotine and nicotinic acid (NA) are both considered to be representatives of N-heterocyclic aromatic compounds, and their degradation pathways have been revealed in Pseudomonas species. However, the co-occurrence of these two pathways is only observed in Pseudomonas sp. JY-Q. Nicotine pyrrolidine catabolism pathway of strain JY-Q consists of function modules Nic1, Spm, and Nic2. The module enzyme, 3-succinoylpyridine monooxygenases (Spm), catalyzes transformation of 3-succinoyl-pyridine (SP) to 6-hydroxy-3-succinoyl-pyridine (HSP). There exist two homologous but not identical Spm (namely Spm1 and Spm2) in JY-Q. However, when spm1 and spm2 both were in-frame deleted, the mutant still grew well in the basic salt medium supplemented with nicotine as the sole carbon/nitrogen nutrition, suggesting that there exists an alternative pathway responsible for SP catabolism in JY-Q. NicAB, an enzyme accounting for NA hydroxylation, contains re-organized domains similar to that of Spm. When JY-Q_nicAB gene was introduced into another Pseudomonas strain, which is unable to degrade NA, the resultant recombinant strain exhibited the ability transforming SP to HSP except the ability NA metabolism. Here, we ccan conclude that NicAB in strain JY-Q exhibits an additional role of SP transforming. The other genes in NA cluster, NicXDFE (Nic2 homolog), then also exhibit the role of HSP subsequent metabolism for energy yield. This finding also suggests that the co-occurrence of nicotine and NA degradation genes in strain JY-Q represents a contribution for JY-Q making it more effective and flexible for the degradation of nicotine. Importance 3-Succinoyl-pyridine (SP) and 6-hydroxy-3-succinoyl-pyridine (HSP) both are valuable chemical precursors to produce insecticides and hypotensive agents. SP and HSP could be renewable through the nicotine microbial degradation pathway in which 3-succinoylpyridine monooxygenases (Spm) account for transforming SP into HSP in Pseudomonas sp. JY-Q. However, when two homologous Spm genes (spm1 and spm2) were knocked out, the mutant kept the ability to degrade nicotine. Thus, in addition to Spm, JY-Q should have alternative pathway for SP conversion. In this research, we showed that JY-Q_NicAB was responsible for this alternative SP conversion. Both of the primary functions for nicotinic acid dehydrogenation and the additional function for SP metabolism were detected in a recombinant strain harboring JY-Q_NicAB. As a result, both nicotinic acid and nicotine degradation pathways in JY-Q contribute to its remarkable nicotine tolerance and nicotine degradation availability. These findings also provide one more metabolic engineering strategy for accumulation for value-added intermediates.

Differential effect of homologous NicR2A/NicR2Bs and endogenous ectopic strong promoters on nicotine metabolism in Pseudomonas sp. JY-Q

Nicotine is a toxic environmental pollutant that widely exists in tobacco wastes. As a natural nicotine-degrading strain, Pseudomonas sp. JY-Q still has difficulties to degrade high-concentrations of nicotine. In this study, we investigated the effect of two homologous transcriptional regulators and endogenous ectopic strong promoters on the efficiency of nicotine degradation. Comparative genomics analysis showed that two homologous transcriptional regulators NicR2A and NicR2Bs can repress nicotine-degrading genes expression. When both of nicR2A and nicR2Bs were deleted, the resulting mutant QΔnicR2AΔnicR2B1ΔnicR2B2 exhibit 17% higher nicotine degradation efficiency than wide type JY-Q. The RNA-seq analysis showed that the transcription level (FPKM value) of six genes was particularly higher than the other genes in JY-Q. Based on the genetic organization of these genes, three putative promoters, PRS28250, PRS09985 and PRS24685, were identified. Their promoter activities were evaluated by comparing their expression levels using RT-qPCR. We found that the transcription levels of RS28250, RS09985 and RS24685 were 16.8-, 2.6-, and 1.6-times higher than that of hspB2, encoding 6-hydroxy-3-succinylpyridine hydroxylase involved in nicotine degradation. Thus, two strong endogenous promoters PRS28250 and PRS09985 were selected to replace the original promoters of Nic2 gene clusters. The effect of endogenous ectopic promoter was also related to the replaced position of target gene clusters. When the promoter PRS28250 replaced the promoter of hspB2, the resultant mutant, QΔABs-ΔPhspB2::PRS28250, exhibited 69% higher nicotine degrading efficiency than the JY-Q. This research suggests a feasible strategy to enhance strain ability by removal of repressing regulatory proteins and replacing target promoter with strong endogenous ectopic promoters. IMPORTANCE This study evaluated the differential effects of homologous NicR2A/NicR2Bs and endogenous ectopic strong promoters on nicotine metabolism in Pseudomonas sp. JY-Q. Based on our differential analysis, a feasible strategy is presented to modify wild type strain JY-Q by removing repressing regulatory proteins, NicR2A/NicR2Bs, and replacing the target promoter with strong endogenous ectopic promoters. The resulting mutants exhibited high tolerance and degradation of nicotine. These findings should be beneficial for improving pollutant-degrading capacity of naturally strains through genomic modification.

Molecular Deceleration Regulates Toxicant Release to Prevent Cell Damage in Pseudomonas putida S16 (DSM 28022)

ABSTRACT The underlying molecular mechanisms of flavin-dependent amine oxidases remain relatively poorly understood, even though many of these enzymes have been reported. The nicotine oxidoreductase NicA2 is a crucial enzyme for the first step of nicotine degradation in Pseudomonas putida S16 (DSM 28022). Here, we present the crystal structure of a ternary complex comprising NicA2 residues 21 to 482, flavin adenine dinucleotide (FAD), and nicotine at 2.25 Å resolution. Unlike other, related structures, NicA2 does not have an associated diacyl glycerophospholipid, wraps its substrate more tightly, and has an intriguing exit passage in which nine bulky amino acid residues occlude the release of its toxic product, pseudooxynicotine (PN). The replacement of these bulky residues by amino acids with small side chains effectively increases the catalytic turnover rate of NicA2. Our results indicate that the passage in wild-type NicA2 effectively controls the rate of PN release and thus prevents its rapid intracellular accumulation. It gives ample time for PN to be converted to less-harmful substances by downstream enzymes such as pseudooxynicotine amine oxidase (Pnao) before its accumulation causes cell damage or even death. The temporal metabolic regulation mode revealed in this study may shed light on the production of cytotoxic compounds. IMPORTANCE Flavin-dependent amine oxidases have received extensive attention because of their importance in drug metabolism, Parkinson’s disease, and neurotransmitter catabolism. However, the underlying molecular mechanisms remain relatively poorly understood. Here, combining the crystal structure of NicA2 (an enzyme in the first step of the bacterial nicotine degradation pathway in Pseudomonas putida S16 (DSM 28022)), biochemical analysis, and mutant construction, we found an intriguing exit passage in which bulky amino acid residues occlude the release of the toxic product of NicA2, in contrast to other, related structures. The selective product exportation register for NicA2 has proven to be beneficial to cell growth. Those seeking to produce cytotoxic compounds could greatly benefit from the use of such an export register mechanism.

Functional analysis of the ocnE gene involved in nicotine-degradation pathways in Ochrobactrum intermedium SCUEC4 and its enzymatic properties

The SCUEC4 strain of Ochrobactrum intermedium is a newly isolated bacterium that degrades nicotine can use nicotine as the sole carbon source via a series of enzymatic catalytic processes. The mechanisms underlying nicotine degradation in this bacterium and the corresponding functional genes remain unclear. Here, we analyzed the function and biological properties of the ocnE gene involved in the nicotine-degradation pathways in strain SCUEC4. The ocnE gene was cloned by PCR with total DNA of strain SCUEC4 and used to construct the recombinant plasmid pET28a-ocnE. The overexpression of the OcnE protein was detected by SDS–PAGE analysis, and study of the function of this protein was spectrophotometrically carried out by monitoring the changes of 2,5-dihydroxypyridine. Moreover, the effects of temperature, pH, and metal ions on the biological activities of the OcnE protein were analyzed. The optimal conditions for the biological activities of OcnE, a protein of approximately 37.6 kDa, were determined to be 25 °C, pH 7.0, and 25 μmol/L Fe2+, and the suitable storage conditions for the OcnE protein were 0 °C and pH 7.0. In conclusion, the ocnE gene is responsible for the ability of 2,5-dihydroxypyridine dioxygenase. These findings will be beneficial in clarifying the mechanisms of nicotine degradation in O. intermedium SCUEC4.