Development of a chemically defined medium for Paenibacillus polymyxa by parallel online monitoring of the respiration activity in microtiter plates.

BACKGROUND: One critical parameter in microbial cultivations is the composition of the cultivation medium. Nowadays, the application of chemically defined media increases, due to a more defined and reproducible fermentation performance than in complex media. In order, to improve cost-effectiveness of fermentation processes using chemically defined media, the media should not contain nutrients in large excess. Additionally, to obtain high product yields, the nutrient concentrations should not be limiting. Therefore, efficient medium optimization techniques are required which adapt medium compositions to the specific nutrient requirements of microorganisms. RESULTS: Since most Paenibacillus cultivation protocols so far described in literature are based on complex ingredients, in this study, a chemically defined medium for an industrially relevant Paenibacillus polymyxa strain was developed. A recently reported method, which combines a systematic experimental procedure in combination with online monitoring of the respiration activity, was applied and extended to identify growth limitations for Paenibacillus polymyxa. All cultivations were performed in microtiter plates. By systematically increasing the concentrations of different nutrient groups, nicotinic acid was identified as a growth-limiting component. Additionally, an insufficient buffer capacity was observed. After optimizing the growth in the chemically defined medium, the medium components were systematically reduced to contain only nutrients relevant for growth. Vitamins were reduced to nicotinic acid and biotin, and amino acids to methionine, histidine, proline, arginine, and glutamate. Nucleobases/-sides could be completely left out of the medium. Finally, the cultivation in the reduced medium was reproduced in a laboratory fermenter. CONCLUSION: In this study, a reliable and time-efficient high-throughput methodology was extended to investigate limitations in chemically defined media. The interpretation of online measured respiration activities agreed well with the growth performance of samples measured in parallel via offline analyses. Furthermore, the cultivation in microtiter plates was validated in a laboratory fermenter. The results underline the benefits of online monitoring of the respiration activity already in the early stages of process development, to avoid limitations of medium components, oxygen limitation and pH inhibition during the scale-up.

Improvement of multicatalytic properties of nitrilase from Paraburkholderia graminis for efficient biosynthesis of 2-chloronicotinic acid.

Nitrilases are promising biocatalysts to produce high-value-added carboxylic acids through hydrolysis of nitriles. However, since the enzymes always show low activity and sometimes with poor reaction specificity toward 2-chloronicotinonitrile (2-CN), very few robust nitrilases have been reported for efficient production of 2-chloronicotinic acid (2-CA) from 2-CN. Herein, a nitrilase from Paraburkholderia graminis (PgNIT) was engineered to improve its catalytic properties. We identified the beneficial residues via computational analysis and constructed the mutant library. The positive mutants were obtained and the activity of the "best" mutant F164G/I130L/N167Y/A55S/Q260C/T133I/R199Q toward 2-CN was increased from 0.14 × 10-3  to 4.22 U/mg. Its reaction specificity was improved with elimination of hydration activity. Molecular docking and molecular dynamics simulation revealed that the conformational flexibility, the nucleophilic attack distance, as well as the interaction forces between the enzyme and substrate were the main reason alternating the catalytic properties of PgNIT. With the best mutant as biocatalyst, 150 g/L 2-CN was completely converted, resulting in 2-CA accumulated to 169.7 g/L. When the substrate concentration was increased to 200 g/L, 203.1 g/L 2-CA was obtained with yield of 85.7%. The results laid the foundation for industrial production of 2-CA with the nitrilase-catalyzed route.

Cryo-EM structure provides insights into the dimer arrangement of the O-linked ß-N-acetylglucosamine transferase OGT.

The O-linked ß-N-acetylglucosamine modification is a core signalling mechanism, with erroneous patterns leading to cancer and neurodegeneration. Although thousands of proteins are subject to this modification, only a single essential glycosyltransferase catalyses its installation, the O-GlcNAc transferase, OGT. Previous studies have provided truncated structures of OGT through X-ray crystallography, but the full-length protein has never been observed. Here, we report a 5.3 Å cryo-EM model of OGT. We show OGT is a dimer, providing a structural basis for how some X-linked intellectual disability mutations at the interface may contribute to disease. We observe that the catalytic section of OGT abuts a 13.5 tetratricopeptide repeat unit region and find the relative positioning of these sections deviate from the previously proposed, X-ray crystallography-based model. We also note that OGT exhibits considerable heterogeneity in tetratricopeptide repeat units N-terminal to the dimer interface with repercussions for how OGT binds protein ligands and partners.

Two new lanthanide complexes with 5-(Pyrazol-1-yl)nicotinic acid: Structures and their anti-cancer properties.

Two new lanthanide complexes [PrL2(EA)2]NO3 (complex 1) and [SmL2(EA)2]NO3 (complex 2) (H2L = 5-(Pyrazol-1-yl)nicotinic acid, EA = CH3CH2OH) were synthesized. The structures were characterized by single crystal X-ray and elemental analysis. The interaction between the complex and fish sperm DNA(FS-DNA) was monitored using ultraviolet and fluorescence spectroscopy, and the binding constants were determined. Both complexes showed the ability to effectively bind DNA, and the molecular docking technology was used to simulate the binding of the complex and DNA. In addition, through the annexin V-Fluorescein Isothiocyanate(FITC)/ Propidium Iodide (PI) test experiment, tetrazollium [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) in vitro test, and cell morphology apoptosis studies, it was shown that the complex can effectively induce HeLa tumor cell apoptosis. Compared with cisplatin and complex, complex 1 shows significant cancer cell inhibition, and we hope that this new type of complex will open up new ways for the next generation of drugs in biomedical applications.

Exploration of Structure-Activity Relationship of Aromatic Aldehydes Bearing Pyridinylmethoxy-Methyl Esters as Novel Antisickling Agents.

Aromatic aldehydes elicit their antisickling effects primarily by increasing the affinity of hemoglobin (Hb) for oxygen (O2). However, challenges related to weak potency and poor pharmacokinetic properties have hampered their development to treat sickle cell disease (SCD). Herein, we report our efforts to enhance the pharmacological profile of our previously reported compounds. These compounds showed enhanced effects on Hb modification, Hb-O2 affinity, and sickling inhibition, with sustained pharmacological effects in vitro. Importantly, some compounds exhibited unusually high antisickling activity despite moderate effects on the Hb-O2 affinity, which we attribute to an O2-independent antisickling activity, in addition to the O2-dependent activity. Structural studies are consistent with our hypothesis, which revealed the compounds interacting strongly with the polymer-stabilizing αF-helix could potentially weaken the polymer. In vivo studies with wild-type mice demonstrated significant pharmacologic effects. Our structure-based efforts have identified promising leads to be developed as novel therapeutic agents for SCD.

Structural mechanism for regulation of DNA binding of BpsR, a Bordetella regulator of biofilm formation, by 6-hydroxynicotinic acid.

Bordetella bacteria are respiratory pathogens of humans, birds, and livestock. Bordetella pertussis the causative agent of whopping cough remains a significant health issue. The transcriptional regulator, BpsR, represses a number of Bordetella genes relating to virulence, cell adhesion, cell motility, and nicotinic acid metabolism. DNA binding of BpsR is allosterically regulated by interaction with 6-hydroxynicotinic acid (6HNA), the first product in the nicotinic acid degradation pathway. To understand the mechanism of this regulation, we have determined the crystal structures of BpsR and BpsR in complex with 6HNA. The structures reveal that BpsR binding of 6HNA induces a conformational change in the protein to prevent DNA binding. We have also identified homologs of BpsR in other Gram negative bacteria in which the amino acids involved in recognition of 6HNA are conserved, suggesting a similar mechanism for regulating nicotinic acid degradation.

Stability indicating liquid chromatographic method for simultaneous quantification of betamethasone valerate and tazarotene in in vitro and ex vivo studies of complex nanoformulation.

Low-potency corticosteroid betamethasone valerate and vitamin-A tazarotene are used in combination for effective treatment of psoriasis. There is no robust high-performance liquid chromatography analytical technique available for simultaneous estimation of betamethasone valerate and tazarotene in conventional and nanocarriers based formulations. A simple, accurate, robust isocratic high-performance liquid chromatography method was developed for simultaneous estimation of betamethasone valerate and tazarotene in topical pharmaceutical formulations. The developed method was validated as per the regulatory guidelines. The validated method was linear over the concentration range of 150-6000 ng/mL (r2  > 0.999) at 239 nm wavelength. Limits of detection and quantification of two analytes were 50 and 150 ng/mL, respectively. The %relative standard deviation for intraday and interday precision was less than 2%. The method was also evaluated in the presence of forced degradation conditions. The developed method was successfully applied for in vitro and ex vivo drug release studies of in-house designed nanoformulations.

Identification of N-Methyl Nicotinamide and N-Methyl Pyridazine-3-Carboxamide Pseudokinase Domain Ligands as Highly Selective Allosteric Inhibitors of Tyrosine Kinase 2 (TYK2).

As a member of the Janus (JAK) family of nonreceptor tyrosine kinases, TYK2 plays an important role in mediating the signaling of pro-inflammatory cytokines including IL-12, IL-23, and type 1 interferons. The nicotinamide 4, identified by a SPA-based high-throughput screen targeting the TYK2 pseudokinase domain, potently inhibits IL-23 and IFNα signaling in cellular assays. The described work details the optimization of this poorly selective hit (4) to potent and selective molecules such as 47 and 48. The discoveries described herein were critical to the eventual identification of the clinical TYK2 JH2 inhibitor (see following report in this issue). Compound 48 provided robust inhibition in a mouse IL-12-induced IFNγ pharmacodynamic model as well as efficacy in an IL-23 and IL-12-dependent mouse colitis model. These results demonstrate the ability of TYK2 JH2 domain binders to provide a highly selective alternative to conventional TYK2 orthosteric inhibitors.

Adsorption and degradation of imazapic in soils under different environmental conditions.

Imazapic is widely used in peanut production, and its residues can cause damage to succeeding crops planted in the following year. The planting area of peanut is large in Henan province. Inceptisol is the main soil type in Henan Province and was used in laboratory experiments that were conducted to investigate imazapic degradation in soil under various environmental conditions. The results indicated that the imazapic degradation rate increased with an increase in temperature, soil pH, and soil moisture, and decreased with organic matter content. The use of biogas slurry as a soil amendment accelerated imazapic degradation. The half-life of imazapic in sterilized soil (364.7 d) was longer than in unsterilized soil (138.6 d), which suggested that there was a significant microbial contribution to imazapic degradation. Imazapic adsorption was also examined and was found to be well described by the Freundlich isotherm. The results indicate that soil has a certain adsorption capacity for imazapic.

Tissue-specific and maturity-dependent distribution of pyridine alkaloids in Areca triandra.

Areca nuts (seeds of Areca catechu L.) are a traditional and popular masticatory in India, Bangladesh, Malaysia, certain parts of China, and some other countries. Four related pyridine alkaloids (arecoline, arecaidine, guvacoline, and guvacine) are considered being the main functional ingredients in areca nut. Until now, A. catechu is the only known species producing these alkaloids in the Arecaceae family. In the present study, we investigated alkaloid contents in 12 Arecaceae species and found that only Areca triandra Roxb. contained these pyridine alkaloids. We further analyzed in more detail tissue-specific and development-related distribution of these alkaloids in leaves, male and female flowers and fruits in different stages of maturity in A. triandra by ultra-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry. Results revealed that the alkaloids were most abundant in young leaves, the pericarp of ripe fruits and the endosperm of unripe fruits in developmental stage 2. Abundance of the 4 different alkaloids in A. triandra fruits varied during maturation. Pericarps of ripe fruits had the highest arecaidine concentration (4.45 mg g-1) and the lowest guvacoline concentration (0.0175 mg g-1), whereas the endosperm of unripe fruits of developmental stage 2 contained the highest guvacoline concentration (3.39 mg g-1) and the lowest guvacine concentration (0.245 mg g-1). We conclude that A. triandra is useful in future as a further valuable source of Areca alkaloids.

Development of a simple method for simultaneous determination of tazarotene and betamethasone dipropionate and their metabolites using LC-MS method and its application to dermatopharmacokinetic study.

In our study, a method for the determination for tazarotene and betamethasone dipropionate in human tissue-engineered skin was established. Tazarotene gel, betamethasone dipropionate cream or a combination cream was administered to the skin. Then the skin was taken off at 0.25, 0.75, 1.75, 3, 5, 8, 12, 24, 36, 48 h time points after the residual drug was removed. The concentrations of tazarotene, betamethasone dipropionate and their major metabolites in skin were determined by LC-MS. Tazarotene and tazarotenic acid were detected in the concentration range of 2-200 µg/mL with an LLOQ of 2 µg/mL. Betamethasone dipropionate was detected in the concentration range 0.5-300 µg/mL with an LLOQ of 0.5 µg/mL, and betamethasone was detected at 2-200 µg/mL with an LLOQ of 2 µg/mL. The intra- and inter-day precisions of the four analytes in the skin homogenate were all <15% (RSD, %). The results showed that tazarotene could be metabolized to tazarotenic acid and betamethasone dipropionate could be metabolized to betamethasone in tissue-engineered skin. The results also revealed that this method was suitable for the simultaneous determination of tazarotene, betamethasone dipropionate and their metabolites in tissue-engineered skin.

Metabolism and excretion of (S)-6-(3-cyclopentyl-2-(4-trifluoromethyl)-1H-imidazol-1-yl)propanamido)nicotinic acid (PF-04991532), a hepatoselective glucokinase activator, in humans: confirmation of the MIST potential noted in first-in-Human metabolite scouting studies.

1. The absorption, metabolism, and excretion of a single oral 450-mg dose of [14C]-(S)-6-(3-cyclopentyl-2-(4-trifluoromethyl)-1H-imidazol-1-yl)propanamido)nicotinic acid (PF-04991532), a hepatoselective glucokinase activator, was investigated in humans. Mass balance was achieved with ∼94.6% of the administered dose recovered in urine and feces. The total administered radioactivity excreted in feces and urine was 70.6% and 24.1%, respectively. Unchanged PF-04991532 collectively accounted for ∼47.2% of the dose excreted in feces and urine, suggestive of moderate metabolic elimination in humans. 2. The biotransformation pathways involved acyl glucuronidation (M1), amide bond hydrolysis (M3), and CYP3A4-mediated oxidative metabolism on the cyclopentyl ring in PF-04991532 yielding monohydroxylated isomers (M2a-d). Unchanged PF-04991532 was the major circulating component (64.4% of total radioactivity) whereas M2a-d collectively represented 28.9% of the total plasma radioactivity. 3. Metabolites M2a-d were not detected systemically in rats and dogs, the preclinical species for the toxicological evaluation of PF-04991532. In contrast, cynomologus monkeys dosed orally with unlabeled PF-04991532 revealed M2a-d in circulation, whose UV abundance was comparable to the profile in humans. This observation suggested that monkeys could potentially serve as a non-rodent alternative for studying the toxicity of PF-04991532 and its metabolites M2a-d. 4. The present results are in excellent agreement with our previously generated metabolite scouting data, which provided preliminary evidence for the disproportionate metabolism of PF-04991532 in humans.

Mechanism of 6-Hydroxynicotinate 3-Monooxygenase, a Flavin-Dependent Decarboxylative Hydroxylase Involved in Bacterial Nicotinic Acid Degradation.

6-Hydroxynicotinate 3-monooxygenase (NicC) is a Group A FAD-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of 6-hydroxynicotinic acid (6-HNA) to 2,5-dihydroxypyridine (2,5-DHP) with concomitant oxidation of NADH in nicotinic acid degradation by aerobic bacteria. Two mechanisms for the decarboxylative hydroxylation half-reaction have been proposed [Hicks, K., et al. (2016) Biochemistry 55, 3432-3446]. Results with Bordetella bronchiseptica RB50 NicC here show that a homocyclic analogue of 6-HNA, 4-hydroxybenzoic acid (4-HBA), is decarboxylated and hydroxylated by NicC with a 420-fold lower catalytic efficiency than is 6-HNA. The 13( V/ K), measured with wild-type NicC by isotope ratio mass spectrometry following the natural abundance of 13C in the CO2 product, is inverse for both 6-HNA (0.9989 ± 0.0002) and 4-HBA (0.9942 ± 0.0004) and becomes negligible (0.9999 ± 0.0004) for 5-chloro-6-HNA, an analogue that is 10-fold more catalytically efficient than 6-HNA. Covalently bound 6-HNA complexes of NicC are not observed by mass spectrometry. Comparative steady-state kinetic and Kd6HNA analyses of active site NicC variants (C202A, H211A, H302A, H47E, Y215F, and Y225F) identify Tyr215 and His47 as critical determinants both of 6-HNA binding ( KdY215F/ KdWT > 240; KdH47E/ KdWT > 350) and in coupling rates of 2,5-DHP and NAD+ product formation ([2,5-DHP]/[NAD+] = 1.00 (WT), 0.005 (Y215F), and 0.07 (H47E)]. Results of these functional analyses are in accord with an electrophilic aromatic substitution reaction mechanism in which His47-Tyr215 may serve as the general base to catalyze substrate hydroxylation and refine the structural model for substrate binding by NicC.

Comparison of the Photodegradation of Imazethapyr in Aqueous Solution, on Epicuticular Waxes, and on Intact Corn (Zea Mays) and Soybean (Glycine Max) Leaves.

A direct, controlled comparison of the photodegradation of imazethapyr has been made between imazethapyr in aqueous solutions, imazethapyr on the surface of epicuticular waxes of corn and soybean plants, and imazethapyr on the surface of intact corn and soybean plant leaves. In some experiments, the imazethapyr solutions were allowed to evaporate partially or fully after application to better model environmental conditions. The photodegradation of imazethapyr was fastest in aqueous solutions (k = 0.16 ± 0.02 h-1) and slowest on the surface of corn and soybean plants (kcorn = 0.00048 ± 0.001 h-1 and ksoy = 0.00054 ± 0.003 h-1). Experiments allowing evaporation during irradiation have intermediate rate constants (e.g., kcorn = 0.082 ± 0.005 h-1). Finally, identification of photoproducts was also examined on epicuticular waxes of corn and soybean plants for the first time.

Manganese tolerance in Verbascum olympicum Boiss. affecting elemental uptake and distribution: changes in nicotinic acid levels under stress conditions.

A multielemental determination methodology in conjunction with an organic acid analysis that were supplemented with other stress parameters and an ultrastructural analysis used herein to study Verbascum olympicum Boiss. (Scrophulariaceae) under Mn stress. Uptake and accumulation characteristics of B, Cu, Fe, Mn, Mo, and Zn were evaluated in 8-week-old seedlings grown in Hoagland's nutrient solution and exposed to 5 (CK), 50, and 200 µM MnSO4 for 7 days. Hydrogen peroxide levels were determined to evaluate oxidative stress, and changes in compatible substance levels (total phenolic contents, glutathione and glutathione disulfide levels) were determined to assess antioxidant defense mechanisms. The distribution of manganese on the root surface was characterized by scanning electron microscopy images and energy-dispersive X-ray spectroscopy analysis. The levels of nicotinic acid, which is involved in nicotinamide adenine dinucleotide biosynthesis, were determined in roots and leaves to assess tolerance mechanisms. V. olympicum exhibited the ability to cope with oxidative stress originating from excessive Mn, while increased Mn concentrations were observed in both roots and leaves. The translocation factor of B was the most affected among other studied elements under the experimental conditions. Total nicotinic acid levels exhibited a trend of reduction in the roots and leaves, which could be attributed to the appropriate metabolic progress associated with oxidative stress based on the nicotinamide adenine dinucleotide cycle that may reach glutathione in response to manganese stress during plant growth.

MeNA, Controlled by Reversible Methylation of Nicotinate, Is an NAD Precursor that Undergoes Long-Distance Transport in Arabidopsis.

Nicotinamide adenine dinucleotide (NAD) biosynthesis, including synthesis from aspartate via the de novo pathway and from nicotinate (NA) via the Preiss-Handler pathway, is conserved in land plants. Diverse species of NA conjugates, which are mainly involved in NA detoxification, were also found in all tested land plants. Among these conjugates, MeNA (NA methyl ester) has been widely detected in angiosperm plants, although its physiological function and the underlying mechanism for its production in planta remain largely unknown. Here, we show that MeNA is an NAD precursor undergoing more efficient long-distance transport between organs than NA and nicotinamide in Arabidopsis. We found that Arabidopsis has one methyltransferase (designated AtNaMT1) capable of catalyzing carboxyl methylation of NA to yield MeNA and one methyl esterase (MES2) predominantly hydrolyzing MeNA back to NA. We further uncovered that the transfer of [14C]MeNA from the root to leaf was significantly increased in both MES2 knockdown and NaMT1-overexpressing lines, suggesting that both NaMT1 and MES2 fine-tune the long-distance transport of MeNA, which is ultimately utilized for NAD production. Abiotic stress (salt, abscisic acid, and mannitol) treatments, which are known to exacerbate NAD degradation, induce the expression of NaMT1 but suppress MES2 expression, suggesting that MeNA may play a role in stress adaption. Collectively, our study indicates that reversible methylation of NA controls the biosynthesis of MeNA in Arabidopsis, which presumably functions as a detoxification form of free NA for efficient long-distance transport and eventually NAD production especially under abiotic stress, providing new insights into the relationship between NAD biosynthesis and NA conjugation in plants.

Hyperthermophilic Archaeon Thermococcus kodakarensis Utilizes a Four-Step Pathway for NAD+ Salvage through Nicotinamide Deamination.

Many organisms possess pathways that regenerate NAD+ from its degradation products, and two pathways are known to salvage NAD+ from nicotinamide (Nm). One is a four-step pathway that proceeds through deamination of Nm to nicotinic acid (Na) by Nm deamidase and phosphoribosylation to nicotinic acid mononucleotide (NaMN), followed by adenylylation and amidation. Another is a two-step pathway that does not involve deamination and directly proceeds with the phosphoribosylation of Nm to nicotinamide mononucleotide (NMN), followed by adenylylation. Judging from genome sequence data, the hyperthermophilic archaeon Thermococcus kodakarensis is supposed to utilize the four-step pathway, but the fact that the adenylyltransferase encoded by TK0067 recognizes both NMN and NaMN also raises the possibility of a two-step salvage mechanism. Here, we examined the substrate specificity of the recombinant TK1676 protein, annotated as nicotinic acid phosphoribosyltransferase. The TK1676 protein displayed significant activity toward Na and phosphoribosyl pyrophosphate (PRPP) and only trace activity with Nm and PRPP. We further performed genetic analyses on TK0218 (quinolinic acid phosphoribosyltransferase) and TK1650 (Nm deamidase), involved in de novo biosynthesis and four-step salvage of NAD+, respectively. The ΔTK0218 mutant cells displayed growth defects in a minimal synthetic medium, but growth was fully restored with the addition of Na or Nm. The ΔTK0218 ΔTK1650 mutant cells did not display growth in the minimal medium, and growth was restored with the addition of Na but not Nm. The enzymatic and genetic analyses strongly suggest that NAD+ salvage in T. kodakarensis requires deamination of Nm and proceeds through the four-step pathway.IMPORTANCE Hyperthermophiles must constantly deal with increased degradation rates of their biomolecules due to their high growth temperatures. Here, we identified the pathway that regenerates NAD+ from nicotinamide (Nm) in the hyperthermophilic archaeon Thermococcus kodakarensis The organism utilizes a four-step pathway that initially hydrolyzes the amide bond of Nm to generate nicotinic acid (Na), followed by phosphoribosylation, adenylylation, and amidation. Although the two-step pathway, consisting of only phosphoribosylation of Nm and adenylylation, seems to be more efficient, Nm mononucleotide in the two-step pathway is much more thermolabile than Na mononucleotide, the corresponding intermediate in the four-step pathway. Although NAD+ itself is thermolabile, this may represent an example of a metabolism that has evolved to avoid the use of thermolabile intermediates.

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.

Urinary Excretion of Niacin Metabolites in Humans After Coffee Consumption.

SCOPE: Coffee is a major natural source of niacin in the human diet, as it is formed during coffee roasting from the alkaloid trigonelline. The intention of our study was to monitor the urinary excretion of niacin metabolites after coffee consumption under controlled diet. METHODS AND RESULTS: We performed a 4-day human intervention study on the excretion of major niacin metabolites in the urine of volunteers after ingestion of 500 mL regular coffee containing 34.8 µmol nicotinic acid (NA) and 0.58 µmol nicotinamide (NAM). In addition to NA and NAM, the metabolites N1 -methylnicotinamide (NMNAM), N1 -methyl-2-pyridone-5-carboxamide (2-Py), and nicotinuric acid (NUA) were identified and quantified in the collected urine samples by stable isotope dilution analysis (SIVA) using HPLC-ESI-MS/MS. Rapid urinary excretion was observed for the main metabolites (NA, NAM, NMNAM, and 2-Py), with tmax values within the first hour after ingestion. NUA appeared in traces even more rapidly. In sum, 972 nmol h-1 of NA, NAM, NMNAM, and 2-Py were excreted within 12 h after coffee consumption, corresponding to 6% of the ingested NA and NAM. CONCLUSION: The results indicate regular coffee consumption to be a source of niacin in human diet.

Identification and characterization of a new three-component nicotinic acid hydroxylase NahAB1 B2 from Pusillimonas sp. strain T2.

Nicotinic acid (NA) is ubiquitous in nature and its microbial degradation mechanisms are diverse. In this study, Pusillimonas sp. strain T2 was found to be capable of utilizing NA as sole carbon and nitrogen sources. This strain could completely degrade 300 mg l-1 NA within 3·5 h at 30°C and pH 7·0 and one of the degradation intermediate of NA was identified as 6-hydroxynicotinic acid (6HNA). The draft genome sequences of strain T2 were determined to have a total length of 3·3 M bp and 3054 proteins were predicted. The encoding genes of three-component NA hydroxylase (NahAB1 B2 ) genes were identified. The nahAB1 B2 genes were heterologously expressed in the non-NA-degrading Shinella sp. strain HZN7. The recombinant HZN7-pBBR-nahAB1 B2 converted NA into equimolar 6HNA, while the recombinants HZN7-pBBR-nahAB1 (lacking component B2 ) and HZN7-pBBR-nahAB2 (lacking component B1 ) could not convert NA. Cell-free extracts of HZN7-pBBR-nahAB1 B2 exhibited NA hydroxylase activity. After addition of an artificial electron acceptor (such as phenazine methosulphate, PMS), the NA hydroxylase activity was significantly increased. The Km and Vmax values for NA were 65·94 µmol l-1 and 260·80 ± 5·69 mU mg-1 , respectively, using PMS as an electron acceptor. This study provides a novel insight into the NA degradation by bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Nicotinic acid (NA) serves as a model system for the degradation of N-heterocyclic aromatic compounds and the microbial degradation mechanisms are diverse. This is the first time that a three-component hydroxylase has been identified. This study provides a novel insight into the NA degradation by bacteria.