Benzenesulfonamides act as open-channel blockers on KV3.1 potassium channel.

KV3.1 blockers can serve as modulators of the rate of action potential firing in neurons with high rates of firing such as those of the auditory system. We studied the effects of several bioisosteres of N-alkylbenzenesulfonamides, and molecules derived from sulfanilic acid on KV3.1 channels, heterologously expressed in L-929 cells, using the whole-cell patch-clamp technique. Only the N-alkyl-benzenesulfonamides acted as open-channel blockers on KV3.1, while molecules analogous to PABA (p-aminobenzoic acid) and derived from sulfanilic acids did not block the channel. The IC50 of six N-alkyl-benzenesulfonamides ranged from 9 to 55 µM; and the Hill coefficient suggests the binding of two molecules to block KV3.1. Also, the effects of all molecules on KV3.1 were fully reversible. We look for similar features amongst the molecules that effectively blocked the channel and used them to model a blocker prototype. We found that bulkier groups and amino-lactams decreased the effectiveness of the blockage, while the presence of NO2 increased the effectiveness of the blockage. Thus, we propose N-alkylbenzenesulfonamides as a new class of KV3.1 channel blockers.

Starvation- and xenobiotic-related transcriptomic responses of the sulfanilic acid-degrading bacterium, Novosphingobium resinovorum SA1.

Novosphingobium resinovorum SA1 was the first single isolate capable of degrading sulfanilic acid, a widely used representative of sulfonated aromatic compounds. The genome of the strain was recently sequenced, and here, we present whole-cell transcriptome analyses of cells exposed to sulfanilic acid as compared to cells grown on glucose. The comparison of the transcript profiles suggested that the primary impact of sulfanilic acid on the cell transcriptome was a starvation-like effect. The genes of the peripheral, central, and common pathways of sulfanilic acid biodegradation had distinct transcript profiles. The peripheral genes located on a plasmid had very high basal expressions which were hardly upregulated by sulfanilic acid. The genomic context and the codon usage preference of these genes suggested that they were acquired by horizontal gene transfer. The genes of the central pathways were remarkably inducible by sulfanilic acid indicating the presence of a substrate-specific regulatory system in the cells. Surprisingly, the genes of the common part of the metabolic pathway had low and sulfanilic acid-independent transcript levels. The approach applied resulted in the identification of the genes of proteins involved in auxiliary processes such as electron transfer, substrate and iron transports, sulfite oxidases, and sulfite transporters. The whole transcriptome analysis revealed that the cells exposed to xenobiotics had multiple responses including general starvation-like, substrate-specific, and substrate-related effects. From the results, we propose that the genes of the peripheral, central, and common parts of the pathway have been evolved independently.

Hydrophobic Patch of Ubiquitin is Important for its Optimal Activation by Ubiquitin Activating Enzyme E1.

Protein ubiquitination plays a role in essentially every process in eukaryotic cells. The attachment of ubiquitin (Ub) or Ub-like (UBL) proteins to target proteins is achieved by parallel but distinct cascades of enzymatic reactions involving three enzymes: E1, E2, and E3. The E1 enzyme functions at the apex of this pathway and plays a critical role in activating the C-terminus of ubiquitin or UBL, which is an essential step that triggers subsequent downstream transfer to their cognate E2s resulting in the fidelity of the Ub/UBL conjugation machinery. Despite the central role of the E1 enzyme in protein modification, a quantitative method to measure Ub/UBL activation by E1 is lacking. Here, we present a mass spectrometry-based assay to accurately measure the activation of Ub/UBL by E1 independent of the E2/E3 enzymes. Our method does not require radiolabeling of any components and therefore can be used in any biochemical laboratory having access to a mass spectrometer. This method allowed us to dissect the concerted process of E1-E2-catalyzed Ub conjugation in order to separately characterize the process of Ub activation and how it is affected by select mutations and other factors. We found that the hydrophobic patch of Ub is important for the optimal activation of Ub by E1. We further show that the blockers of the Ub-proteasome system such as ubistatin and fullerenol inhibit Ub activation by E1. Interestingly, our data indicate that the phosphorylation of Ub at the S65 position augments its activation by the E1 enzyme.

Evaluation of metabolism of azo dyes and their effects on Staphylococcus aureus metabolome.

Dyes containing one or more azo linkages are widely applied in cosmetics, tattooing, food and drinks, pharmaceuticals, printing inks, plastics, leather, as well as paper industries. Previously we reported that bacteria living on human skin have the ability to reduce some azo dyes to aromatic amines, which raises potential safety concerns regarding human dermal exposure to azo dyes such as those in tattoo ink and cosmetic colorant formulations. To comprehensively investigate azo dye-induced toxicity by skin bacteria activation, it is very critical to understand the mechanism of metabolism of the azo dyes at the systems biology level. In this study, an LC/MS-based metabolomics approach was employed to globally investigate metabolism of azo dyes by Staphylococcus aureus as well as their effects on the metabolome of the bacterium. Growth of S. aureus in the presence of Sudan III or Orange II was not affected during the incubation period. Metabolomics results showed that Sudan III was metabolized to 4-(phenyldiazenyl) aniline (48%), 1-[(4-aminophenyl) diazenyl]-2-naphthol (4%) and eicosenoic acid Sudan III (0.9%). These findings indicated that the azo bond close to naphthalene group of Sudan III was preferentially cleaved compared with the other azo bond. The metabolite from Orange II was identified as 4-aminobenzene sulfonic acid (35%). A much higher amount of Orange II (~90×) was detected in the cell pellets from the active viable cells compared with those from boiled cells incubated with the same concentration of Orange II. This finding suggests that Orange II was primarily transported into the S. aureus cells for metabolism, instead of the theory that the azo dye metabolism occurs extracellularly. In addition, the metabolomics results showed that Sudan III affected energy pathways of the S. aureus cells, while Orange II had less noticeable effects on the cells. In summary, this study provided novel information regarding azo dye metabolism by the skin bacterium, the effects of azo dyes on the bacterial cells and the important role on the toxicity and/or inactivation of these compounds due to microbial metabolism.

Isolation and characterization of Bradyrhizobium sp. 224 capable of degrading sulfanilic acid.

A bacterial strain (strain 224), which has the ability to utilize sulfanilic acid as a sole source of carbon, was isolated from soil. 16S rRNA gene sequence obtained from strain 224 exhibited 100% identical to that of species in the genus Bradyrhizobium. Strain 224 degraded 4.7 mM of sulfanilic acid and released almost the same molar concentration of sulfate ion.

Design, synthesis and application of benzyl-sulfonate biomimetic affinity adsorbents for monoclonal antibody purification from transgenic corn.

The human anti-human immunodeficiency virus (HIV) antibody 2G12 (mAb 2G12) is one of the most broadly neutralizing antibodies against HIV that recognizes a unique epitope on the surface glycoprotein gp120. In the present work, a limited affinity-ligand library was synthesized and evaluated for its ability to bind and purify recombinant mAb 2G12 expressed in transgenic corn. The affinity ligands were structural fragments of polysulfonate triazine dye Cibacron Blue 3GA (CB3GA) and represent novel lead scaffolds for designing synthetic affinity ligands. Solid phase chemistry was used to synthesize variants of CB3GA lead ligand. One immobilized ligand, bearing 4-aminobenzyl sulfonic acid (4ABS) linked on two chlorine atoms of the triazine ring (4ABS-Trz-4ABS), displayed high affinity for mAb 2G12. Absorption equilibrium, 3D molecular modelling and molecular dynamics simulation studies were carried out to provide a detailed picture of the 4ABS-Trz-4ABS interaction with mAb 2G12. This biomimetic affinity ligand was exploited for the development of a facile two-step purification protocol for mAb 2G12. In the first step of the procedure, mAb 2G12 was purified on an S-Sepharose FF cation exchanger, and in the second step, mAb 2G12 was purified using affinity chromatography on 4ABS-Trz-4ABS affinity adsorbent. Analysis of the antibody preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay showed that the mAb 2G12 was fully active and of sufficient purity suitable for analytical applications.

Glutathione-conjugated sulfanylalkanols are substrates for ABCC11 and γ-glutamyl transferase 1: a potential new pathway for the formation of odorant precursors in the apocrine sweat gland.

We have previously shown that precursors of odorous components characteristic of axillary sweat are hardly detectable or undetectable in individuals carrying the 538G > A SNP in the ABCC11 transporter gene. However, it is unclear, whether ABCC11 is directly involved in the transport of these compounds. To approach this question, transport of peptide-conjugated potential precursors of 3-methyl-3-sulfanylhexanol (3M3SH), a key determinant of axillary malodour, was measured using membrane vesicles of Sf9 insect cells overexpressing human ABCC11. Whilst no ABCC11-mediated transport was detected for the dipeptide precursor Cys-Gly-3M3SH, the glutathione conjugate of 3M3SH (SG-3M3SH) was robustly taken up by ABCC11 at a transport rate of 0.47 pmol/mg/min. Collectively, these results illuminate SG-3M3SH as a putative precursor of 3M3SH, which then may undergo intra-vesicular maturation to generate Cys-Gly-3M3SH. Critically, the apocrine sweat gland was demonstrated to express γ-glutamyl transferase 1 (GGT1) protein, which is known to catalyse the deglutamylation of glutathionyl conjugates. Additionally, we provide evidence that recombinant and isolated hepatic human GGT1 is capable of transforming SG-3M3SH to Cys-Gly-3M3SH in vitro. To sum up, we demonstrate that the functionality of ABCC11 is likely to play an important role in the generation of axillary malodour. Furthermore, we identify GGT1 as a key enzyme involved in the biosynthesis of Cys-Gly-3M3SH.

Ammonium-oxidizing bacteria facilitate aerobic degradation of sulfanilic acid in activated sludge.

Sulfanilic acid (SA) is a toxic sulfonated aromatic amine commonly found in anaerobically treated azo dye contaminated effluents. Aerobic acclimatization of SA-degrading mixed microbial culture could lead to co-enrichment of ammonium-oxidizing bacteria (AOB) because of the concomitant release of ammonium from SA oxidation. To what extent the co-enriched AOB would affect SA oxidation at various ammonium concentrations was unclear. Here, a series of batch kinetic experiments were conducted to evaluate the effect of AOB on aerobic SA degradation in an acclimatized activated sludge culture capable of oxidizing SA and ammonium simultaneously. To account for the effect of AOB on SA degradation, allylthiourea was used to inhibit AOB activity in the culture. The results indicated that specific SA degradation rate of the mixed culture was negatively correlated with the initial ammonium concentration (0-93 mM, R²= 0.99). The presence of AOB accelerated SA degradation by reducing the inhibitory effect of ammonium (≥ 10 mM). The Haldane substrate inhibition model was used to correlate substrate concentration (SA and ammonium) and oxygen uptake rate. This study revealed, for the first time, that AOB could facilitate SA degradation at high concentration of ammonium (≥ 10 mM) in an enriched activated sludge culture.

Effect of various carbon and nitrogen sources on decolorization of textile dye remazol golden yellow using bacterial species.

Textile dyes with different chemical structures are consistently used in textile industries and they are being recalcitrant xenobiotic in nature. The aim of present research is directed to finding the preference of striking carbon and nitrogen sources on remazol golden yellow decolorization. Bacterial strains were isolated, screened and tested for dye degradation of remazol golden yellow in basal medium amended with different carbon and nitrogen sources. This study was carried out for the period of 12 d at 37 degrees C. Among various carbon and nitrogen sources, starch and yeast extracts promote maximum decolorization in the medium inoculated with Bacillus. sp. (ESL-52). Nevertheless, the rate of decolorization was less in the medium amended with various carbon and nitrogen sources in the presence of Bacillus sp. (TSL-9), Micrococcus sp. (TSL-7), Pseudomonas sp. (M-1) and Staphylococcus sp. (ES-37) respectively. The results clearly showed that addition of significant organic carbon and nitrogen sources are only desirable co-substrates for bacterial dye decolorization process.

Degradation of a model azo dye in submerged anaerobic membrane bioreactor (SAMBR) operated with powdered activated carbon (PAC).

This work investigated the anaerobic degradation of the model azo dye Remazol Yellow Gold RNL in an upflow anaerobic sludge blanket reactor (UASB) and two submerged anaerobic membrane (SAMBR) bioreactors, one of which (SAMBR-1) was operated with powdered activated carbon (PAC) in its interior. The reactors were operated at 35 °C with a hydraulic retention time of 24 h in three operational phases, aimed to assess the effect of external sources of carbon (glucose) or redox mediator (yeast extract) on the removal or color and organic matter. The results showed that removal efficiencies of COD (73-94%) and color (90-94%) were higher for SAMBR-1 when compared to SAMBR-2 (operated without PAC) and UASB reactors. In addition, the presence of PAC in SAMBR-1 increased reactor stability, thereby leading to a lower accumulation of volatile fatty acids (VFA). The microfiltration membrane was responsible for an additional removal of ~50% of soluble residual COD in the form of VFA, thus improving permeate quality. On its turn, PAC exhibited the ability to adsorb byproducts (aromatic amines) of azo dye degradation as well as to act as source of immobilized redox mediator (quinone groups on its surface), thereby enhancing color removal.

Genome sequence of Ralstonia sp. strain PBA, a bacterium involved in the biodegradation of 4-aminobenzenesulfonate.

Ralstonia sp. strain PBA was isolated from textile wastewater in a coculture with Hydrogenophaga sp. strain PBC. Here we present the assembly and annotation of its genome, which may provide further insights into the mechanism of its interaction with strain PBC during 4-aminobenzenesulfonate degradation.

Genome sequence of Hydrogenophaga sp. strain PBC, a 4-aminobenzenesulfonate-degrading bacterium.

Hydrogenophaga sp. strain PBC is an effective degrader of 4-aminobenzenesulfonate isolated from textile wastewater. Here we present the assembly and annotation of its genome, which may provide further insights into its metabolic potential. This is the first announcement of the draft genome sequence of a strain from the genus Hydrogenophaga.

Cloning and functional analysis of the genes coding for 4-aminobenzenesulfonate 3,4-dioxygenase from Hydrogenophaga sp. PBC.

The gene coding for the oxygenase component, sadA, of 4-aminobenzenesulfonate (4-ABS) 3,4-dioxygenase in Hydrogenophaga sp. PBC was previously identified via transposon mutagenesis. Expression of wild-type sadA in trans restored the ability of the sadA mutant to grow on 4-ABS. The inclusion of sadB and sadD, coding for a putative glutamine-synthetase-like protein and a plant-type ferredoxin, respectively, further improved the efficiency of 4-ABS degradation. Transcription analysis using the gfp promoter probe plasmid showed that sadABD was expressed during growth on 4-ABS and 4-sulfocatechol. Heterologous expression of sadABD in Escherichia coli led to the biotransformation of 4-ABS to a metabolite which shared a similar retention time and UV/vis profile with 4-sulfocatechol. The putative reductase gene sadC was isolated via degenerate PCR and expression of sadC and sadABD in E. coli led to maximal 4-ABS biotransformation. In E. coli, the deletion of sadB completely eliminated dioxygenase activity while the deletion of sadC or sadD led to a decrease in dioxygenase activity. Phylogenetic analysis of SadB showed that it is closely related to the glutamine-synthetase-like proteins involved in the aniline degradation pathway. This is the first discovery, to our knowledge, of the functional genetic components for 4-ABS aromatic ring hydroxylation in the bacterial domain.

Sulfonated diiron complexes as water-soluble models of the [Fe-Fe]-hydrogenase enzyme active site.

A series of diiron complexes developed as fundamental models of the two-iron subsite in the [FeFe]-hydrogenase enzyme active site show water-solubility by virtue of a sulfonate group incorporated into the -SCH(2)NRCH(2)S- dithiolate unit that bridges two Fe(I)(CO)(2)L moieties. The sulfanilic acid group imparts even greater water solubility in the presence of ß-cyclodextrin, ß-CyD, for which NMR studies suggest aryl-sulfonate inclusion into the cyclodextrin cavity as earlier demonstrated in the X-ray crystal structure of 1Na·2 ß-CyD clathrate, where 1Na = Na(+)(µ-SCH(2)N(C(6)H(4)SO(3)(-))CH(2)S-)[Fe(CO)(3)](2), (Singleton et al., J. Am. Chem. Soc.2010, 132, 8870). Electrochemical analysis of the complexes for potential as electrocatalysts for proton reduction to H(2) finds the presence of ß-CyD to diminish response, possibly reflecting inhibition of structural rearrangements required of the diiron unit for a facile catalytic cycle. Advantages of the aryl sulfonate approach include entry into a variety of water-soluble derivatives from the well-known (µ-SRS)[Fe(CO)(3)](2) parent biomimetic, that are stable in O(2)-free aqueous solutions.

Enhanced biodegradation of 4-aminobenzenesulfonate in membrane bioreactor by Pannonibacter sp. W1.

4-Aminobenzenesulfonate (4-ABS), an aromatic amine and recalcitrant toxic pollutant, is widely used in the dye and pharmaceutical industry. Pannonibactersp. W1 is a specialized microbial strain which can efficiently degrade 4-ABS. This study shows the feasibility of using the specialized strain in an MBR system to treat synthetic wastewater containing large amount of 4-ABS. Due to membrane retention, the biomass concentration is able to reach 5 g/L within two months of continuous operation. Pannonibacter sp. W1 is able to adapt to the high loading rate of 1000 mg 4-ABS/L and achieve a remarkable 4-ABS removal efficiency of 99% within 6 h. Strain W1 grows well under the MBR continuous operation and remains as the dominant bacterium at the end of 60 days continuous operation. Minor membrane fouling has been detected within 40 days of operating at 15 LMH. At a flux of 25 LMH, the system experiences the 'TMP jump'. The high organic removal rate and low membrane fouling results illustrate the excellent performance of the bioaugmented MBR system in 4-ABS wastewater treatment.

Whole-cell fungal transformation of precursors into dyes.

BACKGROUND: Chemical methods of producing dyes involve extreme temperatures and unsafe toxic compounds. Application of oxidizing enzymes obtained from fungal species, for example laccase, is an alternative to chemical synthesis of dyes. Laccase can be replaced by fungal biomass acting as a whole-cell biocatalyst with properties comparable to the isolated form of the enzyme. The application of the whole-cell system simplifies the transformation process and reduces the time required for its completion. In the present work, four fungal strains with a well-known ability to produce laccase were tested for oxidation of 17 phenolic and non-phenolic precursors into stable and non-toxic dyes. RESULTS: An agar-plate screening test of the organic precursors was carried out using four fungal strains: Trametes versicolor, Fomes fomentarius, Abortiporus biennis, and Cerrena unicolor. Out of 17 precursors, nine were transformed into coloured substances in the presence of actively growing fungal mycelium. The immobilized fungal biomass catalyzed the transformation of 1 mM benzene and naphthalene derivatives in liquid cultures yielding stable and non-toxic products with good dyeing properties. The type of fungal strain had a large influence on the absorbance of the coloured products obtained after 48-hour transformation of the selected precursors, and the most effective was Fomes fomentarius (FF25). Whole-cell transformation of AHBS (3-amino-4-hydroxybenzenesulfonic acid) into a phenoxazinone dye was carried out in four different systems: in aqueous media comprising low amounts of carbon and nitrogen source, in buffer, and in distilled water. CONCLUSIONS: This study demonstrated the ability of four fungal strains belonging to the ecological type of white rot fungi to transform precursors into dyes. This paper highlights the potential of fungal biomass for replacing isolated enzymes as a cheaper industrial-grade biocatalyst for the synthesis of dyes and other commercially important products. The use of immobilized fungal biomass limits free migration of cells and facilitates their reuse in a continuous system for precursor transformation.

Application of novel bacterial consortium for biodegradation of aromatic amine 2-ABS using response surface methodology.

There is a strong need to develop purification methods for textile industrial wastewater containing toxic azo dyes. The reductive cleavage of azo dyes can be made by anaerobic bacteria, but the products of aromatic amines require an aerobic process. In this study a novel bacterial dye degrading consortium (DDC) of five isolated strains identified with 16S rRNA sequence: Proteus mirabilis (KR732288), Bacillus anthracis (KR732289), Enterobacter hormaechei (KR732290), Pseudomonas aeruginosa (KR732293) and Serratia rubidaea (KR732296) were used to aerobically decompose metabolite 2-aminobenxenesulfonic acid (2-ABS), as a model compound. The effect of three variables: temperature (28-42 °C), pH (5.0-8.0) and initial concentration of 2-ABS (5-40 ppm) was investigated in terms of degradation and chemical oxygen demand (COD) removal. Central composite design matrixand response surface methodology (RSM) were used for experimental design to evaluate theinteraction of the three process variables. The results show that up to 95% degradation and COD 90% removal are possible at optimal values of 32.4 ppm 2-ABS, pH 6.6 and a temperature of 35.7 °C. The theoretical response variables predicted by the developed RSM model was supported the experimental results. The optimized degradation of 2-ABS and COD removal were further confirmed by UV-HPLC analysis.

Degradation pathway of an anthraquinone dye catalyzed by a unique peroxidase DyP from Thanatephorus cucumeris Dec 1.

The reactants produced by action of a purified unique dye-decolorizing peroxidase, DyP, on a commercial anthraquinone dye, Reactive Blue 5, were investigated using electrospray ionization mass spectrometry (ESI-MS), thin-layer chromatography (TLC), and (1)H- and (13)C- nuclear magnetic resonance (NMR). The results of ESI-MS analysis showed that phthalic acid, a Product 2 (molecular weight 472.5), and a Product 3 (molecular weight 301.5), were produced. Product 2 and Product 3 were generated by usual peroxidase reaction, whereas phthalic acid was generated by hydrolase- or oxygenase-catalyzed reaction. One potential associated product, o-aminobenzene sulfonic acid, was found to be converted to 2,2'-disulfonyl azobenzene by ESI-MS and NMR analyses. From these results, we propose, for the first time, the degradation pathway of an anthraquinone dye by the enzyme DyP.

Mutagenicity and genotoxicity of acid yellow 17 and its biodegradation products.

Acid yellow 17 (AY17), a very important commercial azo dye used in the textile industry, was degraded by Pseudomonas putida mt-2 at a concentration of up to 200 mg/L. High-performance liquid chromatography analysis of the biodegradation media revealed the presence of 4-aminobenzensulfonic acid (4-ABS) derived from AY17 azoreduction, which attests the expression of an azoreductase by this bacterium. This amine was identified only in the medium of static incubation, which is consistent with its biotransformation under shaken incubation (i.e., aerobic conditions). The mutagenicity of AY17 and its biodegradation products was evaluated by using Salmonella typhimurium TA102 and TA104. No mutagenicity was observed in the presence or absence of a metabolic activation system (S9). In addition, the ability of tested compounds to induce DNA damage in vitro with the DNA strand scission assay was evaluated. Results showed that only static decolorization culture of AY17 showed a significant ability to induce the pKS plasmid DNA opening. The present study showed that P. putida mt-2, cultivated under aerobic conditions, was able to decolorize, and especially to detoxify, AY17.

Structural basis of malodour precursor transport in the human axilla.

Mammals produce volatile odours that convey different types of societal information. In Homo sapiens, this is now recognised as body odour, a key chemical component of which is the sulphurous thioalcohol, 3-methyl-3-sulfanylhexan-1-ol (3M3SH). Volatile 3M3SH is produced in the underarm as a result of specific microbial activity, which act on the odourless dipeptide-containing malodour precursor molecule, S-Cys-Gly-3M3SH, secreted in the axilla (underarm) during colonisation. The mechanism by which these bacteria recognise S-Cys-Gly-3M3SH and produce body odour is still poorly understood. Here we report the structural and biochemical basis of bacterial transport of S-Cys-Gly-3M3SH by Staphylococcus hominis, which is converted to the sulphurous thioalcohol component 3M3SH in the bacterial cytoplasm, before being released into the environment. Knowledge of the molecular basis of precursor transport, essential for body odour formation, provides a novel opportunity to design specific inhibitors of malodour production in humans.