Nicotine Catabolism Research Articles

Rational Design of a Flavoenzyme for Aerobic Nicotine Catabolism.

Enzymatic therapy with nicotine-degrading enzyme is a new strategy in treating nicotine addiction, which can reduce nicotine concentrations and weaken withdrawal in the rat model. However, when O2 is used as the electron acceptor, no satisfactory performance has been achieved with one of the most commonly studied and efficient nicotine-catabolizing enzymes, NicA2. To obtain more efficient nicotine-degrading enzyme, we rationally designed and engineered a flavoenzyme Pnao, which shares high structural similarity with NicA2 (RMSD = 1.143 Å) and efficiently catalyze pseudooxynicotine into 3-succinoyl-semialdehyde pyridine using O2. Through amino acid alterations with NicA2, five Pnao mutants were generated, which can degrade nicotine in Tris-HCl buffer and retained catabolic activity on its natural substrate. Nicotine-1'-N-oxide was identified as one of the reaction products. Four of the derivative mutants showed activity in rat serum and Trp220 and Asn224 were found critical for enzyme specificity. Our findings offer a novel avenue for research into aerobic nicotine catabolism and provides a promising method of generating additional nicotine-catalytic enzymes.

Cigarette Smoking and Intracranial Aneurysms: A Pilot Analysis of SNPs in the CYP2A6 Gene in the Italian Population

Cigarette smoking is a modifiable risk factor associated with formation and rupture of intracranial aneurysms (IAs). Cytochrome P450 2A6 (CYP2A6) is the main enzyme implied in catabolism of nicotine and xenobiotics, giving rise to oxidative stress products. Our study investigated the associations between specific single-nucleotide polymorphisms (SNPs) in the CYP2A6 gene and the presence of sporadic IAs in a cluster of Italian patients, as well as their rupture regarding cigarette smoking habit. Three hundred and thirty-one Italian patients with sporadic IAs were recruited in a single institution. We recorded data on clinical onset with subarachnoid hemorrhage (SAH) and smoking habit. Genetic analysis was performed with a standard procedure on peripheral blood samples: CYP2A6 ∗1B2, CYP2A6 ∗2, and CYP2A6 ∗14 SNPs were analyzed in the study group along with 150 healthy control subjects. Statistical analysis was conducted according to genetic association study guidelines. In the patient cohort, the frequency of aSAH was significantly higher in current smokers (P < 0.001; OR=17.45), regardless of the pattern of CYP2A6 SNPs. There was a correlation between IA rupture and cigarette smoking in patients with the heterozygous CYP2A6 ∗1B2 allele (P < 0.001; OR=15.47). All patients carrying the heterozygous CYP2A6 ∗14 allele had an aSAH event (100%), regardless of smoking habit, although this correlation was not statistically significant (P= 1). According to our findings, a cigarette smoker carrying a fully active CYP2A6 enzyme (heterozygous ∗1B2 allele) may have an increased risk of IA rupture compared to those with functionally less active variants: further investigation on a larger sample is needed to verify this result. The role of the heterozygous CYP2A6 ∗14 allele in aSAH is yet to be clarified.

Impact of Adolescent Nicotine Exposure in Pre- and Post-natal Oxycodone Exposed Offspring.

Perinatal exposure to prescription opioids pose a critical public health risk. Notably, research has found significant neurodevelopmental and behavioral deficits between in utero (IUO) and postnatal (PNO) oxycodone-exposed offspring but there is a notable gap in knowledge regarding the interaction of these groups to other drug exposure, particularly nicotine exposure. Nicotine's widespread use represents a ubiquitous clinical interaction that current research does not address. Children often experiment with drugs and risky behavior; therefore, adolescence is a key timepoint to characterize. This study employed an integrated systems approach to investigate escalating nicotine exposure in adolescence and subsequent nicotine withdrawal in the IUO- and PNO-offspring. Western blot analysis found synaptic protein alterations, especially upregulation of synaptophysin in IUO-withdrawal animals. RT-qPCR further validated immune dysfunction in the central nervous system (CNS). Peripheral nicotine metabolism was consistent with increased catabolism of nicotine concerning IUO animals. Lastly, behavioral assays found subtle deficits to withdrawal in nociception and anxiety-like behavior. This study showed, for the first time, the vulnerabilities of PNO- and IUO-exposed groups concerning nicotine use during early adolescence and withdrawal. Graphical Abstract.

Characterization of a novel nornicotine-degrading strain Mycolicibacterium sp. SMGY-1XX from a nornicotine-degrading consortium and preliminary elucidation of its biodegradation pathway by multi-omics analysis

Nicotine and nornicotine are all toxic alkaloids involved in the formation of carcinogenic tobacco-specific nitrosamines. Microbes play an important role in removing these toxic alkaloids and their derivatives from tobacco-polluted environments. By now, microbial degradation of nicotine has been well studied. However, limited information is available on the microbial catabolism of nornicotine. In the present study, a nornicotine-degrading consortium was enriched from a river sediment sample and characterized by metagenomic sequencing using a combination of Illumina and Nanopore technologies. The metagenomic sequencing analysis demonstrated that Achromobacter, Azospirillum, Mycolicibacterium, Terrimonas, and Mycobacterium were the dominant genera in the nornicotine-degrading consortium. A total of 7 morphologically distinct bacterial strains were isolated from the nornicotine-degrading consortium. These 7 bacterial strains were characterized by whole genome sequencing and examined for their ability to degrade nornicotine. Based on a combination of 16 S rRNA gene similarity comparisons, 16 S rRNA gene-based phylogenetic analysis, and ANI analysis, the accurate taxonomies of these 7 isolated strains were identified. These 7 strains were identified as Mycolicibacterium sp. strain SMGY-1XX, Shinella yambaruensis strain SMGY-2XX, Sphingobacterium soli strain SMGY-3XX, Runella sp. strain SMGY-4XX, Chitinophagaceae sp. strain SMGY-5XX, Terrimonas sp. strain SMGY-6XX, Achromobacter sp. strain SMGY-8XX. Among these 7 strains, Mycolicibacterium sp. strain SMGY-1XX, which has not been reported previously to have the ability to degrade nornicotine or nicotine, was found to be capable of degrading nornicotine, nicotine as well as myosmine. The degradation intermediates of nornicotine and myosmine by Mycolicibacterium sp. strain SMGY-1XX were determined and the nornicotine degradation pathway in strain SMGY-1XX was proposed. Three novel intermediates, myosmine, pseudooxy-nornicotine, and γ-aminobutyrate, were identified during the nornicotine degradation process. Further, the most likely candidate genes responsible for nornicotine degradation in Mycolicibacterium sp. strain SMGY-1XX were identified by integrating genomic analysis, transcriptomic analysis, and proteomic analysis. The findings in this study will help to expand our understanding on the microbial catabolism of nornicotine and nicotine and provide new insights into the nornicotine degradation mechanism by consortia and pure culture, laying a foundation for the application of strain SMGY-1XX for the removal, biotransformation, or detoxification of nornicotine.

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.

Guardian, Intermediary, or Perpetrator? New Insights Into Environmental Exposure, the Gut Microbiome, and Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) affects more than 25% of the population, and is now the leading cause of liver-related morbidity and mortality worldwide. Its evolution represents the net sum of interactions between genetic and environmental factors. Two ubiquitous exposures that confer added risk for NAFLD progression include cigarette smoke and ethanol exposure. What has remained enigmatic, however, is the degree, directionality, and mechanisms by which gut microbial interactions can modify NAFLD pathophysiology. Two recent studies bring new and intriguing light to this issue. Chen B, Sun L, Zeng G, et al. Gut bacteria alleviate smoking-related NASH by degrading gut nicotine. Nature 2022;610(7932):562–568. Chen et al show that nicotine accumulates in the terminal ileum of smokers vs nonsmokers, and in mice exposed to cigarette smoke or nicotine. They found that nicotine accumulation promotes ileal ceramide formation by activating AMPKα-dependent stabilization of the ceramide biosynthetic enzyme, sphingomyelin phosphodiesterase 3. Next, they identified a nicotine-metabolizing bacterial species, Bacteroides xylanisolvens, which expresses a nicotine oxidoreductase homolog, nicX. Colonizing mice with B xylanisolvens reduced intestinal nicotine, intestinal ceramide content, and nicotine-exacerbated hepatosteatosis. Finally, B xylanisolvens correlated inversely with nicotine, intestinal ceramides, and advanced liver disease in patients with NAFLD. Although the human microbiota is diverse, and the full spectrum of implicated species and nicotine-modulatory pathways are unlikely to be represented here, this work mechanistically links NAFLD pathophysiology with gut nicotine catabolism and identifies a potential therapeutic target for smokers with NAFLD through manipulation of nicotine-degrading microbes. Meijnikman AS, Davids M, Herrema H, et al. Microbiome-derived ethanol in nonalcoholic fatty liver disease. Nat Med 2022;28:2100–2106. Conversely, new data from Meijnikman et al highlight an exacerbating role for host-microbial interactions in NAFLD progression. NAFLD, by definition, precludes “significant” exogenous ethanol exposure. The gut microbiome produces endogenous ethanol, but first-pass hepatic ethanol clearance has been a barrier to quantify the extent to which microbially derived ethanol exposure contributes to NAFLD pathogenesis. Meijnikman et al address this by quantifying fasting portal blood ethanol in patients with NAFL or nonalcoholic steatohepatitis (NASH) obtained at the time of bariatric surgery. Patients with NAFL had elevated portal venous ethanol compared with subjects without steatosis in the primary cohort, and patients with NASH had elevated portal venous ethanol compared with individuals without steatosis in the external validation cohort. Mixed-meal testing (MMT) in a subsequent prospective cohort then demonstrated higher fasting and post-prandial peripheral ethanol levels in 146 body mass index–matched patients with NAFL or NASH vs healthy control subjects, and these differences were abrogated in a subset of patients treated with broad-spectrum antibiotics before MMT. In this prospective cohort, Lactobacillaceae correlated with post-prandial ethanol levels. Although the generalizability of this study is limited by its predominantly female enrollment, it nonetheless shows that hepatic first-pass metabolism previously obscured the extent of hepatic exposure to microbially derived ethanol, and that microbially derived ethanol exposure could drive advanced NAFLD, independently from exogenous exposure. To be human is to comprise at least as many bacterial as somatic cells. To our benefit or detriment, metabolic processes between the kingdoms are intertwined. Together, these studies now highlight ethanol production and nicotine catabolism as novel examples of this paradigm. Modulating these bacterial processes, or the involved species themselves, thus represents a new target to attenuate NAFLD progression. The number and nature and the interactions between our kingdoms continues to expand, and it is likely that further connections will be declared over time. Nonetheless, what should no longer surprise us is that our microbial residents not only modify our response to environmental exposures, but also might even create the exposure itself, and thus provide an opportunity for therapeutic intervention.

The enzyme pseudooxynicotine amine oxidase from Pseudomonas putida S16 is not an oxidase, but a dehydrogenase

The soil-dwelling bacterium Pseudomonas putida S16 can survive on nicotine as its sole carbon and nitrogen source. The enzymes nicotine oxidoreductase (NicA2) and pseudooxynicotine amine oxidase (Pnao), both members of the flavin-containing amine oxidase family, catalyze the first two steps in the nicotine catabolism pathway. Our laboratory has previously shown that, contrary to other members of its enzyme family, NicA2 is actually a dehydrogenase that uses a cytochrome c protein (CycN) as its electron acceptor. The natural electron acceptor for Pnao is unknown; however, within the P. putida S16 genome, pnao forms an operon with cycN and nicA2, leading us to hypothesize that Pnao may also be a dehydrogenase that uses CycN as its electron acceptor. Here we characterized the kinetic properties of Pnao and show that Pnao is poorly oxidized by O2, but can be rapidly oxidized by CycN, indicating that Pnao indeed acts as a dehydrogenase that uses CycN as its oxidant. Comparing steady-state kinetics with transient kinetic experiments revealed that product release primarily limits turnover by Pnao. We also resolved the crystal structure of Pnao at 2.60 Å, which shows that Pnao has a similar structural fold as NicA2. Furthermore, rigid-body docking of the structure of CycN with Pnao and NicA2 identified a potential conserved binding site for CycN on these two enzymes. Taken together, our results demonstrate that although Pnao and NicA2 show a high degree of similarity to flavin containing amine oxidases that use dioxygen directly, both enzymes are actually dehydrogenases.

Tobacco Root Endophytic Arthrobacter Harbors Genomic Features Enabling the Catabolism of Host-Specific Plant Specialized Metabolites.

ABSTRACTPlant roots constitute the primary interface between plants and soilborne microorganisms and harbor microbial communities called the root microbiota. Recent studies have demonstrated a significant contribution of plant specialized metabolites (PSMs) to the assembly of root microbiota. However, the mechanistic and evolutionary details underlying the PSM-mediated microbiota assembly and its contribution to host specificity remain elusive. Here, we show that the bacterial genus Arthrobacter is predominant specifically in the tobacco endosphere and that its enrichment in the tobacco endosphere is partially mediated by a combination of two unrelated classes of tobacco-specific PSMs, santhopine and nicotine. We isolated and sequenced Arthrobacter strains from tobacco roots as well as soils treated with these PSMs and identified genomic features, including but not limited to genes for santhopine and nicotine catabolism, that are associated with the ability to colonize tobacco roots. Phylogenomic and comparative analyses suggest that these genes were gained in multiple independent acquisition events, each of which was possibly triggered by adaptation to particular soil environments. Taken together, our findings illustrate a cooperative role of a combination of PSMs in mediating plant species-specific root bacterial microbiota assembly and suggest that the observed interaction between tobacco and Arthrobacter may be a consequence of an ecological fitting process.

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.

Exploration of Nicotine Metabolism in Paenarthrobacter nicotinovorans pAO1 by Microbial Proteomics.

Proteomics, or the large-scale study of proteins, is a post-genomics field that, together with transcriptomics and metabolomics, has moved the study of bacteria to a new era based on system-wide understanding of bacterial metabolic and regulatory networks. The study of bacterial proteins or microbial proteomics has found a wide array of applications in many fields of microbiology, from food, clinical, and industrial microbiology to microbial ecology and physiology. The current chapter makes a brief technical introduction into the available approaches for the large-scale study of bacterial proteins using mass-spectrometry. Furthermore, the advantages and disadvantages of using bacteria for proteomics studies are indicated as well as several example studies where MS-based bacterial proteomics had a fundamental role in deciphering the scientific question. Finally, the proteomics study of nicotine catabolism in Paenarthrobacter nicotinovorans pAO1 using nanoLC-MS/MS is given as an in-depth example for possible applications of microbial proteomics.The nicotine degradation pathway functioning in Paenarthrobacter nicotinovorans is encoded by the catabolic megaplasmid pAO1 that contains about 40 nicotine-related genes making out the nic-gens cluster. Despite the promising biotechnological potential for the production of green-chemicals, only half of the nic-genes have been experimentally linked to nicotine. In an attempt to systematically identify all the proteins involved in nicotine degradation, a gel-based proteomics approach was used to identify a total of 801 proteins when Paenarthrobacter nicotinovorans was grown on three carbon sources: citrate, nicotine and nicotine and citrate. The differences in protein abundance showed that the bacterium is able to switch between deamination and demethylation in the lower nicotine pathway based on the available C source. Several pAO1 putative genes including a hypothetical polyketide cyclase have been shown to have a nicotine-dependent expression and we hypothesize that the polyketide cyclase would hydrolyze the N1-C6 bond from the pyridine ring with the formation of alpha-keto-glutaramate. Two chromosomal proteins, a malate dehydrogenase, and a D-3-phosphoglycerate dehydrogenase were shown to be strongly upregulated when nicotine was the sole carbon source and could be related to the production of the alpha-keto-glutaramate by the polyketide cyclase.

Proteomics based analysis of the nicotine catabolism in Paenarthrobacter nicotinovorans pAO1

Paenarthrobacter nicotinovorans is a nicotine-degrading microorganism that shows a promising biotechnological potential for the production of compounds with industrial and pharmaceutical importance. Its ability to use nicotine was linked to the presence of the catabolic megaplasmid pAO1. Although extensive work has been performed on the molecular biology of nicotine degradation in this bacterium, only half of the genes putatively involved have been experimentally linked to nicotine. In the current approach, we used nanoLC–MS/MS to identify a total of 801 proteins grouped in 511 non-redundant protein clusters when P. nicotinovorans was grown on citrate, nicotine and nicotine and citrate as the only carbon sources. The differences in protein abundance showed that deamination is preferred when citrate is present. Several putative genes from the pAO1 megaplasmid have been shown to have a nicotine-dependent expression, including a hypothetical polyketide cyclase. We hypothesize that the enzyme would hydrolyze the N1-C6 bond from the pyridine ring with the formation of α-keto- glutaramate. Two chromosomally-encoded proteins, a malate dehydrogenase, and a D-3-phosphoglycerate dehydrogenase were shown to be strongly up-regulated when nicotine was the sole carbon source and could be related to the production the α-keto-glutarate. The data have been deposited to the ProteomeXchange with identifier PXD008756.

NdpT encodes a new protein involved in nicotine catabolism by Sphingomonas melonis TY.

Sphingomonas melonis TY utilizes nicotine as a sole source of carbon, nitrogen, and energy to grow. One of the genes in its ndp catabolic cluster, ndpT, encodes a hypothetical transporter. Since no transporter for nicotine has been identified in microorganisms, we investigated whether NdpT is responsible for nicotine transport. ndpT was induced by nicotine, and gene knockout and complementation studies clearly indicated that ndpT is essential for the catabolism of nicotine in strain TY. NdpT-GFP was located at the periphery of the cells, suggesting that NdpT is a membrane protein. Uptake assays with L-[14C] nicotine illustrated that nicotine uptake in strain TY is mediated by a constitutively synthesized permease with a Km of 0.362 ± 0.07μM and a Vmax of 0.762 ± 0.068μmolmin-1 (mg cell dry weight)-1 and that ndpT may play a role in nicotine exclusion. Hence, we consider NdpT a nicotine catabolism-related protein.

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.

Crystallography Coupled with Kinetic Analysis Provides Mechanistic Underpinnings of a Nicotine-Degrading Enzyme.

Nicotine oxidoreductase (NicA2) is a bacterial flavoenzyme, which catalyzes the first step of nicotine catabolism by oxidizing S-nicotine into N-methyl-myosmine. It has been proposed as a biotherapeutic for nicotine addiction because of its nanomolar substrate binding affinity. The first crystal structure of NicA2 has been reported, establishing NicA2 as a member of the monoamine oxidase (MAO) family. However, substrate specificity and structural determinants of substrate binding and/or catalysis have not been explored. Herein, analysis of the pH-rate profile, single-turnover kinetics, and binding data establish that pH does not significantly affect the catalytic rate and product release is not rate-limiting. The X-ray crystal structure of NicA2 with S-nicotine refined to 2.65 Å resolution reveals a hydrophobic binding site with a solvent exclusive cavity. Hydrophobic interactions predominantly orient the substrate, promoting the binding of a deprotonated species and supporting a hydride-transfer mechanism. Notably, NicA2 showed no activity against neurotransmitters oxidized by the two isoforms of human MAO. To further probe the substrate range of NicA2, enzyme activity was evaluated using a series of substrate analogues, indicating that S-nicotine is the optimal substrate and substitutions within the pyridyl ring abolish NicA2 activity. Moreover, mutagenesis and kinetic analysis of active-site residues reveal that removal of a hydrogen bond between the pyridyl ring of S-nicotine and the hydroxyl group of T381 has a 10-fold effect on KM, supporting the role of this bond in positioning the catalytically competent form of the substrate. Together, crystallography combined with kinetic analysis provides a deeper understanding of this enzyme's remarkable specificity.

Elucidating the Role of a Putative Monooxygenase in the Catabolism of Nicotinic Acid and Nicotine by Bacillus niacini

N‐Heteroaromatic compounds (NHACs) comprise a class of molecules that are commonly found in pharmaceuticals, personal care products, and pesticides and are known to have toxic effects on the environment. Intriguingly, certain species of soil‐dwelling bacteria have the innate ability to degrade particular NHACs. A better understanding of how these soil bacteria degrade NHACs has important implications in the development of bioremediation techniques to remove toxic compounds from the environment. This investigation focuses on developing preexisting hypotheses in regards to how Bacillus niacini, a ubiquitous species of soil bacteria, degrades nicotinic acid (NA) and nicotine, both of which are NHACs. It has been hypothesized that the non‐canonical NA degradation pathway in B. niacini involves a reaction in which 2,6‐dihydroxynicotinic acid undergoes oxidative decarboxylation to form 2,3,6‐trihydroxypyridine by a enzyme termed NicC‐like. The identity of the nicC‐like gene has yet to be established. A novel transformation technique was developed to generate a B. niacini knock out variant lacking the putative nicC gene. Though there are hypotheses regarding the identity of the nicC‐like gene and its involvement in NA catabolism, sequence analysis indicates that the nicC‐like gene is only 21% similar to the nicC gene involved in NA degradation in Pseudomonas putida. In contrast, the nicC‐like gene is 24% similar to HspB, which encodes an enzyme involved in nicotine degradation. Additionally, the structure and the associated catabolic processes of nicotine and NA are similar, suggesting that the novel nicC‐like gene in B. niacini might play a role in either nicotine or NA degradation. Preliminary HPLC evidence indicates that B. niacini does not degrade nicotine. Thus, the genes currently hypothesized to encode the genes necessary for NA catabolism do not code for nicotine degradation enzymes. Characterizing the reaction catalyzed by the putative NicC‐like enzyme and determining whether or not B. niacini catabolizes nicotine has led to an improved understanding about the genes necessary for NA degradation by this particular species of pervasive soil bacteria. Continued contributions to this field may eventually lead to the development of techniques to remove NHACs from the environment.Support or Funding InformationHenry J. Copeland Fund for Independent Study at the College of WoosterThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Catabolism of Nicotinic Acid and Nicotine and the Role of a Putative Monooxygenase in Bacillus niacini

N‐Heteroaromatic compounds (NHACs) comprise a class of molecules that are commonly found in pharmaceuticals, personal care products, and pesticides and are known to have toxic effects on the environment. Intriguingly, certain species of soil‐dwelling bacteria have the innate ability to degrade particular NHACs. A better understanding of how these soil bacteria degrade NHACs has important implications in the development of bioremediation techniques to remove toxic compounds from the environment. This investigation focuses on developing preexisting hypotheses in regards to how Bacillus niacini, a ubiquitous species of soil bacteria, degrades nicotinic acid (NA) and nicotine, both of which are NHACs. It has been hypothesized that the non‐canonical NA degradation pathway in B. niacini involves a reaction in which 2,6‐dihydroxynicotinic acid (2,6‐DHNA) undergoes oxidative decarboxylation to form 2,3,6‐trihydroxypyridine by an enzyme termed NicC‐like. Two methods have been utilized to establish the identity of the nicC‐like gene. First, a novel transformation technique was developed to generate a B. niacini knock out variant lacking the putative nicC gene. Second, the substrate for the NicC‐like enzyme, 2,6‐DHNA, was synthesized with a 60% yield. The substrate was utilized to assess the kinetic activity of the NicC‐like enzyme. Though there are hypotheses regarding the identity of the nicC‐like gene and its involvement in NA catabolism, sequence analysis indicates that the nicC‐like gene is only 21% similar to the nicC gene involved in NA degradation in Pseudomonas putida. In contrast, the nicC‐like gene is 24% similar to HspB, which encodes an enzyme involved in nicotine degradation. Additionally, the structure and the associated catabolic processes of nicotine and NA are similar, suggesting that the novel nicC‐like gene in B. niacini might play a role in either nicotine or NA degradation. Preliminary HPLC evidence indicates that B. niacini does not degrade nicotine. Furthermore, the NicC‐like enzyme is not kinetically active when incubated with the substrate of the HspB enzyme, 6‐Hydroxyl‐3‐succinoyl‐pyridine. Thus, the genes currently hypothesized to encode the enzymes necessary for NA catabolism do not code for nicotine degradation enzymes. Characterizing the reaction catalyzed by the putative NicC‐like enzyme and determining whether or not B. niacini catabolizes nicotine has led to an improved understanding about the genes necessary for NA degradation by this particular species of pervasive soil bacteria. Continued contributions to this field may eventually lead to the development of techniques to remove NHACs from the environment.Support or Funding InformationHenry J. Copeland Fund for Independent Study at the College of WoosterThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Investigating putative key catalytic residues and uncoupled hydroperoxyflavin formation in the mechanism of 6‐hydroxynicotinate‐3‐monooxygenase, a decarboxylative‐hydroxylase in bacterial nicotinate catabolism

Agrochemicals, pharmaceuticals and personal care products (APPCPs) are contaminants of emerging concern that are ineffectively remediated by current methodologies. Many APPCPs contain N‐heterocyclic aromatic constituents (NHACs) that are persistent contaminants in aquatic environments and cause adverse effects in non‐target organisms. Understanding bacterial degradation of NHACs may assist future bioremediation techniques. Aerobic bacterial catabolism of nicotinic acid (NA) serves as a model for understanding the biodegradation of NHACs, and a key enzyme of this pathway is 6‐hydroxynicotinate 3‐monooxygenase (NicC). We recently published the first crystal structure and two postulated mechanisms for NicC. Both mechanisms involve acid‐base catalysis, in which active site residue His211 may serve as a key H‐bond donor. Additionally, the Cys202 active site residue may donate a thiol group, playing a role in one postulated mechanism. Kinetic characterization of the H211A and C202A variants shows that the apparent substrate affinity and catalytic turnover are largely unaffected by mutagenesis of these bases (H211A: KM of 6HNA increases 2.1–fold, KM of NADH increases 2.3–fold, kcat increases 30% compared to WT; C202A: KM of 6HNA increases 2.4–fold, kcat drops 15‐fold). These data suggest that the His211 and Cys202 residues are not critical for catalysis. 6‐Hydroxynicotinaldehyde (6‐HNAH), a competitive inhibitor of NicC with respect to 6‐HNA, exhibits tight binding (KD of 6HNAH = 36 ± 2 μM) compared to 6HNA (KD of 6HNA = 190 ± 25 μM), but shows weak inhibition (Ki of 6HNAH = 8.5 ± 2.4 mM). These results are consistent with the possibility that the formation of hydroperoxyflavin (FADHOOH) is uncoupled from the oxidative decarboxylation reaction, and subsequently produces hydrogen peroxide. Hydrogen peroxide production was detected for reactions of WT+6HNAH and H211A+6HNAH, but not detected for WT+6HNA and H211A+6HNA, suggesting that 6HNAH causes uncoupling and His211 is not a key catalytic residue in the mechanism of NicC.Support or Funding InformationCollege of Wooster Sophomore Research ProgramThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Bordetella bronchiseptica nic locus encodes a nicotinic acid degradation pathway and the 6-hydroxynicotinate-responsive regulator BpsR.

The classical Bordetella species use amino acids as carbon sources and can catabolize organic acids and tricarboxylic acid cycle intermediates. They are also auxotrophic for nicotinamide adenine dinucleotide (NAD) pathway precursors such as nicotinic acid. Bordetellae have a putative nicotinate catabolism gene locus highly similar to that characterized in Pseudomonas putida KT2440. This study determined the distribution of the nic genes among Bordetella species and analyzed the regulation of this nicotinic acid degradation system. Transcription of the Bordetella bronchiseptica nicC gene was repressed by the NicR ortholog, BpsR, previously shown to regulate extracellular polysaccharide synthesis genes. nicC expression was derepressed by nicotinic acid or by the first product of the degradation pathway, 6-hydroxynicotinic acid, which was shown to be the inducer. Results using mutants with either a hyperactivated pathway or an inactivated pathway showed a marked effect on growth on nicotinic acid that indicated this degradation pathway influences NAD biosynthesis. Pathway dysregulation also affected Bordetella BvgAS-mediated virulence gene regulation, demonstrating that fluctuation of intracellular nicotinic acid pools impacts Bvg phase transition responses.

A eukaryotic nicotinate-inducible gene cluster: convergent evolution in fungi and bacteria.

Nicotinate degradation has hitherto been elucidated only in bacteria. In the ascomycete Aspergillus nidulans, six loci, hxnS/AN9178 encoding the molybdenum cofactor-containing nicotinate hydroxylase, AN11197 encoding a Cys2/His2 zinc finger regulator HxnR, together with AN11196/hxnZ, AN11188/hxnY, AN11189/hxnP and AN9177/hxnT, are clustered and stringently co-induced by a nicotinate derivative and subject to nitrogen metabolite repression mediated by the GATA factor AreA. These genes are strictly co-regulated by HxnR. Within the hxnR gene, constitutive mutations map in two discrete regions. Aspergillus nidulans is capable of using nicotinate and its oxidation products 6-hydroxynicotinic acid and 2,5-dihydroxypyridine as sole nitrogen sources in an HxnR-dependent way. HxnS is highly similar to HxA, the canonical xanthine dehydrogenase (XDH), and has originated by gene duplication, preceding the origin of the Pezizomycotina. This cluster is conserved with some variations throughout the Aspergillaceae. Our results imply that a fungal pathway has arisen independently from bacterial ones. Significantly, the neo-functionalization of XDH into nicotinate hydroxylase has occurred independently from analogous events in bacteria. This work describes for the first time a gene cluster involved in nicotinate catabolism in a eukaryote and has relevance for the formation and evolution of co-regulated primary metabolic gene clusters and the microbial degradation of N-heterocyclic 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.